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Bennett, Vann

Overview:

A major interest of our laboratory is in understanding how cells in metazoan organisms manage to target ion channels to physiological sites that optimize their physiological efficiency. Our research began with discovery of the ankyrin family of membrane-adapter proteins, which interact with structurally diverse membrane proteins and couple these proteins to the spectrin-based membrane skeleton. Currently identified ankyrin partners are anion exchangers, the Na/K ATPase, the voltage-dependent sodium channel, and the Na/Ca exchanger. Ankyrin(s) also associate with calcium-release channels including both IP3 and ryanodine receptors. Finally, ankyrins also bind to cell adhesion molecules of the L1 CAM family (L1/neurofascin/NrCAM/NgCAM in vertebrates; neuroglian in Drosophila; LAD-1 in C. elegans). Ankyrins interact with these diverse proteins through a motif known as ANK repeats, which are found in many different proteins and operate in protein recognition for multiple structurally unrelated ligands.
We have recently reported that humans heterozygous for a E1425G loss-of-function mutation in ankyrin-B and mice heterozygous for a null mutation in ankyrin-B have type 4 long QT syndrome, a cardiac arrhythmia associated with sudden cardiac death. We also have discovered that ankyri9n-B mutation results in reduced levels of Na/Ca exchanger, Na/K ATPase, and IP3 R at T-tubule sites in cardiomyocytes and leads to altered Ca2+ signaling and extrasystoles that provide a rationale for the arrhythmia. This work has identified a new mechanism for cardiac arrhythmia due to abnormal co-ordination of multiple functionally related ion channels and transporters. We have also found that conditional knockout of ankyrin-G in the mouse cerebellum results in severe ataxia accompanied by coordinate loss of the sodium channel Nav1.6, neurofascin (a member of the L1CAM family), and beta IV spectrin from axon initial segments. These studies, together with the role of ankyrin-B in type 4 long QT syndrome, establish a physiological requirement for ankyrins in localization of a variety of ion channels in excitable membranes in the heart and nervous system, and suggest a new class of functional channelopathies due to abnormal cellular localization.
Future research will be based on the discovery that ankyrin-B and ankyrin-G have physiological roles as coordinators of multiple functionally related proteins in specialized cell membrane compartments. A major effort will be to understand mechanisms, beginning at a protein level with ankyrin-B structure and function, and including the cellular basis for ankyrin-B-dependent protein sorting in cardiomyocytes. We also plan to study the roles of ankyrins B and G in ion channel organization in the visual system using targeted gene knockouts in rods, retinal ganglion neurons, and retinal pigmented epithelial cells (mice with loss of function may be blind but should be viable).

Positions:

George B. Geller Professor for Research in Molecular Biology in the School of Medicine

Biochemistry
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1975

Ph.D. — Johns Hopkins University

M.D. 1976

M.D. — Johns Hopkins University

News:

Grants:

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2022

Multidisciplinary Heart and Vascular Diseases

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
March 31, 2021

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Control of Excitatory Synapse Formation by Huntingtin

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
May 01, 2017
End Date
April 30, 2020

Interdisciplinary Training Program in Lung Disease

Administered By
Medicine, Pulmonary, Allergy, and Critical Care Medicine
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
July 01, 2009
End Date
March 31, 2020

Deciphering high function autism using mice with human de novo ANK2 mutations

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 18, 2017
End Date
August 31, 2019

Mechanistic Basis for Regulation of the Axon Initial Segment

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 2016
End Date
May 31, 2018

Control of Tripartite Synapse Formation by Astrocytic Neuroligins

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
February 01, 2015
End Date
January 31, 2018

Center for Molecular & Cellular Studies of Ped Disease

Administered By
Pediatrics
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
April 11, 2003
End Date
November 30, 2017

Lightsheet Imaging System

Administered By
Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
March 15, 2016
End Date
March 14, 2017

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Control of Excitatory Synapse Formation and Maturation by Astrocytes

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
July 01, 2013
End Date
June 30, 2015

Institutional Training Grant in Pediatric Cardiology

Administered By
Pediatrics, Cardiology
AwardedBy
National Institutes of Health
Role
Faculty Member
Start Date
April 01, 2009
End Date
June 30, 2015

Fiber Cell Cytoskeletal Scaffolds-Role in Lens Architecture and Function

Administered By
Ophthalmology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
March 01, 1999
End Date
September 29, 2013

The Role of Ankyrin-B Mutations in Premature Senescence

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
August 15, 2011
End Date
July 31, 2013

Nanomechanics of Spiral Proteins

Administered By
Mechanical Engineering and Materials Science
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
March 01, 2008
End Date
February 28, 2013

Interdomain Interactions and Ankyrin-B Specificity

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2005
End Date
November 30, 2007

Cellular Basis for Ankyrin-B-Related Cardiac Arrhythmia

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2005
End Date
October 31, 2005

Zeiss LSM510 META confocal-fluorescence spectroscopy

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
May 01, 2003
End Date
October 30, 2004

Erythrocyte Membrane Cytoskeleton Associations

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1987
End Date
March 31, 2000

Human Erythrocyte Membrane Cytoskeleton Associations

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1995
End Date
March 31, 1999

Human Erythrocyte Membrane Cytoskeleton Associations

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1994
End Date
March 31, 1996

Human Erythrocyte Membrane Cytoskeleton Association

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1991
End Date
March 01, 1996

Protein Associations Of Brain Spectrin And Ankyrin

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1987
End Date
June 01, 1989
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Awards:

Fellow. American Association for the Advancement of Science.

Type
National
Awarded By
American Association for the Advancement of Science
Date
January 01, 2013

Fellow. Association of American Physicians.

Type
National
Awarded By
Association of American Physicians
Date
January 01, 2013

Member. National Academy of Sciences of the USA.

Type
National
Awarded By
National Academy of Sciences of the USA
Date
January 01, 2010

Fellow. American Academy of Arts and Sciences.

Type
National
Awarded By
American Academy of Arts and Sciences
Date
January 01, 2009

Merit Award. NIH.

Type
National
Awarded By
NIH
Date
January 01, 1990

Investigator. Howard Hughes Medical Institute.

Type
National
Awarded By
Howard Hughes Medical Institute
Date
January 01, 1987

Member. American Society of Clinical Investigation.

Type
National
Awarded By
American Society of Clinical Investigation
Date
January 01, 1987

Publications:

Regulation of Pancreatic Beta Cell Function by Ankyrin-B

Authors
Lorenzo, DN; Hostettler, J; Bennett, V
MLA Citation
Lorenzo, DN, Hostettler, J, and Bennett, V. "Regulation of Pancreatic Beta Cell Function by Ankyrin-B." April 2017.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
31
Publish Date
2017

Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes.

Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5β1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5β1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5β1-integrin and likely of other specialized endocytic cargos.

Authors
Qu, F; Lorenzo, DN; King, SJ; Brooks, R; Bear, JE; Bennett, V
MLA Citation
Qu, F, Lorenzo, DN, King, SJ, Brooks, R, Bear, JE, and Bennett, V. "Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes." eLife 5 (October 8, 2016).
PMID
27718357
Source
epmc
Published In
eLife
Volume
5
Publish Date
2016
DOI
10.7554/elife.20417

Common human ANK2 variant confers in vivo arrhythmia phenotypes.

Human ANK2 (ankyrin-B) loss-of-function variants are directly linked with arrhythmia phenotypes. However, in atypical non-ion channel arrhythmia genes such as ANK2 that lack the same degree of robust structure/function and clinical data, it may be more difficult to assign variant disease risk based simply on variant location, minor allele frequency, and/or predictive structural algorithms. The human ankyrin-B p.L1622I variant found in arrhythmia probands displays significant diversity in minor allele frequency across populations.The objective of this study was to directly test the in vivo impact of ankyrin-B p.L1622I on cardiac electrical phenotypes and arrhythmia risk using a new animal model.We tested arrhythmia phenotypes in a new "knock-in" animal model harboring the human ankyrin-B p.L1622I variant.Ankyrin-B p.L1622I displays reduced posttranslational expression in vivo, resulting in reduced cardiac ankyrin-B expression and reduced association with binding-partner Na/Ca exchanger. Ankyrin-B(L1622I/L1622I) mice display changes in heart rate, atrioventricular and intraventricular conduction, and alterations in repolarization. Furthermore, ankyrin-B(L1622I/L1622I) mice display catecholamine-dependent arrhythmias. At the cellular level, ankyrin-B(L1622I/L1622I) myocytes display increased action potential duration and severe arrhythmogenic afterdepolarizations that provide a mechanistic rationale for the arrhythmias.Our findings support in vivo arrhythmogenic phenotypes of an ANK2 variant with unusual frequency in select populations. On the basis of our findings and current clinical data, we support classification of p.L1622I as a "mild" loss-of-function variant that may confer arrhythmia susceptibility in the context of secondary risk factors including environment, medication, and/or additional genetic variation.

Authors
Musa, H; Murphy, NP; Curran, J; Higgins, JD; Webb, TR; Makara, MA; Wright, P; Lancione, PJ; Lubbers, ER; Healy, JA; Smith, SA; Bennett, V; Hund, TJ; Kline, CF; Mohler, PJ
MLA Citation
Musa, H, Murphy, NP, Curran, J, Higgins, JD, Webb, TR, Makara, MA, Wright, P, Lancione, PJ, Lubbers, ER, Healy, JA, Smith, SA, Bennett, V, Hund, TJ, Kline, CF, and Mohler, PJ. "Common human ANK2 variant confers in vivo arrhythmia phenotypes." Heart rhythm 13.9 (September 2016): 1932-1940.
PMID
27298202
Source
epmc
Published In
Heart Rhythm
Volume
13
Issue
9
Publish Date
2016
Start Page
1932
End Page
1940
DOI
10.1016/j.hrthm.2016.06.012

Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics.

Periaxin (Prx), a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how Prx is anchored to the plasma membrane. Here we report that ankyrin-B (AnkB), a well-characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment and decreased compressive stiffness in mouse lenses parallel to the changes observed in Prx null mouse lens. Both AnkB- and Prx-deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization, and biomechanics.

Authors
Maddala, R; Walters, M; Brophy, PJ; Bennett, V; Rao, PV
MLA Citation
Maddala, R, Walters, M, Brophy, PJ, Bennett, V, and Rao, PV. "Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics." American Journal of Physiology - Cell Physiology 310.2 (January 2016): C115-C126.
PMID
26538089
Source
epmc
Published In
American journal of physiology. Cell physiology
Volume
310
Issue
2
Publish Date
2016
Start Page
C115
End Page
C126
DOI
10.1152/ajpcell.00111.2015

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.

Authors
Cadwell, CM; Jenkins, PM; Bennett, V; Kowalczyk, AP
MLA Citation
Cadwell, CM, Jenkins, PM, Bennett, V, and Kowalczyk, AP. "Ankyrin-G Inhibits Endocytosis of Cadherin Dimers." The Journal of biological chemistry 291.2 (January 2016): 691-704.
PMID
26574545
Source
epmc
Published In
The Journal of biological chemistry
Volume
291
Issue
2
Publish Date
2016
Start Page
691
End Page
704
DOI
10.1074/jbc.m115.648386

An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues.

Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that early axon initial segments and epithelial lateral membranes initially were based on spectrin-ankyrin-cell adhesion molecule assemblies and subsequently served as "incubators," where ion transporters independently acquired ankyrin-binding activity through positive selection.

Authors
Bennett, V; Lorenzo, DN
MLA Citation
Bennett, V, and Lorenzo, DN. "An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues." Current topics in membranes 77 (January 2016): 143-184. (Review)
PMID
26781832
Source
epmc
Published In
Current Topis in Membranes
Volume
77
Publish Date
2016
Start Page
143
End Page
184
DOI
10.1016/bs.ctm.2015.10.001

Ankyrin-B is a PI3P effector that promotes polarized alpha 5 beta 1-integrin recycling through recruiting RabGAP1L to early endosomes.

Authors
Qu, F; Lorenzo, D; King, SJ; Brooks, R; Bear, JE; Bennett, V
MLA Citation
Qu, F, Lorenzo, D, King, SJ, Brooks, R, Bear, JE, and Bennett, V. "Ankyrin-B is a PI3P effector that promotes polarized alpha 5 beta 1-integrin recycling through recruiting RabGAP1L to early endosomes." 2016.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
27
Publish Date
2016

An ankyrin-B/beta-2 spectrin-based mechanism regulates axonal organelle transport and axonal growth.

Authors
Lorenzo, DN; Badea, A; Johnson, G; Bennett, V
MLA Citation
Lorenzo, DN, Badea, A, Johnson, G, and Bennett, V. "An ankyrin-B/beta-2 spectrin-based mechanism regulates axonal organelle transport and axonal growth." 2016.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
27
Publish Date
2016

Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis.

Current physical models for plasma membranes emphasize dynamic 10- to 300-nm compartments at thermodynamic equilibrium but subject to thermal fluctuations. However, epithelial lateral membranes contain micrometer-sized domains defined by an underlying membrane skeleton composed of spectrin and its partner ankyrin-G. We demonstrate that these spectrin/ankyrin-G domains exhibit local microtubule-dependent movement on a time scale of minutes and encounter most of the lateral membranes within an hour. Spectrin/ankyrin-G domains exclude clathrin and clathrin-dependent cargo, and inhibit both receptor-mediated and bulk endocytosis. Moreover, inhibition of endocytosis fully restores lateral membrane height in spectrin- or ankyrin-G-depleted cells. These findings support a non-equilibrium cellular-scale model for epithelial lateral membranes, where spectrin/ankyrin-G domains actively patrol the plasma membrane, analogous to "window washers," and promote columnar morphology by blocking membrane uptake.

Authors
Jenkins, PM; He, M; Bennett, V
MLA Citation
Jenkins, PM, He, M, and Bennett, V. "Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis." Science advances 1.8 (September 2015): e1500301-.
PMID
26523289
Source
epmc
Published In
Science Advances
Volume
1
Issue
8
Publish Date
2015
Start Page
e1500301
DOI
10.1126/sciadv.1500301

Ankyrin-B metabolic syndrome combines age-dependent adiposity with pancreatic β cell insufficiency.

Rare functional variants of ankyrin-B have been implicated in human disease, including hereditary cardiac arrhythmia and type 2 diabetes (T2D). Here, we developed murine models to evaluate the metabolic consequences of these alterations in vivo. Specifically, we generated knockin mice that express either the human ankyrin-B variant R1788W, which is present in 0.3% of North Americans of mixed European descent and is associated with T2D, or L1622I, which is present in 7.5% of African Americans. Young AnkbR1788W/R1788W mice displayed primary pancreatic β cell insufficiency that was characterized by reduced insulin secretion in response to muscarinic agonists, combined with increased peripheral glucose uptake and concomitantly increased plasma membrane localization of glucose transporter 4 (GLUT4) in skeletal muscle and adipocytes. In contrast, older AnkbR1788W/R1788W and AnkbL1622I/L1622I mice developed increased adiposity, a phenotype that was reproduced in cultured adipocytes, and insulin resistance. GLUT4 trafficking was altered in animals expressing mutant forms of ankyrin-B, and we propose that increased cell surface expression of GLUT4 in skeletal muscle and fatty tissue of AnkbR1788W/R1788W mice leads to the observed age-dependent adiposity. Together, our data suggest that ankyrin-B deficiency results in a metabolic syndrome that combines primary pancreatic β cell insufficiency with peripheral insulin resistance and is directly relevant to the nearly one million North Americans bearing the R1788W ankyrin-B variant.

Authors
Lorenzo, DN; Healy, JA; Hostettler, J; Davis, J; Yang, J; Wang, C; Hohmeier, HE; Zhang, M; Bennett, V
MLA Citation
Lorenzo, DN, Healy, JA, Hostettler, J, Davis, J, Yang, J, Wang, C, Hohmeier, HE, Zhang, M, and Bennett, V. "Ankyrin-B metabolic syndrome combines age-dependent adiposity with pancreatic β cell insufficiency." The Journal of clinical investigation 125.8 (August 2015): 3087-3102.
PMID
26168218
Source
epmc
Published In
Journal of Clinical Investigation
Volume
125
Issue
8
Publish Date
2015
Start Page
3087
End Page
3102
DOI
10.1172/jci81317

Evolution in action: giant ankyrins awake.

Reporting in Developmental Cell, Stephan et al. (2015) demonstrate critical axonal and presynaptic functions from acquisition of an enormous exon by the Drosophila ank2 gene. They propose that highly elongated ank2-XL molecules, associated with the plasma membrane through spectrin and ank2-L, extend deep into the axoplasm to promote microtubule organization.

Authors
Bennett, V; Walder, K
MLA Citation
Bennett, V, and Walder, K. "Evolution in action: giant ankyrins awake." Developmental cell 33.1 (April 2015): 1-2.
PMID
25850669
Source
epmc
Published In
Developmental Cell
Volume
33
Issue
1
Publish Date
2015
Start Page
1
End Page
2
DOI
10.1016/j.devcel.2015.03.017

Giant ankyrin-G: a critical innovation in vertebrate evolution of fast and integrated neuronal signaling.

Axon initial segments (AISs) and nodes of Ranvier are sites of clustering of voltage-gated sodium channels (VGSCs) in nervous systems of jawed vertebrates that facilitate fast long-distance electrical signaling. We demonstrate that proximal axonal polarity as well as assembly of the AIS and normal morphogenesis of nodes of Ranvier all require a heretofore uncharacterized alternatively spliced giant exon of ankyrin-G (AnkG). This exon has sequence similarity to I-connectin/Titin and was acquired after the first round of whole-genome duplication by the ancestral ANK2/ANK3 gene in early vertebrates before development of myelin. The giant exon resulted in a new nervous system-specific 480-kDa polypeptide combining previously known features of ANK repeats and β-spectrin-binding activity with a fibrous domain nearly 150 nm in length. We elucidate previously undescribed functions for giant AnkG, including recruitment of β4 spectrin to the AIS that likely is regulated by phosphorylation, and demonstrate that 480-kDa AnkG is a major component of the AIS membrane "undercoat' imaged by platinum replica electron microscopy. Surprisingly, giant AnkG-knockout neurons completely lacking known AIS components still retain distal axonal polarity and generate action potentials (APs), although with abnormal frequency. Giant AnkG-deficient mice live to weaning and provide a rationale for survival of humans with severe cognitive dysfunction bearing a truncating mutation in the giant exon. The giant exon of AnkG is required for assembly of the AIS and nodes of Ranvier and was a transformative innovation in evolution of the vertebrate nervous system that now is a potential target in neurodevelopmental disorders.

Authors
Jenkins, PM; Kim, N; Jones, SL; Tseng, WC; Svitkina, TM; Yin, HH; Bennett, V
MLA Citation
Jenkins, PM, Kim, N, Jones, SL, Tseng, WC, Svitkina, TM, Yin, HH, and Bennett, V. "Giant ankyrin-G: a critical innovation in vertebrate evolution of fast and integrated neuronal signaling." Proceedings of the National Academy of Sciences of the United States of America 112.4 (January 2015): 957-964.
PMID
25552556
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
4
Publish Date
2015
Start Page
957
End Page
964
DOI
10.1073/pnas.1416544112

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors.

GABAA-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABAA receptors, and requires a newly described interaction with GABARAP, a GABAA receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.

Authors
Tseng, WC; Jenkins, PM; Tanaka, M; Mooney, R; Bennett, V
MLA Citation
Tseng, WC, Jenkins, PM, Tanaka, M, Mooney, R, and Bennett, V. "Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors." Proceedings of the National Academy of Sciences of the United States of America 112.4 (January 2015): 1214-1219.
PMID
25552561
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
4
Publish Date
2015
Start Page
1214
End Page
1219
DOI
10.1073/pnas.1417989112

Altered Axonal Properties in Mice Lacking Autism-Associated 440kDa Ankyrin-B

Authors
Walder, KK; Hostettler, JD; Bennett, V
MLA Citation
Walder, KK, Hostettler, JD, and Bennett, V. "Altered Axonal Properties in Mice Lacking Autism-Associated 440kDa Ankyrin-B." 2015.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
26
Publish Date
2015

Structural basis of diverse membrane target recognitions by ankyrins

Authors
Wang, C; Wei, Z; Chen, K; Ye, F; Yu, C; Bennett, V; Zhang, M
MLA Citation
Wang, C, Wei, Z, Chen, K, Ye, F, Yu, C, Bennett, V, and Zhang, M. "Structural basis of diverse membrane target recognitions by ankyrins." 2015.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
26
Publish Date
2015

An ankyrin-B based mechanism for polarized alpha 5 beta 1-integrin transport is required for haptotaxis.

Authors
Qu, F; Lorenzo, D; King, SJ; Bear, JE; Bennett, V
MLA Citation
Qu, F, Lorenzo, D, King, SJ, Bear, JE, and Bennett, V. "An ankyrin-B based mechanism for polarized alpha 5 beta 1-integrin transport is required for haptotaxis." 2015.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
26
Publish Date
2015

Ankyrin-B deficiency causes cell-autonomous increase in adiposity and triggers the onset of a metabolic syndrome in mice

Authors
Lorenzo, DN; Healy, JA; Hostettler, JD; Bennett, V
MLA Citation
Lorenzo, DN, Healy, JA, Hostettler, JD, and Bennett, V. "Ankyrin-B deficiency causes cell-autonomous increase in adiposity and triggers the onset of a metabolic syndrome in mice." 2015.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
26
Publish Date
2015

Developmental mechanism of the periodic membrane skeleton in axons.

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites.

Authors
Zhong, G; He, J; Zhou, R; Lorenzo, D; Babcock, HP; Bennett, V; Zhuang, X
MLA Citation
Zhong, G, He, J, Zhou, R, Lorenzo, D, Babcock, HP, Bennett, V, and Zhuang, X. "Developmental mechanism of the periodic membrane skeleton in axons." eLife 3 (December 23, 2014).
Website
http://hdl.handle.net/10161/10975
PMID
25535840
Source
epmc
Published In
eLife
Volume
3
Publish Date
2014
DOI
10.7554/elife.04581

A hierarchy of ankyrin-spectrin complexes clusters sodium channels at nodes of Ranvier.

The scaffolding protein ankyrin-G is required for Na(+) channel clustering at axon initial segments. It is also considered essential for Na(+) channel clustering at nodes of Ranvier to facilitate fast and efficient action potential propagation. However, notwithstanding these widely accepted roles, we show here that ankyrin-G is dispensable for nodal Na(+) channel clustering in vivo. Unexpectedly, in the absence of ankyrin-G, erythrocyte ankyrin (ankyrin-R) and its binding partner βI spectrin substitute for and rescue nodal Na(+) channel clustering. In addition, channel clustering is also rescued after loss of nodal βIV spectrin by βI spectrin and ankyrin-R. In mice lacking both ankyrin-G and ankyrin-R, Na(+) channels fail to cluster at nodes. Thus, ankyrin R-βI spectrin protein complexes function as secondary reserve Na(+) channel clustering machinery, and two independent ankyrin-spectrin protein complexes exist in myelinated axons to cluster Na(+) channels at nodes of Ranvier.

Authors
Ho, TS-Y; Zollinger, DR; Chang, K-J; Xu, M; Cooper, EC; Stankewich, MC; Bennett, V; Rasband, MN
MLA Citation
Ho, TS-Y, Zollinger, DR, Chang, K-J, Xu, M, Cooper, EC, Stankewich, MC, Bennett, V, and Rasband, MN. "A hierarchy of ankyrin-spectrin complexes clusters sodium channels at nodes of Ranvier." Nature neuroscience 17.12 (December 2014): 1664-1672.
PMID
25362473
Source
epmc
Published In
Nature Neuroscience
Volume
17
Issue
12
Publish Date
2014
Start Page
1664
End Page
1672
DOI
10.1038/nn.3859

Glial ankyrins facilitate paranodal axoglial junction assembly.

Neuron-glia interactions establish functional membrane domains along myelinated axons. These include nodes of Ranvier, paranodal axoglial junctions and juxtaparanodes. Paranodal junctions are the largest vertebrate junctional adhesion complex, and they are essential for rapid saltatory conduction and contribute to assembly and maintenance of nodes. However, the molecular mechanisms underlying paranodal junction assembly are poorly understood. Ankyrins are cytoskeletal scaffolds traditionally associated with Na(+) channel clustering in neurons and are important for membrane domain establishment and maintenance in many cell types. Here we show that ankyrin-B, expressed by Schwann cells, and ankyrin-G, expressed by oligodendrocytes, are highly enriched at the glial side of paranodal junctions where they interact with the essential glial junctional component neurofascin 155. Conditional knockout of ankyrins in oligodendrocytes disrupts paranodal junction assembly and delays nerve conduction during early development in mice. Thus, glial ankyrins function as major scaffolds that facilitate early and efficient paranodal junction assembly in the developing CNS.

Authors
Chang, K-J; Zollinger, DR; Susuki, K; Sherman, DL; Makara, MA; Brophy, PJ; Cooper, EC; Bennett, V; Mohler, PJ; Rasband, MN
MLA Citation
Chang, K-J, Zollinger, DR, Susuki, K, Sherman, DL, Makara, MA, Brophy, PJ, Cooper, EC, Bennett, V, Mohler, PJ, and Rasband, MN. "Glial ankyrins facilitate paranodal axoglial junction assembly." Nature neuroscience 17.12 (December 2014): 1673-1681.
PMID
25362471
Source
epmc
Published In
Nature Neuroscience
Volume
17
Issue
12
Publish Date
2014
Start Page
1673
End Page
1681
DOI
10.1038/nn.3858

A PIK3C3-ankyrin-B-dynactin pathway promotes axonal growth and multiorganelle transport.

Axon growth requires long-range transport of organelles, but how these cargoes recruit their motors and how their traffic is regulated are not fully resolved. In this paper, we identify a new pathway based on the class III PI3-kinase (PIK3C3), ankyrin-B (AnkB), and dynactin, which promotes fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes. We show that dynactin associates with cargo through AnkB interactions with both the dynactin subunit p62 and phosphatidylinositol 3-phosphate (PtdIns(3)P) lipids generated by PIK3C3. AnkB knockout resulted in shortened axon tracts and marked reduction in membrane association of dynactin and dynein, whereas it did not affect the organization of spectrin-actin axonal rings imaged by 3D-STORM. Loss of AnkB or of its linkages to either p62 or PtdIns(3)P or loss of PIK3C3 all impaired organelle transport and particularly retrograde transport in hippocampal neurons. Our results establish new functional relationships between PIK3C3, dynactin, and AnkB that together promote axonal transport of organelles and are required for normal axon length.

Authors
Lorenzo, DN; Badea, A; Davis, J; Hostettler, J; He, J; Zhong, G; Zhuang, X; Bennett, V
MLA Citation
Lorenzo, DN, Badea, A, Davis, J, Hostettler, J, He, J, Zhong, G, Zhuang, X, and Bennett, V. "A PIK3C3-ankyrin-B-dynactin pathway promotes axonal growth and multiorganelle transport." The Journal of cell biology 207.6 (December 2014): 735-752.
PMID
25533844
Source
epmc
Published In
The Journal of Cell Biology
Volume
207
Issue
6
Publish Date
2014
Start Page
735
End Page
752
DOI
10.1083/jcb.201407063

Scaling up through teamwork: Ankyrin-G microdomains control large-scale cellular patterning through opposing endocytosis.

Authors
Jenkins, PM; He, M; Tseng, W; Bennett, V
MLA Citation
Jenkins, PM, He, M, Tseng, W, and Bennett, V. "Scaling up through teamwork: Ankyrin-G microdomains control large-scale cellular patterning through opposing endocytosis." December 2014.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
25
Publish Date
2014

A PIK3C3/Ankyrin-B/Dynactin pathway promotes axonal growth and multi-organelle transport.

Authors
Lorenzo, DN; Badea, A; Davis, J; Hostettler, JD; He, J; Zhong, G; Zhuang, X; Bennett, V
MLA Citation
Lorenzo, DN, Badea, A, Davis, J, Hostettler, JD, He, J, Zhong, G, Zhuang, X, and Bennett, V. "A PIK3C3/Ankyrin-B/Dynactin pathway promotes axonal growth and multi-organelle transport." December 2014.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
25
Publish Date
2014

Ankyrin-B is required for directed cell migration and targeted recycling of beta 1-integrin

Authors
Qu, F; Lorenzo, DN; Bennett, V
MLA Citation
Qu, F, Lorenzo, DN, and Bennett, V. "Ankyrin-B is required for directed cell migration and targeted recycling of beta 1-integrin." December 2014.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
25
Publish Date
2014

Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo.

Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined.Define the in vivo mechanisms underlying Nav1.5 membrane regulation.Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G(-/-) mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin-dependent kinase II to the intercalated disc. Mechanistically, βIV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin-dependent kinase II-δ; however, βIV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation.Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.

Authors
Makara, MA; Curran, J; Little, SC; Musa, H; Polina, I; Smith, SA; Wright, PJ; Unudurthi, SD; Snyder, J; Bennett, V; Hund, TJ; Mohler, PJ
MLA Citation
Makara, MA, Curran, J, Little, SC, Musa, H, Polina, I, Smith, SA, Wright, PJ, Unudurthi, SD, Snyder, J, Bennett, V, Hund, TJ, and Mohler, PJ. "Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo." Circulation research 115.11 (November 2014): 929-938.
PMID
25239140
Source
epmc
Published In
Circulation Research
Volume
115
Issue
11
Publish Date
2014
Start Page
929
End Page
938
DOI
10.1161/circresaha.115.305154

Ankyrin-G palmitoylation and βII-spectrin binding to phosphoinositide lipids drive lateral membrane assembly.

Ankyrin-G and βII-spectrin colocalize at sites of cell-cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and βII-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that βII-spectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and βII-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G-βII-spectrin interaction or βII-spectrin-phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with βII-spectrin, and βII-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells.

Authors
He, M; Abdi, KM; Bennett, V
MLA Citation
He, M, Abdi, KM, and Bennett, V. "Ankyrin-G palmitoylation and βII-spectrin binding to phosphoinositide lipids drive lateral membrane assembly." The Journal of cell biology 206.2 (July 2014): 273-288.
PMID
25049274
Source
epmc
Published In
The Journal of Cell Biology
Volume
206
Issue
2
Publish Date
2014
Start Page
273
End Page
288
DOI
10.1083/jcb.201401016

Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo

© 2014 American Heart Association, Inc. Rationale: Na < inf > v < /inf > 1.5 (SCN5A) is the primary cardiac voltage-gated Na < inf > v < /inf > channel. Na < inf > v < /inf > 1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Na < inf > v < /inf > 1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Na < inf > v < /inf > 1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Na < inf > v < /inf > 1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective: Define the in vivo mechanisms underlying Na < inf > v < /inf > 1.5 membrane regulation. Methods and Results: Here, we define the molecular basis of an Na < inf > v < /inf > channel regulatory platform in heart. Using new cardiac-selective ankyrin-G < sup > -/- < /sup > mice (conditional knock-out mouse), we report that ankyrin-G targets Na < inf > v < /inf > 1.5 and its regulatory protein calcium/calmodulin-dependent kinase II to the intercalated disc. Mechanistically, β < inf > IV < /inf > -spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin-dependent kinase II-δ however, β < inf > IV < /inf > -spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Na < inf > v < /inf > 1.5 expression/membrane localization and reduced I < inf > Na < /inf > associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Na < inf > v < /inf > channel antagonists. Moreover, we report that ankyrin-G links Na < inf > v < /inf > channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation. Conclusions: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Na < inf > v < /inf > 1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Na < inf > v < /inf > 1.5. (Circ Res. 2014;115:929-938.)

Authors
Makara, MA; Curran, J; Little, SC; Musa, H; Polina, I; Smith, SA; Wright, PJ; Unudurthi, SD; Snyder, J; Bennett, V; Hund, TJ; Mohler, PJ
MLA Citation
Makara, MA, Curran, J, Little, SC, Musa, H, Polina, I, Smith, SA, Wright, PJ, Unudurthi, SD, Snyder, J, Bennett, V, Hund, TJ, and Mohler, PJ. "Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo." Circulation Research 115.11 (January 1, 2014): 929-938.
Source
scopus
Published In
Circulation Research
Volume
115
Issue
11
Publish Date
2014
Start Page
929
End Page
938
DOI
10.1161/CIRCRESAHA.115.305154

Structural basis of diverse membrane target recognitions by ankyrins.

Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins' membrane targets, with implications for other proteins containing extended ANK repeat domains.

Authors
Wang, C; Wei, Z; Chen, K; Ye, F; Yu, C; Bennett, V; Zhang, M
MLA Citation
Wang, C, Wei, Z, Chen, K, Ye, F, Yu, C, Bennett, V, and Zhang, M. "Structural basis of diverse membrane target recognitions by ankyrins." eLife 3 (January 2014).
PMID
25383926
Source
epmc
Published In
eLife
Volume
3
Publish Date
2014
DOI
10.7554/elife.04353

'ANKYRIN' THE PARANODE

Authors
Chang, K-J; Zollinger, D; Susuki, K; Ho, T; Cooper, EC; Mohler, PJ; Bennett, V; Rasband, MN
MLA Citation
Chang, K-J, Zollinger, D, Susuki, K, Ho, T, Cooper, EC, Mohler, PJ, Bennett, V, and Rasband, MN. "'ANKYRIN' THE PARANODE." July 2013.
Source
wos-lite
Published In
Glia
Volume
61
Publish Date
2013
Start Page
S17
End Page
S18

The roles of ankyrin-G in the formation and the maintenance of nodes of Ranvier in vivo

Authors
Ho, TS; Bennett, V; Rasband, MN
MLA Citation
Ho, TS, Bennett, V, and Rasband, MN. "The roles of ankyrin-G in the formation and the maintenance of nodes of Ranvier in vivo." May 2013.
Source
wos-lite
Published In
Journal of Neurochemistry
Volume
125
Publish Date
2013
Start Page
202
End Page
202

Paranodal ankyrins: enigmatic glial anchors?

Authors
Chang, KJ; Ho, TS; Susuki, K; Zollinger, DR; Bennett, V; Mohler, PJ; Rasband, MN
MLA Citation
Chang, KJ, Ho, TS, Susuki, K, Zollinger, DR, Bennett, V, Mohler, PJ, and Rasband, MN. "Paranodal ankyrins: enigmatic glial anchors?." May 2013.
Source
wos-lite
Published In
Journal of Neurochemistry
Volume
125
Publish Date
2013
Start Page
198
End Page
198

Dynamic micron-scale ankyrin-G/beta-2 spectrin patches stabilize the MDCK cell lateral membrane

Authors
Jenkins, PM; He, M; Bennett, V
MLA Citation
Jenkins, PM, He, M, and Bennett, V. "Dynamic micron-scale ankyrin-G/beta-2 spectrin patches stabilize the MDCK cell lateral membrane." MOLECULAR BIOLOGY OF THE CELL 24 (2013).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
24
Publish Date
2013

Somato-dendritic axon initial segment outposts dependent on 480 kDa ankyrin-G organize GABAergic synapses in hippocampal neurons

Authors
Tseng, W; Jenkins, PM; Walder, KK; Bennett, V
MLA Citation
Tseng, W, Jenkins, PM, Walder, KK, and Bennett, V. "Somato-dendritic axon initial segment outposts dependent on 480 kDa ankyrin-G organize GABAergic synapses in hippocampal neurons." MOLECULAR BIOLOGY OF THE CELL 24 (2013).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
24
Publish Date
2013

A vertebrate- and neural-specific giant exon of 480 kDa ankyrin-G is required for assembly of the axon initial segment in vitro and in vivo.

Authors
Jenkins, PM; Walder, KK; Tseng, W; Bennett, V
MLA Citation
Jenkins, PM, Walder, KK, Tseng, W, and Bennett, V. "A vertebrate- and neural-specific giant exon of 480 kDa ankyrin-G is required for assembly of the axon initial segment in vitro and in vivo." MOLECULAR BIOLOGY OF THE CELL 24 (2013).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
24
Publish Date
2013

Ankyrin-B is the physiological membrane adaptor for dynactin and regulates polarized intracellular organelle transport in axons and fibroblasts

Authors
Lorenzo, DN; Hostlettler, J; Qu, F; Badea, A; Bennett, V
MLA Citation
Lorenzo, DN, Hostlettler, J, Qu, F, Badea, A, and Bennett, V. "Ankyrin-B is the physiological membrane adaptor for dynactin and regulates polarized intracellular organelle transport in axons and fibroblasts." MOLECULAR BIOLOGY OF THE CELL 24 (2013).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
24
Publish Date
2013

E-cadherin polarity is determined by a multifunction motif mediating lateral membrane retention through ankyrin-G and apical-lateral transcytosis through clathrin

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Jenkins, PM; Vasavda, C; Hostettler, J; Davis, JQ; Abdi, K; Bennett, V
MLA Citation
Jenkins, PM, Vasavda, C, Hostettler, J, Davis, JQ, Abdi, K, and Bennett, V. "E-cadherin polarity is determined by a multifunction motif mediating lateral membrane retention through ankyrin-G and apical-lateral transcytosis through clathrin." Journal of Biological Chemistry 288.20 (2013): 14018-14031.
PMID
23530049
Source
scival
Published In
The Journal of biological chemistry
Volume
288
Issue
20
Publish Date
2013
Start Page
14018
End Page
14031
DOI
10.1074/jbc.M113.454439

A single divergent exon inhibits ankyrin-B association with the plasma membrane

Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU52-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
He, M; Tseng, W-C; Bennett, V
MLA Citation
He, M, Tseng, W-C, and Bennett, V. "A single divergent exon inhibits ankyrin-B association with the plasma membrane." Journal of Biological Chemistry 288.21 (2013): 14769-14779.
PMID
23569209
Source
scival
Published In
The Journal of biological chemistry
Volume
288
Issue
21
Publish Date
2013
Start Page
14769
End Page
14779
DOI
10.1074/jbc.M113.465328

Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons

Axons are subdivided into functionally organized microdomains, which are required for generation and propagation of action potentials (APs). In the central nervous system (CNS), APs are generated near the soma in the axon initial segment (AIS) and propagated by nodes of Ranvier (noR). The crucial role of the membrane adapter proteins ankyrin-B and ankyrin-G as organizers of AIS and noR is now well established. By comparison, little is known on the localization and function of these proteins in sensory axon terminals of the peripheral nervous systems (PNS). Here, we tested the hypothesis that somatosensory PNS terminals are organized by distinct members of the ankyrin protein family. We discovered a specific distribution of ankyrin-B in somatosensory axon terminals of skin and muscle. Specifically, ankyrin-B was localized along the membrane of axons innervating Meissner corpuscles, Pacinian corpuscles and hair follicle receptors. Likewise, proprioceptive terminals of muscle spindles exhibited prominent ankyrin-B expression. Furthermore, ankyrin-B expression extended into nociceptive and thermoceptive intraepidermal nerve fibers. Interestingly, all studied somatosensory terminals were largely devoid of ankyrin-G, indicating that this scaffolding protein does not contribute to organization of mechanoelectric transduction zones in peripheral somatosensory neurons. Instead, we propose that ankyrin-B serves as a major membrane organizer in mechanoreceptive and nociceptive terminals of the PNS. © 2012 Springer-Verlag.

Authors
Engelhardt, M; Vorwald, S; Sobotzik, J-M; Bennett, V; Schultz, C
MLA Citation
Engelhardt, M, Vorwald, S, Sobotzik, J-M, Bennett, V, and Schultz, C. "Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons." Brain Structure and Function 218.4 (2013): 1005-1016.
Source
scival
Published In
Brain Structure and Function
Volume
218
Issue
4
Publish Date
2013
Start Page
1005
End Page
1016
DOI
10.1007/s00429-012-0443-0

Spectrin- and ankyrin-based membrane domains and the evolution of vertebrates.

Spectrin and ankyrin are membrane skeletal proteins that contribute to mechanical support of plasma membranes and micron-scale organization of diverse membrane-spanning proteins. This chapter provides a plausible scenario for the evolution of ankyrin- and spectrin-based membrane domains with a focus on vertebrates. The analysis integrates recent phylogenetic information with functional analyses of spectrin and ankyrin in erythrocytes, axon initial segments and nodes of Ranvier in neurons, T-tubules and intercalated disks of cardiomyocytes, lateral membrane domains of epithelial cells, and costameres of striated muscle. A core spectrin-ankyrin mechanism for coordinating membrane-spanning proteins and mechanically stabilizing membrane bilayers was expanded in vertebrates by gene duplication events, insertion of giant alternately spliced exons of axonal ankyrins, and a versatile peptide-binding fold of ANK repeats that facilitated acquisition of new protein partners. Cell adhesion molecules (CAM), including dystroglycan, L1 CAM family members, and cadherins, are the earliest examples of membrane-spanning proteins with ankyrin-binding motifs and were all present in urochordates. In contrast, ion channels have continued to evolve ankyrin-binding sites in vertebrates. These considerations suggest a model where proto-domains formed through interaction of ankyrin and spectrin with CAMs. These proto-domains then became populated with ion channels that developed ankyrin-binding activity with selective pressure provided by optimization of physiological function. The best example is the axon initial segment where ankyrin-binding activity evolved sequentially and independently first in L1 CAMs, then in voltage-gated sodium channels, and finally in KCNQ2/3 channels, with the selective advantage of fast and precisely regulated signaling.

Authors
Bennett, V; Lorenzo, DN
MLA Citation
Bennett, V, and Lorenzo, DN. "Spectrin- and ankyrin-based membrane domains and the evolution of vertebrates." Curr Top Membr 72 (2013): 1-37.
PMID
24210426
Source
pubmed
Published In
Current Topis in Membranes
Volume
72
Publish Date
2013
Start Page
1
End Page
37
DOI
10.1016/B978-0-12-417027-8.00001-5

Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats. (vol 287, pg 19115, 2012)

Authors
Lee, W; Struempfer, J; Bennett, V; Schulten, K; Marszalek, PE
MLA Citation
Lee, W, Struempfer, J, Bennett, V, Schulten, K, and Marszalek, PE. "Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats. (vol 287, pg 19115, 2012)." JOURNAL OF BIOLOGICAL CHEMISTRY 287.42 (October 12, 2012): 35539-35539.
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
287
Issue
42
Publish Date
2012
Start Page
35539
End Page
35539

Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats.

The conserved TPLH tetrapeptide motif of ankyrin repeats (ARs) plays an important role in stabilizing AR proteins, and histidine (TPLH)-to-arginine (TPLR) mutations in this motif have been associated with a hereditary human anemia, spherocytosis. Here, we used a combination of atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations to examine the mechanical effects of His → Arg substitutions in TPLH motifs in a model AR protein, NI6C. Our molecular dynamics results show that the mutant protein is less mechanically stable than the WT protein. Our atomic force microscopy results indicate that the mechanical energy input necessary to fully unfold the mutant protein is only half of that necessary to unfold the WT protein (53 versus 106 kcal/mol). In addition, the ability of the mutant to generate refolding forces is also reduced. Moreover, the mutant protein subjected to cyclic stretch-relax measurements displays mechanical fatigue, which is absent in the WT protein. Taken together, these results indicate that the His → Arg substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of hereditary spherocytosis may be related to mechanical failure of ARs.

Authors
Lee, W; Strümpfer, J; Bennett, V; Schulten, K; Marszalek, PE
MLA Citation
Lee, W, Strümpfer, J, Bennett, V, Schulten, K, and Marszalek, PE. "Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats." J Biol Chem 287.23 (June 1, 2012): 19115-19121.
PMID
22514283
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
23
Publish Date
2012
Start Page
19115
End Page
19121
DOI
10.1074/jbc.M112.365569

Mechanical anisotropy of ankyrin repeats.

Red blood cells are frequently deformed and their cytoskeletal proteins such as spectrin and ankyrin-R are repeatedly subjected to mechanical forces. While the mechanics of spectrin was thoroughly investigated in vitro and in vivo, little is known about the mechanical behavior of ankyrin-R. In this study, we combine coarse-grained steered molecular dynamics simulations and atomic force spectroscopy to examine the mechanical response of ankyrin repeats (ARs) in a model synthetic AR protein NI6C, and in the D34 fragment of native ankyrin-R when these proteins are subjected to various stretching geometry conditions. Our steered molecular dynamics results, supported by AFM measurements, reveal an unusual mechanical anisotropy of ARs: their mechanical stability is greater when their unfolding is forced to propagate from the N-terminus toward the C-terminus (repeats unfold at ~60 pN), as compared to the unfolding in the opposite direction (unfolding force ∼ 30 pN). This anisotropy is also reflected in the complex refolding behavior of ARs. The origin of this unfolding and refolding anisotropy is in the various numbers of native contacts that are broken and formed at the interfaces between neighboring repeats depending on the unfolding/refolding propagation directions. Finally, we discuss how these complex mechanical properties of ARs in D34 may affect its behavior in vivo.

Authors
Lee, W; Zeng, X; Rotolo, K; Yang, M; Schofield, CJ; Bennett, V; Yang, W; Marszalek, PE
MLA Citation
Lee, W, Zeng, X, Rotolo, K, Yang, M, Schofield, CJ, Bennett, V, Yang, W, and Marszalek, PE. "Mechanical anisotropy of ankyrin repeats." Biophys J 102.5 (March 7, 2012): 1118-1126.
PMID
22404934
Source
pubmed
Published In
Biophysical Journal
Volume
102
Issue
5
Publish Date
2012
Start Page
1118
End Page
1126
DOI
10.1016/j.bpj.2012.01.046

Micropatterning of plasma membranes of differentiated vertebrate cells.

Authors
Jenkins, PM; Nilsson, K; Vasavda, C; Bennett, V
MLA Citation
Jenkins, PM, Nilsson, K, Vasavda, C, and Bennett, V. "Micropatterning of plasma membranes of differentiated vertebrate cells." MOLECULAR BIOLOGY OF THE CELL 23 (2012).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
23
Publish Date
2012

Ankyrin-B syndrome: Metabolic consequences of human genetic variations in the membrane adaptor ankyrin-B.

Authors
Lorenzo, DN; Nilsson, KR; Hostettler, J; Davis, JQ; Bennett, V
MLA Citation
Lorenzo, DN, Nilsson, KR, Hostettler, J, Davis, JQ, and Bennett, V. "Ankyrin-B syndrome: Metabolic consequences of human genetic variations in the membrane adaptor ankyrin-B." MOLECULAR BIOLOGY OF THE CELL 23 (2012).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
23
Publish Date
2012

Ankyrin-B is required for the regulation of synaptic vesicle transport and for the maintenance of long axonal tracts in neurons.

Authors
Lorenzo, DN; Badea, A; Hostettler, J; Bennett, V
MLA Citation
Lorenzo, DN, Badea, A, Hostettler, J, and Bennett, V. "Ankyrin-B is required for the regulation of synaptic vesicle transport and for the maintenance of long axonal tracts in neurons." MOLECULAR BIOLOGY OF THE CELL 23 (2012).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
23
Publish Date
2012

Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats (Journal of Biological Chemistry (2012) 287, (19115-19121) DOI: 10.1074/jbc.A112.365569)

Authors
Lee, W; Strümpfer, J; Bennett, V; Schulten, K; Marszalek, PE
MLA Citation
Lee, W, Strümpfer, J, Bennett, V, Schulten, K, and Marszalek, PE. "Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats (Journal of Biological Chemistry (2012) 287, (19115-19121) DOI: 10.1074/jbc.A112.365569)." Journal of Biological Chemistry 287.42 (2012): 35539--.
Source
scival
Published In
The Journal of biological chemistry
Volume
287
Issue
42
Publish Date
2012
Start Page
35539-
DOI
10.1074/jbc.A112.365569

Cysteine 70 of ankyrin-G is S-palmitoylated and is required for function of ankyrin-G in membrane domain assembly

Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and β2-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
He, M; Jenkins, P; Bennett, V
MLA Citation
He, M, Jenkins, P, and Bennett, V. "Cysteine 70 of ankyrin-G is S-palmitoylated and is required for function of ankyrin-G in membrane domain assembly." Journal of Biological Chemistry 287.52 (2012): 43995-44005.
PMID
23129772
Source
scival
Published In
The Journal of biological chemistry
Volume
287
Issue
52
Publish Date
2012
Start Page
43995
End Page
44005
DOI
10.1074/jbc.M112.417501

Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons

Axons are subdivided into functionally organized microdomains, which are required for generation and propagation of action potentials (APs). In the central nervous system (CNS), APs are generated near the soma in the axon initial segment (AIS) and propagated by nodes of Ranvier (noR). The crucial role of the membrane adapter proteins ankyrin-B and ankyrin-G as organizers of AIS and noR is now well established. By comparison, little is known on the localization and function of these proteins in sensory axon terminals of the peripheral nervous systems (PNS). Here, we tested the hypothesis that somatosensory PNS terminals are organized by distinct members of the ankyrin protein family. We discovered a specific distribution of ankyrin-B in somatosensory axon terminals of skin and muscle. Specifically, ankyrin-B was localized along the membrane of axons innervating Meissner corpuscles, Pacinian corpuscles and hair follicle receptors. Likewise, proprioceptive terminals of muscle spindles exhibited prominent ankyrin-B expression. Furthermore, ankyrin-B expression extended into nociceptive and thermoceptive intraepidermal nerve fibers. Interestingly, all studied somatosensory terminals were largely devoid of ankyrin-G, indicating that this scaffolding protein does not contribute to organization of mechanoelectric transduction zones in peripheral somatosensory neurons. Instead, we propose that ankyrin-B serves as a major membrane organizer in mechanoreceptive and nociceptive terminals of the PNS. © 2012 Springer-Verlag.

Authors
Engelhardt, M; Vorwald, S; Sobotzik, J-M; Bennett, V; Schultz, C
MLA Citation
Engelhardt, M, Vorwald, S, Sobotzik, J-M, Bennett, V, and Schultz, C. "Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons." Brain Structure and Function (2012): 1-12.
PMID
22886464
Source
scival
Published In
Brain Structure and Function
Publish Date
2012
Start Page
1
End Page
12
DOI
10.1007/s00429-012-0443-0

Nanomechanics of streptavidin hubs for molecular materials.

A new strategy is reported for creating protein-based nanomaterials by genetically fusing large polypeptides to monomeric streptavidin and exploiting the propensity of streptavidin monomers(SM) to self-assemble into stable tetramers. We have characterized the mechanical properties of streptavidin-linked structures and measured, for the first time, the mechanical strength of streptavidin tetramers themselves. Using streptavidin tetramers as molecular hubs offers a unique opportunity to create a variety of well-defined, self-assembled protein-based (nano)materials with unusual mechanical properties.

Authors
Kim, M; Wang, C-C; Benedetti, F; Rabbi, M; Bennett, V; Marszalek, PE
MLA Citation
Kim, M, Wang, C-C, Benedetti, F, Rabbi, M, Bennett, V, and Marszalek, PE. "Nanomechanics of streptavidin hubs for molecular materials." Adv Mater 23.47 (December 15, 2011): 5684-5688.
PMID
22102445
Source
pubmed
Published In
Advanced Materials
Volume
23
Issue
47
Publish Date
2011
Start Page
5684
End Page
5688
DOI
10.1002/adma.201103316

Ank3-dependent SVZ niche assembly is required for the continued production of new neurons.

The rodent subventricular/subependymal zone (SVZ/SEZ) houses neural stem cells (NSCs) that generate olfactory bulb interneurons. It is unclear how the SVZ environment sustains neuronal production into adulthood. We discovered that the adapter molecule Ankyrin-3 (Ank3) is specifically upregulated in ventricular progenitors destined to become ependymal cells, but not in NSCs, and is required for SVZ niche assembly through progenitor lateral adhesion. Furthermore, we found that Ank3 expression is controlled by Foxj1, a transcriptional regulator of multicilia formation, and genetic deletion of this pathway led to complete loss of SVZ niche structure. Interestingly, radial glia continued to transition into postnatal NSCs without this niche. However, inducible deletion of Foxj1-Ank3 from mature SVZ ependyma resulted in dramatic depletion of neurogenesis. Targeting a pathway regulating ependymal organization/assembly and showing its requirement for new neuron production, our results have important implications for environmental control of adult neurogenesis and harvesting NSCs for replacement therapy.

Authors
Paez-Gonzalez, P; Abdi, K; Luciano, D; Liu, Y; Soriano-Navarro, M; Rawlins, E; Bennett, V; Garcia-Verdugo, JM; Kuo, CT
MLA Citation
Paez-Gonzalez, P, Abdi, K, Luciano, D, Liu, Y, Soriano-Navarro, M, Rawlins, E, Bennett, V, Garcia-Verdugo, JM, and Kuo, CT. "Ank3-dependent SVZ niche assembly is required for the continued production of new neurons." Neuron 71.1 (July 14, 2011): 61-75.
PMID
21745638
Source
pubmed
Published In
Neuron
Volume
71
Issue
1
Publish Date
2011
Start Page
61
End Page
75
DOI
10.1016/j.neuron.2011.05.029

Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protection of skeletal muscle from exercise injury

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Ayalon, G; Hostettler, JD; Hoffman, J; Kizhatil, K; Davis, JQ; Bennett, V
MLA Citation
Ayalon, G, Hostettler, JD, Hoffman, J, Kizhatil, K, Davis, JQ, and Bennett, V. "Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protection of skeletal muscle from exercise injury." Journal of Biological Chemistry 286.9 (2011): 7370-7378.
PMID
21186323
Source
scival
Published In
The Journal of biological chemistry
Volume
286
Issue
9
Publish Date
2011
Start Page
7370
End Page
7378
DOI
10.1074/jbc.M110.187831

Full reconstruction of a vectorial protein folding pathway by atomic force microscopy and molecular dynamics simulations

Authors
Lee, W; Zeng, X; Zhou, H-X; Bennett, V; Yang, W; Marszalek, PE
MLA Citation
Lee, W, Zeng, X, Zhou, H-X, Bennett, V, Yang, W, and Marszalek, PE. "Full reconstruction of a vectorial protein folding pathway by atomic force microscopy and molecular dynamics simulations." Journal of Biological Chemistry 286.10 (2011): 8708--.
Source
scival
Published In
The Journal of biological chemistry
Volume
286
Issue
10
Publish Date
2011
Start Page
8708-
DOI
10.1074/jbc.A110.179697

Ankyrin-G Mediates a Conserved yet Versatile Pathway for Assembly of Specialized Membrane Domains in Epithelial Cells and Neurons.

Authors
Jenkins, PM; Nilsson, KR; Vasavda, C; Davis, JQ; Bennett, V
MLA Citation
Jenkins, PM, Nilsson, KR, Vasavda, C, Davis, JQ, and Bennett, V. "Ankyrin-G Mediates a Conserved yet Versatile Pathway for Assembly of Specialized Membrane Domains in Epithelial Cells and Neurons." 2011.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
22
Publish Date
2011

Palmitoylation of Ankyrin-G Promotes Its Polarized Membrane Targeting in Epithelial Cells.

Authors
He, M; Bennett, V
MLA Citation
He, M, and Bennett, V. "Palmitoylation of Ankyrin-G Promotes Its Polarized Membrane Targeting in Epithelial Cells." 2011.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
22
Publish Date
2011

Full reconstruction of a vectorial protein folding pathway by atomic force microscopy and molecular dynamics simulations.

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and is [corrected] under severe conformational constraints [corrected] dictated by the one-dimensional geometry of the tunnel. [corrected] How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.

Authors
Lee, W; Zeng, X; Zhou, H-X; Bennett, V; Yang, W; Marszalek, PE
MLA Citation
Lee, W, Zeng, X, Zhou, H-X, Bennett, V, Yang, W, and Marszalek, PE. "Full reconstruction of a vectorial protein folding pathway by atomic force microscopy and molecular dynamics simulations." J Biol Chem 285.49 (December 3, 2010): 38167-38172.
PMID
20870713
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
49
Publish Date
2010
Start Page
38167
End Page
38172
DOI
10.1074/jbc.M110.179697

Modeling co-translational folding of ribosome-nascent chain using AFM probe and computer simulations

Authors
Zeng, X; Lee, W; Zhou, H-X; Bennett, V; Yang, W; Marszalek, PE
MLA Citation
Zeng, X, Lee, W, Zhou, H-X, Bennett, V, Yang, W, and Marszalek, PE. "Modeling co-translational folding of ribosome-nascent chain using AFM probe and computer simulations." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 240 (August 22, 2010).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
240
Publish Date
2010

Fast and forceful refolding of stretched alpha-helical solenoid proteins.

Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, beta-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed alpha-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10-15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs.

Authors
Kim, M; Abdi, K; Lee, G; Rabbi, M; Lee, W; Yang, M; Schofield, CJ; Bennett, V; Marszalek, PE
MLA Citation
Kim, M, Abdi, K, Lee, G, Rabbi, M, Lee, W, Yang, M, Schofield, CJ, Bennett, V, and Marszalek, PE. "Fast and forceful refolding of stretched alpha-helical solenoid proteins." Biophys J 98.12 (June 16, 2010): 3086-3092.
PMID
20550922
Source
pubmed
Published In
Biophysical Journal
Volume
98
Issue
12
Publish Date
2010
Start Page
3086
End Page
3092
DOI
10.1016/j.bpj.2010.02.054

Cholinergic augmentation of insulin release requires ankyrin-B.

Parasympathetic stimulation of pancreatic islets augments glucose-stimulated insulin secretion by inducing inositol trisphosphate receptor (IP(3)R)-mediated calcium ion (Ca2+) release. Ankyrin-B binds to the IP(3)R and is enriched in pancreatic beta cells. We found that ankyrin-B-deficient islets displayed impaired potentiation of insulin secretion by the muscarinic agonist carbachol, blunted carbachol-mediated intracellular Ca2+ release, and reduced the abundance of IP3R. Ankyrin-B-haploinsufficient mice exhibited hyperglycemia after oral ingestion but not after intraperitoneal injection of glucose, consistent with impaired parasympathetic potentiation of glucose-stimulated insulin secretion. The R1788W mutation of ankyrin-B impaired its function in pancreatic islets and is associated with type 2 diabetes in Caucasians and Hispanics. Thus, defective glycemic regulation through loss of ankyrin-B-dependent stabilization of IP3R is a potential risk factor for type 2 diabetes.

Authors
Healy, JA; Nilsson, KR; Hohmeier, HE; Berglund, J; Davis, J; Hoffman, J; Kohler, M; Li, L-S; Berggren, P-O; Newgard, CB; Bennett, V
MLA Citation
Healy, JA, Nilsson, KR, Hohmeier, HE, Berglund, J, Davis, J, Hoffman, J, Kohler, M, Li, L-S, Berggren, P-O, Newgard, CB, and Bennett, V. "Cholinergic augmentation of insulin release requires ankyrin-B. (Published online)" Sci Signal 3.113 (March 16, 2010): ra19-.
PMID
20234002
Source
pubmed
Published In
Science Signaling
Volume
3
Issue
113
Publish Date
2010
Start Page
ra19
DOI
10.1126/scisignal.2000771

Single-Molecule Atomic-Force Spectroscopy Captures a Novel Class of Molecular Nanosprings with Robust Stepwise Refolding Properties

Authors
Kim, M; Abdi, K; Lee, G; Rabbi, M; Lee, W; Yang, M; Schofield, CJ; Bennett, V; Marszalek, PE
MLA Citation
Kim, M, Abdi, K, Lee, G, Rabbi, M, Lee, W, Yang, M, Schofield, CJ, Bennett, V, and Marszalek, PE. "Single-Molecule Atomic-Force Spectroscopy Captures a Novel Class of Molecular Nanosprings with Robust Stepwise Refolding Properties." BIOPHYSICAL JOURNAL 98.3 (January 2010): 593A-594A.
Source
wos-lite
Published In
Biophysical Journal
Volume
98
Issue
3
Publish Date
2010
Start Page
593A
End Page
594A

Science signaling podcast: 16 March 2010

Authors
Bennett, V; Vanhook, AM
MLA Citation
Bennett, V, and Vanhook, AM. "Science signaling podcast: 16 March 2010." Science Signaling 3.113 (2010): pc6-.
Source
scival
Published In
Science Signaling
Volume
3
Issue
113
Publish Date
2010
Start Page
pc6
DOI
10.1126/scisignal.3113pc6

Ankyrin-based patterning of membrane microdomains: new insights into a novel class of cardiovascular diseases.

The organization of membrane-spanning proteins within discrete microdomains is critical for their physiologic function. This is especially important in the heart, where ion transporter and force-transducing microdomains are responsible for excitation-contraction coupling, anisotropic depolarization, and mechanotransduction. The following review will discuss recent advances in our understanding of the patterning of ion channel and force-transmitting membrane microdomains in cardiomyocytes, focusing on the T-tubule and intercalated disc.

Authors
Nilsson, KR; Bennett, V
MLA Citation
Nilsson, KR, and Bennett, V. "Ankyrin-based patterning of membrane microdomains: new insights into a novel class of cardiovascular diseases." J Cardiovasc Pharmacol 54.2 (August 2009): 106-115. (Review)
PMID
19636256
Source
pubmed
Published In
Journal of Cardiovascular Pharmacology
Volume
54
Issue
2
Publish Date
2009
Start Page
106
End Page
115
DOI
10.1097/FJC.0b013e3181b2b6ed

Ankyrin-G promotes cyclic nucleotide-gated channel transport to rod photoreceptor sensory cilia.

Cyclic nucleotide-gated (CNG) channels localize exclusively to the plasma membrane of photosensitive outer segments of rod photoreceptors where they generate the electrical response to light. Here, we report the finding that targeting of CNG channels to the rod outer segment required their interaction with ankyrin-G. Ankyrin-G localized exclusively to rod outer segments, coimmunoprecipitated with the CNG channel, and bound to the C-terminal domain of the channel beta1 subunit. Ankyrin-G depletion in neonatal mouse retinas markedly reduced CNG channel expression. Transgenic expression of CNG channel beta-subunit mutants in Xenopus rods showed that ankyrin-G binding was necessary and sufficient for targeting of the beta1 subunit to outer segments. Thus, ankyrin-G is required for transport of CNG channels to the plasma membrane of rod outer segments.

Authors
Kizhatil, K; Baker, SA; Arshavsky, VY; Bennett, V
MLA Citation
Kizhatil, K, Baker, SA, Arshavsky, VY, and Bennett, V. "Ankyrin-G promotes cyclic nucleotide-gated channel transport to rod photoreceptor sensory cilia." Science 323.5921 (March 20, 2009): 1614-1617.
PMID
19299621
Source
pubmed
Published In
Science
Volume
323
Issue
5921
Publish Date
2009
Start Page
1614
End Page
1617
DOI
10.1126/science.1169789

Membrane domains based on ankyrin and spectrin associated with cell-cell interactions.

Nodes of Ranvier and axon initial segments of myelinated nerves, sites of cell-cell contact in early embryos and epithelial cells, and neuromuscular junctions of skeletal muscle all perform physiological functions that depend on clustering of functionally related but structurally diverse ion transporters and cell adhesion molecules within microdomains of the plasma membrane. These specialized cell surface domains appeared at different times in metazoan evolution, involve a variety of cell types, and are populated by distinct membrane-spanning proteins. Nevertheless, recent work has shown that these domains all share on their cytoplasmic surfaces a membrane skeleton comprised of members of the ankyrin and spectrin families. This review will summarize basic features of ankyrins and spectrins, and will discuss emerging evidence that these proteins are key players in a conserved mechanism responsible for assembly and maintenance of physiologically important domains on the surfaces of diverse cells.

Authors
Bennett, V; Healy, J
MLA Citation
Bennett, V, and Healy, J. "Membrane domains based on ankyrin and spectrin associated with cell-cell interactions." Cold Spring Harbor perspectives in biology 1.6 (2009): a003012-.
PMID
20457566
Source
scival
Published In
Cold Spring Harbor perspectives in biology
Volume
1
Issue
6
Publish Date
2009
Start Page
a003012
DOI
10.1101/cshperspect.a003012

AnkyrinG is required to maintain axo-dendritic polarity in vivo

Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG-/- mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG-/- mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.

Authors
Sobotzik, J-M; Sie, JM; Politi, C; Turco, DD; Bennett, V; Deller, T; Schultz, C
MLA Citation
Sobotzik, J-M, Sie, JM, Politi, C, Turco, DD, Bennett, V, Deller, T, and Schultz, C. "AnkyrinG is required to maintain axo-dendritic polarity in vivo." Proceedings of the National Academy of Sciences of the United States of America 106.41 (2009): 17564-17569.
PMID
19805144
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
41
Publish Date
2009
Start Page
17564
End Page
17569
DOI
10.1073/pnas.0909267106

Ankyrin-B is required for coordinated expression of beta-2-spectrin, the Na/K-ATPase and the Na/Ca exchanger in the inner segment of rod photoreceptors

Rod photoreceptors are highly polarized cells whose exquisite sensitivity to light depends on precise compartmentalization of ion channels/transporters within specialized membrane domains. Here, we report evidence for an ankyrin-B based mechanism for coordinated expression of the beta-2-spectrin-based membrane skeleton, and the Na/K-ATPase and Na/Ca exchanger in the inner segment of rod photoreceptors. We first discovered that ankyrin-B localizes to the inner segments but not outer segments of rod photoreceptors in vertebrates including humans, mice, and frogs. We found that haploinsufficiency of ankyrin-B in mice is accompanied by 50% reduction in inner segments of membrane proteins, including the Na/K-ATPase and the Na/Ca exchanger, as well as beta-2-spectrin, which is a component of the spectrin-actin membrane skeleton. These results are consistent with a mechanism where ankyrin-B is required to restrict the Na/K-ATPase and Na/Ca exchanger to the inner segment of rod photoreceptors by tethering these membrane proteins to beta-2-spectrin. © 2008 Elsevier Ltd. All rights reserved.

Authors
Kizhatil, K; Sandhu, NK; Peachey, NS; Bennett, V
MLA Citation
Kizhatil, K, Sandhu, NK, Peachey, NS, and Bennett, V. "Ankyrin-B is required for coordinated expression of beta-2-spectrin, the Na/K-ATPase and the Na/Ca exchanger in the inner segment of rod photoreceptors." Experimental Eye Research 88.1 (2009): 57-64.
PMID
19007774
Source
scival
Published In
Experimental Eye Research
Volume
88
Issue
1
Publish Date
2009
Start Page
57
End Page
64
DOI
10.1016/j.exer.2008.09.022

Localization and structure of the ankyrin-binding site on β 2-spectrin

Spectrins are tetrameric actin-cross-linking proteins that form an elastic network, termed the membrane skeleton, on the cytoplasmic surface of cellular membranes. At the plasma membrane, the membrane skeleton provides essential support, preventing loss of membrane material to environmental shear stresses. The skeleton also controls the location, abundance, and activity of membrane proteins that are critical to cell and tissue function. The ability of the skeleton to modulate membrane stability and function requires adaptor proteins that bind the skeleton to membranes. The principal adaptors are the ankyrin proteins, which bind to the β-subunit of spectrin and to the cytoplasmic domains of numerous integral membrane proteins. Here, we present the crystal structure of the ankyrin-binding domain of human β 2-spectrin at 1.95 Å resolution together with mutagenesis data identifying the binding surface for ankyrins on β 2-spectrin. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Davis, L; Abdi, K; Machius, M; Brautigam, C; Tomchick, DR; Bennett, V; Michaely, P
MLA Citation
Davis, L, Abdi, K, Machius, M, Brautigam, C, Tomchick, DR, Bennett, V, and Michaely, P. "Localization and structure of the ankyrin-binding site on β 2-spectrin." Journal of Biological Chemistry 284.11 (2009): 6982-6987.
PMID
19098307
Source
scival
Published In
The Journal of biological chemistry
Volume
284
Issue
11
Publish Date
2009
Start Page
6982
End Page
6987
DOI
10.1074/jbc.M809245200

An Ankyrin-Based Mechanism for Functional Organization of Dystrophin and Dystroglycan

β-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an ankyrin-based mechanism for sarcolemmal localization of dystrophin and β-DG. Dystrophin binds ankyrin-B and ankyrin-G, while β-DG binds ankyrin-G. Dystrophin and β-DG require ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and β-DG remain intracellular in ankyrin-B-depleted muscle, where β-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires ankyrin-B for localization of dystrophin/utrophin and β-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4. © 2008 Elsevier Inc. All rights reserved.

Authors
Ayalon, G; Davis, JQ; Scotland, PB; Bennett, V
MLA Citation
Ayalon, G, Davis, JQ, Scotland, PB, and Bennett, V. "An Ankyrin-Based Mechanism for Functional Organization of Dystrophin and Dystroglycan." Cell 135.7 (2008): 1189-1200.
PMID
19109891
Source
scival
Published In
Cell
Volume
135
Issue
7
Publish Date
2008
Start Page
1189
End Page
1200
DOI
10.1016/j.cell.2008.10.018

Cell differentiation

Authors
Bennett, V
MLA Citation
Bennett, V. "Cell differentiation." Current Opinion in Cell Biology 20.6 (2008): 607-608.
PMID
18973808
Source
scival
Published In
Current Opinion in Cell Biology
Volume
20
Issue
6
Publish Date
2008
Start Page
607
End Page
608
DOI
10.1016/j.ceb.2008.10.006

Adducin promotes micrometer-scale organization of β2-spectrin in lateral membranes of bronchial epithelial cells

Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with β2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete α-adducin from confluent HBE cells. Depletion of α-adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of β2-spectrin resulted in loss of adducin from the lateral membrane. siRNA-resistant α-adducin prevented loss of lateral membrane, but only if α-adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phosphomimetic versions of adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 μm. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells. © 2008 by The American Society for Cell Biology.

Authors
Abdi, KM; Bennett, V
MLA Citation
Abdi, KM, and Bennett, V. "Adducin promotes micrometer-scale organization of β2-spectrin in lateral membranes of bronchial epithelial cells." Molecular Biology of the Cell 19.2 (2008): 536-545.
PMID
18003973
Source
scival
Published In
Molecular Biology of the Cell
Volume
19
Issue
2
Publish Date
2008
Start Page
536
End Page
545
DOI
10.1091/mbc.E07-08-0818

Being there: Cellular targeting of voltage-gated sodium channels in the heart

Voltage-gated sodium (Nav) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Nav 1.5, the principal Nav channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Nav 1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Nav 1.5 and ankyrin-G is necessary for the expression of Nav 1.5 at the cardiomyocyte cell surface. © The Rockefeller University Press.

Authors
Bennett, V; Healy, J
MLA Citation
Bennett, V, and Healy, J. "Being there: Cellular targeting of voltage-gated sodium channels in the heart." Journal of Cell Biology 180.1 (2008): 13-15.
PMID
18180365
Source
scival
Published In
The Journal of Cell Biology
Volume
180
Issue
1
Publish Date
2008
Start Page
13
End Page
15
DOI
10.1083/jcb.200712098

Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin

Ankyrin and spectrin were first discovered as binding partners in the membrane skeleton of human erythrocytes. Mutations in genes encoding these proteins cause hereditary spherocytosis. Recent advances reveal that ankyrin and spectrin are required for organization of a surprisingly diverse set of proteins, including ion channels and cell adhesion molecules that are localized in specialized membrane domains in many cell types. New insights into the cell biology of ankyrin and spectrin reveal that these proteins actively participate in assembly of specialized membrane domains in addition to their conventional maintenance role as scaffolding proteins. Recently described inherited human diseases due to defects in spectrin or ankyrin include spinocerebellar ataxia type 5 and a cardiac arrhythmia, termed sick sinus syndrome with bradycardia or ankyrin-B syndrome. Together, these studies identify an emerging paradigm for pathogenesis of human disease where failure in cellular localization of membrane-spanning proteins results in loss of physiological function. © 2007 Elsevier Ltd. All rights reserved.

Authors
Bennett, V; Healy, J
MLA Citation
Bennett, V, and Healy, J. "Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin." Trends in Molecular Medicine 14.1 (2008): 28-36.
PMID
18083066
Source
scival
Published In
Trends in Molecular Medicine
Volume
14
Issue
1
Publish Date
2008
Start Page
28
End Page
36
DOI
10.1016/j.molmed.2007.11.005

Ankyrin-B syndrome: enhanced cardiac function balanced by risk of cardiac death and premature senescence.

Here we report the unexpected finding that specific human ANK2 variants represent a new example of balanced human variants. The prevalence of certain ANK2 (encodes ankyrin-B) variants range from 2 percent of European individuals to 8 percent in individuals from West Africa. Ankyrin-B variants associated with severe human arrhythmia phenotypes (eg E1425G, V1516D, R1788W) were rare in the general population. Variants associated with less severe clinical and in vitro phenotypes were unexpectedly common. Studies with the ankyrin-B(+/-) mouse reveal both benefits of enhanced cardiac contractility, as well as costs in earlier senescence and reduced lifespan. Together these findings suggest a constellation of traits that we term "ankyrin-B syndrome", which may contribute to both aging-related disorders and enhanced cardiac function.

Authors
Mohler, PJ; Healy, JA; Xue, H; Puca, AA; Kline, CF; Allingham, RR; Kranias, EG; Rockman, HA; Bennett, V
MLA Citation
Mohler, PJ, Healy, JA, Xue, H, Puca, AA, Kline, CF, Allingham, RR, Kranias, EG, Rockman, HA, and Bennett, V. "Ankyrin-B syndrome: enhanced cardiac function balanced by risk of cardiac death and premature senescence. (Published online)" PLoS One 2.10 (October 17, 2007): e1051-.
PMID
17940615
Source
pubmed
Published In
PloS one
Volume
2
Issue
10
Publish Date
2007
Start Page
e1051
DOI
10.1371/journal.pone.0001051

The mechanical behavior of beta-catenin captured by an atomic force microscopy

Authors
Lee, GR; Abdi, K; Bennett, V; Marszalek, PE
MLA Citation
Lee, GR, Abdi, K, Bennett, V, and Marszalek, PE. "The mechanical behavior of beta-catenin captured by an atomic force microscopy." Biophysical Journal (2007): 659A-659A. (Academic Article)
Source
manual
Published In
Biophysical Journal
Publish Date
2007
Start Page
659A
End Page
659A

Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos

E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and β-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of β-catenin. Ankyrin-G also recruits β-2-spectrin to E-cadherin-β-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and β-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and β-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or β-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and β-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and β-2-spectrin collaborate in biogenesis of the lateral membrane (Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Kizhatil, K; Davis, JQ; Davis, L; Hoffman, J; Hogan, BLM; Bennett, V
MLA Citation
Kizhatil, K, Davis, JQ, Davis, L, Hoffman, J, Hogan, BLM, and Bennett, V. "Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos." Journal of Biological Chemistry 282.36 (2007): 26552-26561.
PMID
17620337
Source
scival
Published In
The Journal of biological chemistry
Volume
282
Issue
36
Publish Date
2007
Start Page
26552
End Page
26561
DOI
10.1074/jbc.M703158200

Ankyrin-G and β2-spectrin collaborate in biogenesis of lateral membrane of human bronchial epithelial cells

Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of β2-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and β2-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Kizhatil, K; Yoon, W; Mohler, PJ; Davis, LH; Hoffman, JA; Bennett, V
MLA Citation
Kizhatil, K, Yoon, W, Mohler, PJ, Davis, LH, Hoffman, JA, and Bennett, V. "Ankyrin-G and β2-spectrin collaborate in biogenesis of lateral membrane of human bronchial epithelial cells." Journal of Biological Chemistry 282.3 (2007): 2029-2037.
PMID
17074766
Source
scival
Published In
The Journal of biological chemistry
Volume
282
Issue
3
Publish Date
2007
Start Page
2029
End Page
2037
DOI
10.1074/jbc.M608921200

Nanospring behaviour of ankyrin repeats.

Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.

Authors
Lee, G; Abdi, K; Jiang, Y; Michaely, P; Bennett, V; Marszalek, PE
MLA Citation
Lee, G, Abdi, K, Jiang, Y, Michaely, P, Bennett, V, and Marszalek, PE. "Nanospring behaviour of ankyrin repeats." Nature 440.7081 (March 9, 2006): 246-249.
PMID
16415852
Source
pubmed
Published In
Nature
Volume
440
Issue
7081
Publish Date
2006
Start Page
246
End Page
249
DOI
10.1038/nature04437

A common ankyrin-G-based mechanism retains KCNQ and Na V channels at electrically active domains of the axon

KCNQ (K V7) potassium channels underlie subthreshold M-currents that stabilize the neuronal resting potential and prevent repetitive firing of action potentials. Here, antibodies against four different KCNQ2 and KCNQ3 polypeptide epitopes show these subunits concentrated at the axonal initial segment (AIS) and node of Ranvier. AIS concentration ofKCNQ2and KCNQ3, like that of voltage-gated sodium (Na V) channels, is abolished in ankyrin-G knock-out mice. A short motif, common to KCNQ2 and KCNQ3, mediates both in vivo ankyrin-G interaction and retention of the subunits at the AIS. This KCNQ2/KCNQ3 motif is nearly identical to the sequence on Na V α subunits that serves these functions. All identified Na V and KCNQ genes of worms, insects, and molluscs lack the ankyrin-G binding motif. In contrast, vertebrate orthologs of Na V α subunits, KCNQ2, and KCNQ3 (including from bony fish, birds, and mammals) all possess the motif. Thus, concerted ankyrin-G interaction with KCNQ and Na V channels appears to have arisen through convergent molecular evolution, after the division between invertebrate and vertebrate lineages, but before the appearance of the lastcommonjawed vertebrate ancestor. This includes the historical period when myelin also evolved. Copyright © 2006 Society for Neuroscience.

Authors
Pan, Z; Kao, T; Horvath, Z; Lemos, J; Sul, J-Y; Cranstoun, SD; Bennett, V; Scherer, SS; Cooper, EC
MLA Citation
Pan, Z, Kao, T, Horvath, Z, Lemos, J, Sul, J-Y, Cranstoun, SD, Bennett, V, Scherer, SS, and Cooper, EC. "A common ankyrin-G-based mechanism retains KCNQ and Na V channels at electrically active domains of the axon." Journal of Neuroscience 26.10 (2006): 2599-2613.
PMID
16525039
Source
scival
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
26
Issue
10
Publish Date
2006
Start Page
2599
End Page
2613
DOI
10.1523/JNEUROSCI.4314-05.2006

Isoform specificity of ankyrin-B: A site in the divergent C-terminal domain is required for intramolecular association

Ankyrins contain significant amino acid identity and are co-expressed in many cell types yet maintain unique functions in vivo. Recent studies have identified the highly divergent C-terminal domain in ankyrin-B as the key domain for driving ankyrin-B-specific functions in cardiomyocytes. Here we identify an intramolecular interaction between the C-terminal domain and the membrane-binding domain of ankyrin-B using pure proteins in solution and the yeast two-hybrid assay. Through extensive deletion and alanine-scanning mutagenesis we have mapped key residues for interaction in both domains. Amino acids 1597EED1599 located in the ankyrin-B C-terminal domain and amino acids Arg37/Arg40 located inANKrepeat 1 are necessary for inter-domain interactions in yeast two-hybrid assays. Furthermore, conversion of amino acidsEED1597 to AAA1597 leads to a loss of function in the localization of inositol 1,4,5-trisphosphate receptors in ankyrin-B mutant cardiomyocytes. Physical properties of the ankyrin-B C-terminal domain determined by circular dichroism spectroscopy and hydrodynamic parameters reveal it is unstructured and highly extended in solution. Similar structural studies performed on full-length 220-kDa ankyrin-B harboring alanine substitutions, 1597AAA1599, reveal a more extended conformation compared with wild-type ankyrin-B. Taken together these results suggest a model of an extended and unstructured C-terminal domain folding back to bind and potentially regulate the membrane-binding domain of ankyrin-B. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Abdi, KM; Mohler, PJ; Davis, JQ; Bennett, V
MLA Citation
Abdi, KM, Mohler, PJ, Davis, JQ, and Bennett, V. "Isoform specificity of ankyrin-B: A site in the divergent C-terminal domain is required for intramolecular association." Journal of Biological Chemistry 281.9 (2006): 5741-5749.
PMID
16368689
Source
scival
Published In
The Journal of biological chemistry
Volume
281
Issue
9
Publish Date
2006
Start Page
5741
End Page
5749
DOI
10.1074/jbc.M506697200

Ankyrin-G regulates inactivation gating of the neuronal sodium channel, Nav1.6

Ankyrin-G, a modular protein, plays a critical role in clustering voltage-gated sodium channels (Nav channels) in nodes of Ranvier and initial segments of mammalian neurons. However, direct effects of ankyrin-G on electrophysiological properties of Nav channels remain elusive. In this study, we explored whether ankyrin-G has a role in modifying gating properties of the neuronal Nav1.6 channel that is predominantly localized at nodes of Ranvier and initial segments. TsA201 cells transfected with the human Nav1.6 cDNA alone exhibited significant persistent sodium current (Ina-p). On the other hand, Ina-p was barely detected on co-expression with ankyrin-G. Ankyrin-B, another ankyrin, did not show such an effect. Expression of chimeras between the two isoforms of ankyrin suggests that the membrane-binding domain of ankyrin-G is critical for reducing the Ina-p of Nav1.6. These results suggest that ankyrin-G regulates neuronal excitability not only through clustering Nav channels but also by directly modifying their channel gating. Copyright © 2006 The American Physiological Society.

Authors
Shirahata, E; Iwasaki, H; Takagi, M; Lin, C; Bennett, V; Okamura, Y; Hayasaka, K
MLA Citation
Shirahata, E, Iwasaki, H, Takagi, M, Lin, C, Bennett, V, Okamura, Y, and Hayasaka, K. "Ankyrin-G regulates inactivation gating of the neuronal sodium channel, Nav1.6." Journal of Neurophysiology 96.3 (2006): 1347-1357.
PMID
16775201
Source
scival
Published In
Journal of neurophysiology
Volume
96
Issue
3
Publish Date
2006
Start Page
1347
End Page
1357
DOI
10.1152/jn.01264.2005

A common sequence motif mediates the concentration of voltage gated Na+ and KCNQ2/3 K+ channels at axon initial segments

Authors
Pan, ZM; Lemos, J; Kao, TC; Horvath, Z; Scherer, SS; Bennett, V; Cooper, EC
MLA Citation
Pan, ZM, Lemos, J, Kao, TC, Horvath, Z, Scherer, SS, Bennett, V, and Cooper, EC. "A common sequence motif mediates the concentration of voltage gated Na+ and KCNQ2/3 K+ channels at axon initial segments." January 2005.
Source
wos-lite
Published In
Biophysical Journal
Volume
88
Issue
1
Publish Date
2005
Start Page
20A
End Page
21A

Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP 3 receptor in a cardiac T-tubule/SR microdomain

We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP 3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B +/- cardiomyocytes, which may explain elevated [Ca2+] i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation. © 2005 Mohler et al.

Authors
Mohler, PJ; Davis, JQ; Bennett, V
MLA Citation
Mohler, PJ, Davis, JQ, and Bennett, V. "Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP 3 receptor in a cardiac T-tubule/SR microdomain." PLoS Biology 3.12 (2005): 1-10.
PMID
16292983
Source
scival
Published In
PLoS biology
Volume
3
Issue
12
Publish Date
2005
Start Page
1
End Page
10
DOI
10.1371/journal.pbio.0030423

Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain.

We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B(+/-) cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

Authors
Mohler, PJ; Davis, JQ; Bennett, V
MLA Citation
Mohler, PJ, Davis, JQ, and Bennett, V. "Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain." PLoS biology 3.12 (2005): e423-.
Source
scival
Published In
PLoS biology
Volume
3
Issue
12
Publish Date
2005
Start Page
e423
DOI
10.1371/journal.pbio.0030423

Ankyrin-based cardiac arrhythmias: A new class of channelopathies due to loss of cellular targeting

Purpose of review: This review addresses a new mechanism for arrhythmia due to abnormal cellular localization of membrane ion channels and transporters. The focus is on ankyrins, a family of proteins that localize diverse membrane ion channels and transporters, and recent evidence that mutations affecting functions of ankyrins result in cardiac arrhythmia. Recent findings: A loss-of-function mutation of ankyrin-B in humans and a null mutation in mice result in a dominantly-inherited fatal cardiac arrhythmia initially classified as type 4 long QT syndrome. Characterization of additional probands suggests ankyrin-B mutations cause a new cardiac arrhythmia syndrome associated with sinus node dysfunction that is distinct from long QT syndrome. Ankyrin-B mutation results in elevated calcium transients in cardiomyocytes accompanied by loss of cellular targeting of Na/K ATPase, Ha/Ca exchanger, and InsP 3 receptor (all ankyrin-binding proteins) to cardiomyocyte membrane domains. The principal voltage-gated Na channel in heart, Nav1.5, is directly associated with ankyrin-G, which is encoded by a distinct gene from ankyrin-B. Mutation of Nav1.5 causing loss of binding to ankyrin-G results in Brugada syndrome and loss of targeting of Nav1.5 to the cell surface of cardiomyocytes. Summary: Ankyrin-B and ankyrin-G are recently recognized constituents of the heart that target diverse ion channels/pumps/transporters to physiologic sites of action in cardiomyocytes. Mutations of ankyrin-B cause a newly defined cardiac arrhythmia syndrome associated with abnormal calcium homeostasis in a mouse model. Ankyrin-G associates with the principal voltage-gated Na channel in the heart, and loss of this interaction due to mutation of Nav1.5 results in Brugada syndrome. © 2005 Lippincott Williams & Wilkins.

Authors
Mohler, PJ; Bennett, V
MLA Citation
Mohler, PJ, and Bennett, V. "Ankyrin-based cardiac arrhythmias: A new class of channelopathies due to loss of cellular targeting." Current Opinion in Cardiology 20.3 (2005): 189-193.
PMID
15861006
Source
scival
Published In
Current Opinion in Cardiology
Volume
20
Issue
3
Publish Date
2005
Start Page
189
End Page
193
DOI
10.1097/01.hco.0000160372.95116.3e

The ammonium transporter RhBG: Requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations, (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Lopez, C; Métral, S; Eladari, D; Drevensek, S; Gane, P; Chambrey, R; Bennett, V; Cartron, J-P; Kim, CLV; Colin, Y
MLA Citation
Lopez, C, Métral, S, Eladari, D, Drevensek, S, Gane, P, Chambrey, R, Bennett, V, Cartron, J-P, Kim, CLV, and Colin, Y. "The ammonium transporter RhBG: Requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells." Journal of Biological Chemistry 280.9 (2005): 8221-8228.
PMID
15611082
Source
scival
Published In
Journal of Biological Chemistry
Volume
280
Issue
9
Publish Date
2005
Start Page
8221
End Page
8228
DOI
10.1074/jbc.M413351200

Defects in ankyrin-based cellular pathways in metazoan physiology

Ankyrins are a ubiquitously expressed family of membrane-adaptor proteins found in most vertebrate tissues. Since the first ankyrin polypeptide was identified over 25 years ago (1), studies in humans, mice, and lower organisms have implicated critical roles for ankyrins in normal metazoan physiology. This review will provide an overview of the ankyrin family and highlight seminal findings in the field which have linked dysfunction in ankyrin-based pathways with defects in metazoan physiology and human disease.

Authors
Mohler, PJ; Bennett, V
MLA Citation
Mohler, PJ, and Bennett, V. "Defects in ankyrin-based cellular pathways in metazoan physiology." Frontiers in Bioscience 10.SUPPL. 2 (2005): 2832-2840.
PMID
15970537
Source
scival
Published In
Frontiers in Bioscience
Volume
10
Issue
SUPPL. 2
Publish Date
2005
Start Page
2832
End Page
2840

A molecular mechanism for axonal targeting of KCNQ2/KCNQ3 potassium channels that are mutated in BFNC

Authors
Pan, ZM; Kao, TC; Horvath, Z; Cranstoun, SD; Scherer, SS; Bennett, V; Cooper, EC
MLA Citation
Pan, ZM, Kao, TC, Horvath, Z, Cranstoun, SD, Scherer, SS, Bennett, V, and Cooper, EC. "A molecular mechanism for axonal targeting of KCNQ2/KCNQ3 potassium channels that are mutated in BFNC." 2005.
Source
wos-lite
Published In
Epilepsia
Volume
46
Publish Date
2005
Start Page
11
End Page
11

Interaction between 190 kDa ankyrin-G and beta-2-spectrin is required for the formation of the lateral membrane domain in human bronchial epithelial cells

Authors
Kizhatil, K; Yoon, W; Bennett, V
MLA Citation
Kizhatil, K, Yoon, W, and Bennett, V. "Interaction between 190 kDa ankyrin-G and beta-2-spectrin is required for the formation of the lateral membrane domain in human bronchial epithelial cells." November 2004.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
15
Publish Date
2004
Start Page
317A
End Page
317A

L1-dependent neuritogenesis involves ankyrin(B) that mediates L1 coupling with retrograde actin flow

Authors
Nishimura, K; Yoshihara, F; Tojima, T; Ooashi, N; Yoon, W; Mikoshiba, K; Bennett, V; Kamiguchi, H
MLA Citation
Nishimura, K, Yoshihara, F, Tojima, T, Ooashi, N, Yoon, W, Mikoshiba, K, Bennett, V, and Kamiguchi, H. "L1-dependent neuritogenesis involves ankyrin(B) that mediates L1 coupling with retrograde actin flow." CELL STRUCTURE AND FUNCTION 29 (May 2004): 21-21.
Source
wos-lite
Published In
Cell structure and function
Volume
29
Publish Date
2004
Start Page
21
End Page
21

Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the of cardiomyocytes

We identify a human mutation (E1053K) in the ankyrin-binding motif of Nav1.5 that is associated with Brugada syndrome, a fatal cardiac arrhythmia caused by altered function of Nav1.5. The E1053K mutation abolishes binding of Nav1.5 to ankyrin-G, and also prevents accumulation of Nav1.5 at cell surface sites in ventricular cardiomyocytes. Ankyrin-G and Nav1.5 are both localized at intercalated disc and T-tubule membranes in cardiomyocytes, and Na v1.5 coimmunoprecipitates with 190-kDa ankyrin-G from detergent-soluble lysates from rat heart. These data suggest that Na v1.5 associates with ankyrin-G and that ankyrin-G is required for Nav1.5 localization at excitable membranes in cardiomyocytes. Together with previous work in neurons, these results in cardiomyocytes suggest that ankyrin-G participates in a common pathway for localization of voltage-gated Nav channels at sites of function in multiple excitable cell types.

Authors
Mohler, PJ; Rivolta, I; Napolitano, C; LeMaillet, G; Lambert, S; Priori, SG; Bennett, V
MLA Citation
Mohler, PJ, Rivolta, I, Napolitano, C, LeMaillet, G, Lambert, S, Priori, SG, and Bennett, V. "Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the of cardiomyocytes." Proceedings of the National Academy of Sciences of the United States of America 101.50 (2004): 17533-17538.
PMID
15579534
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
101
Issue
50
Publish Date
2004
Start Page
17533
End Page
17538
DOI
10.1073/pnas.0403711101

A cardiac arrhythmia syndrome caused by loss of ankyrin-B function

220-kDa ankyrin-B is required for coordinated assembly of Na/Ca exchanger, Na/K ATPase, and inositol trisphosphate (InsP3) receptor at transverse-tubule/ sarcoplasmic reticulum sites in cardiomyocytes. A loss-of-function mutation of ankyrin-B identified in an extended kindred causes a dominantly inherited cardiac arrhythmia, initially described as type 4 long QT syndrome. Here we report the identification of eight unrelated probands harboring ankyrin-B loss-of-function mutations, including four previously undescribed mutations, whose clinical features distinguish the cardiac phenotype associated with loss of ankyrin-B activity from classic long QT syndromes. Humans with ankyrin-B mutations display varying degrees of cardiac dysfunction including bradycardia, sinus arrhythmia, idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, and risk of sudden death. However, a prolonged rate-corrected QT interval was not a consistent feature, indicating that ankyrin-B dysfunction represents a clinical entity distinct from classic long QT syndromes. The mutations are localized in the ankyrin-B regulatory domain, which distinguishes function of ankyrin-B from ankyrin-G in cardiomyocytes. All mutations abolish ability of ankyrin-B to restore abnormal Ca2+ dynamics and abnormal localization and expression of Na/Ca exchanger, Na/K ATPase, and InsP3R in ankyrin-B+/- cardiomyocytes. This study, considered together with the first description of ankyrin-B mutation associated with cardiac dysfunction, supports a previously undescribed paradigm for human disease due to abnormal coordination of multiple functionally related ion channels and transporters, in this case the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor.

Authors
Mohler, PJ; Splawski, I; Napolitano, C; Bottelli, G; Sharpe, L; Timothy, K; Priori, SG; Keating, MT; Bennett, V
MLA Citation
Mohler, PJ, Splawski, I, Napolitano, C, Bottelli, G, Sharpe, L, Timothy, K, Priori, SG, Keating, MT, and Bennett, V. "A cardiac arrhythmia syndrome caused by loss of ankyrin-B function." Proceedings of the National Academy of Sciences of the United States of America 101.24 (2004): 9137-9142.
PMID
15178757
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
101
Issue
24
Publish Date
2004
Start Page
9137
End Page
9142
DOI
10.1073/pnas.0402546101

Ankyrin-based subcellular gradient of neurofascin, an immunoglobulin family protein, directs GABAergic innervation at Purkinje axon initial segment

Distinct classes of GABAergic synapses are segregated into subcellular domains (i.e., dendrite, soma, and axon initial segment-AIS), thereby differentially regulating the input, integration, and output of principal neurons. In cerebellum, for example, basket interneurons make exquisitely precise "pinceau synapses" on AIS of Purkinje neurons, but the underlying mechanism is unknown. Using BAC transgenic reporter mice, we found that basket axons always contacted Purkinje soma before innervating AIS. This synapse targeting process followed the establishment of a subcellular gradient of neurofascin186 (NF186), an L1 family immunoglobulin cell adhesion molecule (L1CAM), along the Purkinje AIS-soma axis. This gradient was dependent on ankyrinG, an AIS-restricted membrane adaptor protein that recruits NF186. In the absence of neurofascin gradient, basket axons lost directional growth along Purkinje neurons and precisely followed NF186 to ectopic locations. Disruption of NF186-ankyrinG interactions at AIS reduced pinceau synapse formation. These results implicate ankyrin-based localization of L1CAMs in subcellular organization of GABAergic synapses.

Authors
Ango, F; Cristo, GD; Higashiyama, H; Bennett, V; Wu, P; Huang, ZJ
MLA Citation
Ango, F, Cristo, GD, Higashiyama, H, Bennett, V, Wu, P, and Huang, ZJ. "Ankyrin-based subcellular gradient of neurofascin, an immunoglobulin family protein, directs GABAergic innervation at Purkinje axon initial segment." Cell 119.2 (2004): 257-272.
PMID
15479642
Source
scival
Published In
Cell
Volume
119
Issue
2
Publish Date
2004
Start Page
257
End Page
272
DOI
10.1016/j.cell.2004.10.004

Ankyrin-B targets β2-spectrin to an intracellular compartment in neonatal cardiomyocytes

Ankyrin-B is a spectrin-binding protein that is required for localization of inositol 1,4,5-trisphosphate receptor and ryanodine receptor in neonatal cardiomyocytes. This work addresses the interaction between ankyrin-B and β2-spectrin in these cells. Ankyrin-B and β2- spectrin are colocalized in an intracellular striated compartment overlying the M-line and distinct from T-tubules, sarcoplasmic reticulum, Golgi, endoplasmic reticulum, lysosomes, and endosomes. β2-Spectrin is absent in ankyrin-B-null cardiomyocytes and is restored to a normal striated pattern by rescue with green fluorescent protein-220-kDa ankyrin-B. We identified two mutants (A1000P and DAR976AAA) located in the ZU5 domain which eliminate spectrin binding activity of ankyrin-B. Ankyrin-B mutants lacking spectrin binding activity are normally targeted but do not reestablish β2-spectrin in ankyrin-B+/- cardiomyocytes. However, both mutant forms of ankyrin-B are still capable of restoring inositol 1,4,5-trisphosphate receptor localization and normal contraction frequency of cardiomyocytes. Therefore, direct binding of β2-spectrin to ankyrin-B is required for the normal targeting of β2-spectrin in neonatal cardiomyocytes. In contrast, ankyrin-B localization and function are independent of β2-spectrin. In summary, this work demonstrates that interaction between members of the ankyrin and β-spectrin families previously established in erythrocytes and axon initial segments also occurs in neonatal cardiomyocytes with ankyrin-B and β2-spectrin. This work also establishes a functional hierarchy in which ankyrin-B determines the localization of β2-spectrin and operates independently of β2-spectrin in its role in organizing membrane-spanning proteins.

Authors
Mohler, PJ; Yoon, W; Bennett, V
MLA Citation
Mohler, PJ, Yoon, W, and Bennett, V. "Ankyrin-B targets β2-spectrin to an intracellular compartment in neonatal cardiomyocytes." Journal of Biological Chemistry 279.38 (2004): 40185-40193.
PMID
15262991
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
38
Publish Date
2004
Start Page
40185
End Page
40193
DOI
10.1074/jbc.M406018200

Isoform specificity among ankyrins: An amphipathic α-helix in the divergent regulatory domain of ankyrin-B interacts with the molecular co-chaperone Hdj1/Hsp40

Ankyrins-R, -B, and -G are a family of membrane-associated adaptors required for localization of structurally diverse proteins to specialized membrane domains, including axon initial segments, cardiomyocyte T-tubules, and epithelial cell lateral membranes. Ankyrins are often co-expressed in the same cells and, although structurally similar, have non-overlapping functions. We previously determined that the regulatory domain of ankyrin-B defines specificity between ankyrins B and G in cardiomyocytes. Here, we identify key residues on the surface of an ampitipathic α-helix unique to the regulatory domain of ankyrin-B that are essential for the function of ankyrin-B in cardiomyocytes. Using circular dichroism, we determined that a peptide representing the predicted helix folds as a helix in solution. Alanine-scanning mutagenesis revealed that residues 1773, 1777, 1780, 1784, and 1788 located in a patch on one surface the helix are critical for ankyrin-B function in cardiomyocytes. In a parallel set of experiments we determined that the molecular co-chaperone human DnaJ homologue 1 (Hdj1)/Hsp40 interacts with the ankyrin-B regulatory domain. Moreover, interaction of Hdj1/Hsp40 with the regulatory domain was mapped by random mutagenesis to same surface of the α-helix that is required for ankyrin-B function. These results provide new insight into the molecular basis for specificity between ankyrin-based pathways by defining a key α-helix structure in the divergent regulatory domain of ankyrin-B as well as interaction of the helix with Hdj1/Hsp40, the first downstream target for ankyrin-B-specific function.

Authors
Mohler, PJ; Hoffman, JA; Davis, JQ; Abdi, KM; Kim, C-R; Jones, SK; Davis, LH; Roberts, KF; Bennett, V
MLA Citation
Mohler, PJ, Hoffman, JA, Davis, JQ, Abdi, KM, Kim, C-R, Jones, SK, Davis, LH, Roberts, KF, and Bennett, V. "Isoform specificity among ankyrins: An amphipathic α-helix in the divergent regulatory domain of ankyrin-B interacts with the molecular co-chaperone Hdj1/Hsp40." Journal of Biological Chemistry 279.24 (2004): 25798-25804.
PMID
15075330
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
24
Publish Date
2004
Start Page
25798
End Page
25804
DOI
10.1074/jbc.M401296200

Lateral Membrane Biogenesis in Human Bronchial Epithelial Cells Requires 190-kDa Ankyrin-G

Ankyrin-G polypeptides are required for restriction of voltage-gated sodium channels, L1 cell adhesion molecules, and β IV spectrin to axon initial segments and are believed to couple the Na/K-ATPase to the spectrin-actin network at the lateral membrane in epithelial cells. We report here that depletion of 190-kDa ankyrin-G in human bronchial epithelial cells by small interfering RNA results in nearly complete loss of lateral plasma membrane in interphase cells, and also blocks de novo lateral membrane biogenesis following mitosis. Loss of the lateral membrane domain is accompanied by an expansion of apical and basal plasma membranes and preservation of apical-basal polarity. Expression of rat 190-kDa ankyrin-G, which is resistant to human small interfering RNA, prevents loss of the lateral membrane following depletion of human 190-kDa ankyrin-G. Human 220-kDa ankyrin-B, a closely related ankyrin isoform, is incapable of preserving the lateral membrane following 190-kDa ankyrin-G depletion. Moreover, analysis of rat 190-kDa ankyrin G/ankyrin B chimeras shows that all three domains of 190-kDa ankyrin-G are required for preservation of the lateral membrane. These results demonstrate that 190-kDa ankyrin-G plays a pleiotropic role in assembly of lateral membranes of bronchial epithelial cells.

Authors
Kizhatil, K; Bennett, V
MLA Citation
Kizhatil, K, and Bennett, V. "Lateral Membrane Biogenesis in Human Bronchial Epithelial Cells Requires 190-kDa Ankyrin-G." Journal of Biological Chemistry 279.16 (2004): 16706-16714.
PMID
14757759
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
16
Publish Date
2004
Start Page
16706
End Page
16714
DOI
10.1074/jbc.M314296200

Inositol 1,4,5-Trisphosphate Receptor Localization and Stability in Neonatal Cardiomyocytes Requires Interaction with Ankyrin-B

The molecular mechanisms required for inositol 1,4,5-trisphosphate receptor (InsP3R) targeting to specialized endoplasmic reticulum membrane domains are unknown. We report here a direct, high affinity interaction between InsP3R and ankyrin-B and demonstrate that this association is critical for InsP3R post-translational stability and localization in cultures of neonatal cardiomyocytes. Recombinant ankyrin-B membrane-binding domain directly interacts with purified cerebellar InsP 3R (Kd = 2 nM). 220-kDa ankyrin-B co-immunoprecipitates with InsP3R in tissue extracts from brain, heart, and lung. Alanine-scanning mutagenesis of the ankyrin-B ANK (ankyrin repeat) repeat β-hairpin loop tips revealed that consecutive ANK repeat β-hairpin loop tips (repeats 22-24) are required for InsP3R interaction, thus providing the first detailed evidence of how ankyrin polypeptides associate with membrane proteins. Pulse-chase biosynthesis experiments demonstrate that reduction or loss of ankyrin-B in ankyrin-B (+/-) or ankyrin-B (-/-) neonatal cardiomyocytes leads to ∼3-fold reduction in half-life of newly synthesized InsP3R. Furthermore, interactions with ankyrin-B are required for InsP3R stability as abnormal InsP3R phenotypes, including mis-localization, and reduced half-life in ankyrin-B (+/-) cardiomyocytes can be rescued by green fluorescent protein (GFP)-220-kDa ankyrin-B but not by GFP-220-kDa ankyrin-B mutants, which do not associate with InsP3R. These new results provide the first physiological evidence of a molecular partner required for early post-translational stability of InsP3R.

Authors
Mohler, PJ; Davis, JQ; Davis, LH; Hoffman, JA; Michaely, P; Bennett, V
MLA Citation
Mohler, PJ, Davis, JQ, Davis, LH, Hoffman, JA, Michaely, P, and Bennett, V. "Inositol 1,4,5-Trisphosphate Receptor Localization and Stability in Neonatal Cardiomyocytes Requires Interaction with Ankyrin-B." Journal of Biological Chemistry 279.13 (2004): 12980-12987.
PMID
14722080
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
13
Publish Date
2004
Start Page
12980
End Page
12987
DOI
10.1074/jbc.M313979200

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

Authors
Mohler, PJ; Schott, J-J; Gramolini, AO; Dilly, KW; Guatimosim, S; duBell, WH; Song, L-S; Haurogné, K; Kyndt, F; Ali, ME; Rogers, TB; Lederer, WJ; Escande, D; Le Marec, H; Bennett, V
MLA Citation
Mohler, PJ, Schott, J-J, Gramolini, AO, Dilly, KW, Guatimosim, S, duBell, WH, Song, L-S, Haurogné, K, Kyndt, F, Ali, ME, Rogers, TB, Lederer, WJ, Escande, D, Le Marec, H, and Bennett, V. "Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death." Nature 421.6923 (February 6, 2003): 634-639.
PMID
12571597
Source
pubmed
Published In
Nature
Volume
421
Issue
6923
Publish Date
2003
Start Page
634
End Page
639
DOI
10.1038/nature01335

L1-dependent neuritogenesis involves ankyrinB that mediates L1-CAM coupling with retrograde actin flow

The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin-actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD-ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.

Authors
Nishimura, K; Yoshihara, F; Tojima, T; Ooashi, N; Yoon, W; Mikoshiba, K; Bennett, V; Kamiguchi, H
MLA Citation
Nishimura, K, Yoshihara, F, Tojima, T, Ooashi, N, Yoon, W, Mikoshiba, K, Bennett, V, and Kamiguchi, H. "L1-dependent neuritogenesis involves ankyrinB that mediates L1-CAM coupling with retrograde actin flow." Journal of Cell Biology 163.5 (2003): 1077-1088.
PMID
14657231
Source
scival
Published In
Journal of Cell Biology
Volume
163
Issue
5
Publish Date
2003
Start Page
1077
End Page
1088
DOI
10.1083/jcb.200303060

Kv3.1b is a novel component of CNS nodes

We herein demonstrate that Kv3.1b subunits are present at nodes of Ranvier in the CNS of both rats and mice. Kv3.1b colocalizes with voltage-gated Na+ channels in a subset of nodes in the spinal cord, particularly those of large myelinated axons. Kv3.1b is abundantly expressed in the gray matter of the spinal cord, but does not colocalize with Na+ channels in initial segments. In the PNS, few nodes are Kv3.1b-positive. During the development of the CNS, Kv3.1b clustering at nodes occurs later than that of Na+ channels, but precedes the juxtaparanodal clustering of Kv1.2. Moreover, in myelin-deficient rats, which have severe CNS dysmyelination, node-like clusters of Kv3.1b and Na+ channels are observed even in regions devoid of oligodendrocytes. Ankyrin G coimmunoprecipitates Kv3.1b in vivo, indicating that these two proteins may interact in the CNS at nodes. 4-Aminopyridine, a K+ channel blocker, broadened the compound action potential recorded from adult rat optic nerve and spinal cord, but not from the sciatic nerve. These effects were also observed in Kv3.1-deficient mice. In conclusion, Kv3.1b is the first K+ channel subunit to be identified in CNS nodes; but Kv3.1b does not account for the effects of 4-aminopyridine on central myelinated tracts.

Authors
Devaux, J; Alcaraz, G; Grinspan, J; Bennett, V; Joho, R; Crest, M; Scherer, SS
MLA Citation
Devaux, J, Alcaraz, G, Grinspan, J, Bennett, V, Joho, R, Crest, M, and Scherer, SS. "Kv3.1b is a novel component of CNS nodes." Journal of Neuroscience 23.11 (2003): 4509-4518.
PMID
12805291
Source
scival
Published In
Journal of Neuroscience
Volume
23
Issue
11
Publish Date
2003
Start Page
4509
End Page
4518

α-adducin dissociates from F-actin and spectrin during platelet activation

Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. α-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcγRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form.

Authors
Barkalow, KL; Jr, JEI; Chou, DE; Matsuoka, Y; Bennett, V; Hartwig, JH
MLA Citation
Barkalow, KL, Jr, JEI, Chou, DE, Matsuoka, Y, Bennett, V, and Hartwig, JH. "α-adducin dissociates from F-actin and spectrin during platelet activation." Journal of Cell Biology 161.3 (2003): 557-570.
PMID
12743105
Source
scival
Published In
Journal of Cell Biology
Volume
161
Issue
3
Publish Date
2003
Start Page
557
End Page
570
DOI
10.1083/jcb.200211122

Mutation in the ankyrin-B gene causes long QT syndrome and sinus node dysfunction

Authors
Schott, JJ; Mohler, PJ; Gramolini, AO; Haurogne, W; Kyndt, F; Escande, D; Bennett, V; Le Marec, H
MLA Citation
Schott, JJ, Mohler, PJ, Gramolini, AO, Haurogne, W, Kyndt, F, Escande, D, Bennett, V, and Le Marec, H. "Mutation in the ankyrin-B gene causes long QT syndrome and sinus node dysfunction." November 5, 2002.
Source
wos-lite
Published In
Circulation
Volume
106
Issue
19
Publish Date
2002
Start Page
308
End Page
308

A 160 kD ankyrin-B isoform is associated with a novel myosin Va-containing vesicle compartment distinct from Golgi and lysosomes

Authors
Mohler, PJ; Bennett, V
MLA Citation
Mohler, PJ, and Bennett, V. "A 160 kD ankyrin-B isoform is associated with a novel myosin Va-containing vesicle compartment distinct from Golgi and lysosomes." November 2002.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
13
Publish Date
2002
Start Page
55A
End Page
56A

A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin.

Doublecortin is a cytoplasmic protein mutated in the neuronal migration disorder X-linked lissencephaly. This study describes a novel activity of doublecortin in recognition of the FIGQY-phosphotyrosine motif present in the cytoplasmic domain of the L1 cell adhesion molecule neurofascin. Phospho-FIGQY-neurofascin (186 kDa) coimmunoprecipitated with doublecortin from detergent extracts of embryonic brain membranes, and this doublecortin-phospho-FIGQY neurofascin complex was disassociated by a synthetic phospho-FIGQY neurofascin peptide but not by a dephospho-FIGQY peptide. Doublecortin specifically recognized the phospho-FIGQY tyrosine in the context of a synthetic phospho-FIGQY neurofascin peptide and in phospho-FIGQY neurofascin isolated from cells treated with pervanadate. Mutations of doublecortin causing lissencephaly (R59H, D62N, and G253D) abolished binding to the phospho-FIGQY peptide and to phospho-FIGQY neurofascin. Finally, phospho-FIGQY neurofascin and doublecortin colocalize in developing axon tracts and in zones enriched in migrating neurons in the embryonic cerebral cortex. In the adult rostral migratory stream, doublecortin colocalizes in migrating neurons with a phospho-FIGQY bearing L1 CAM different from neurofascin. The finding that doublecortin associates with FIGQY-phosphorylated neurofascin provides the first connection of doublecortin with the plasma membrane and could be important for a function of doublecortin in directing neuronal migration.

Authors
Kizhatil, K; Wu, Y-X; Sen, A; Bennett, V
MLA Citation
Kizhatil, K, Wu, Y-X, Sen, A, and Bennett, V. "A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin." J Neurosci 22.18 (September 15, 2002): 7948-7958.
PMID
12223548
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
22
Issue
18
Publish Date
2002
Start Page
7948
End Page
7958

Sexual desire in post-menopausal women.

Authors
Bennett, V
MLA Citation
Bennett, V. "Sexual desire in post-menopausal women." Aust Fam Physician 31.7 (July 2002): 603-. (Letter)
PMID
12143319
Source
pubmed
Published In
Australian family physician
Volume
31
Issue
7
Publish Date
2002
Start Page
603

Ankyrins: Molecular keys to the cellular code for directing ion channels to sites of physiological function

Authors
Bennett, V; Mohler, P; Gramolini, A
MLA Citation
Bennett, V, Mohler, P, and Gramolini, A. "Ankyrins: Molecular keys to the cellular code for directing ion channels to sites of physiological function." JOURNAL OF GENERAL PHYSIOLOGY 120.1 (July 2002): 5A-6A.
Source
wos-lite
Published In
The Journal of General Physiology
Volume
120
Issue
1
Publish Date
2002
Start Page
5A
End Page
6A

Ankyrins.

Authors
Mohler, PJ; Gramolini, AO; Bennett, V
MLA Citation
Mohler, PJ, Gramolini, AO, and Bennett, V. "Ankyrins." J Cell Sci 115.Pt 8 (April 15, 2002): 1565-1566.
PMID
11950874
Source
pubmed
Published In
Journal of cell science
Volume
115
Issue
Pt 8
Publish Date
2002
Start Page
1565
End Page
1566

The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate and ryanodine receptor distribution in ankyrin-B (-/-) neonatal cardiomyocytes.

Ankyrins are a closely related family of membrane adaptor proteins that are believed to participate in targeting diverse membrane proteins to specialized domains in the plasma membrane and endoplasmic reticulum. This study addresses the question of how individual ankyrin isoforms achieve functional specificity when co-expressed in the same cell. Cardiomyocytes from ankyrin-B (-/-) mice display mis-localization of inositol 1,4,5-trisphosphate receptors and ryanodine receptors along with reduced contraction rates that can be rescued by expression of green fluorescent protein (GFP)-ankyrin-B but not GFP-ankyrin-G. We developed chimeric GFP expression constructs containing all combinations of the three major domains of ankyrin-B and ankyrin-G to determine which domain(s) of ankyrin-B are required for ankyrin-B-specific functions. The death/C-terminal domain of ankyrin-B determined activity of ankyrin-B/G chimeras in localization in a striated pattern in cardiomyocytes and in restoration of a normal striated distribution of both ryanodine and inositol 1,4,5-trisphosphate receptors as well as normal beat frequency of contracting cardiomyocytes. Further deletions within the death/C-terminal domain demonstrated that the C-terminal domain determines ankyrin-B activity, whereas deletion of the death domain had no effect. C-terminal domains are the most divergent between ankyrin isoforms and are candidates to encode the signal(s) that enable ankyrins to selectively target proteins to diverse cellular sites.

Authors
Mohler, PJ; Gramolini, AO; Bennett, V
MLA Citation
Mohler, PJ, Gramolini, AO, and Bennett, V. "The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate and ryanodine receptor distribution in ankyrin-B (-/-) neonatal cardiomyocytes." J Biol Chem 277.12 (March 22, 2002): 10599-10607.
PMID
11781319
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
12
Publish Date
2002
Start Page
10599
End Page
10607
DOI
10.1074/jbc.M110958200

Developing nodes of Ranvier are defined by ankyrin-G clustering and are independent of paranodal axoglial adhesion.

Nodes of Ranvier are excitable regions of axonal membranes highly enriched in voltage-gated sodium channels that propagate action potentials. The mechanism of protein clustering at nodes has been a source of controversy. In this study, developmental analysis of nodes of Ranvier in optic nerve axons reveals that early node intermediates are defined by ankyrin-G. Other node components, including beta IV spectrin, voltage-gated sodium channels, and the L1 cell adhesion molecule neurofascin, are subsequently recruited to sites of ankyrin-G clustering. The role of intact paranodes in protein clustering was examined in the dysmyelinating mouse mutant jimpy. Jimpy mice do not have intact paranodal axoglial contacts, which is indicated by a complete lack of neurexin/contactin-associated protein/paranodin clustering in paranodes. In the absence of intact paranodes, ankyrin-G was still able to cluster, although fewer ankyrin clusters were seen in jimpy optic nerves than in wild-type optic nerves. Recruitment of Na(v)1.2, Na(v)1.6, beta IV spectrin, and neurofascin to sites of ankyrin-G clustering is unimpaired in jimpy mice, indicating that node formation occurs independent of intact paranodal axoglial contacts.

Authors
Jenkins, SM; Bennett, V
MLA Citation
Jenkins, SM, and Bennett, V. "Developing nodes of Ranvier are defined by ankyrin-G clustering and are independent of paranodal axoglial adhesion." Proc Natl Acad Sci U S A 99.4 (February 19, 2002): 2303-2308.
PMID
11842202
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
4
Publish Date
2002
Start Page
2303
End Page
2308
DOI
10.1073/pnas.042601799

Ankyrins

Authors
Mohler, PJ; Gramolini, AO; Bennett, V
MLA Citation
Mohler, PJ, Gramolini, AO, and Bennett, V. "Ankyrins." Journal of Cell Science 115.8 (2002): 1565-1566.
Source
scival
Published In
Journal of Cell Science
Volume
115
Issue
8
Publish Date
2002
Start Page
1565
End Page
1566

Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments.

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, betaIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and betaIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. betaIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.

Authors
Jenkins, SM; Bennett, V
MLA Citation
Jenkins, SM, and Bennett, V. "Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments." J Cell Biol 155.5 (November 26, 2001): 739-746.
PMID
11724816
Source
pubmed
Published In
The Journal of Cell Biology
Volume
155
Issue
5
Publish Date
2001
Start Page
739
End Page
746
DOI
10.1083/jcb.200109026

alpha-adducin connects actin filament barbed ends to the platelet membrane skeleton.

Authors
Barkalow, KL; Lee, MK; Italiano, JE; Bennett, V; Hartwig, JH
MLA Citation
Barkalow, KL, Lee, MK, Italiano, JE, Bennett, V, and Hartwig, JH. "alpha-adducin connects actin filament barbed ends to the platelet membrane skeleton." BLOOD 98.11 (November 16, 2001): 787A-787A.
Source
wos-lite
Published In
Blood
Volume
98
Issue
11
Publish Date
2001
Start Page
787A
End Page
787A

FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons.

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.

Authors
Jenkins, SM; Kizhatil, K; Kramarcy, NR; Sen, A; Sealock, R; Bennett, V
MLA Citation
Jenkins, SM, Kizhatil, K, Kramarcy, NR, Sen, A, Sealock, R, and Bennett, V. "FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons." J Cell Sci 114.Pt 21 (November 2001): 3823-3835.
PMID
11719549
Source
pubmed
Published In
Journal of cell science
Volume
114
Issue
Pt 21
Publish Date
2001
Start Page
3823
End Page
3835

Requirement of ankyrin-B in the intracellular targeting of IP3 receptor calcium release channels in diverse tissues

Authors
Gramolini, AO; Mohler, PJ; Ali, ME; Davis, LH; Kontos, CD; Bennett, V
MLA Citation
Gramolini, AO, Mohler, PJ, Ali, ME, Davis, LH, Kontos, CD, and Bennett, V. "Requirement of ankyrin-B in the intracellular targeting of IP3 receptor calcium release channels in diverse tissues." MOLECULAR BIOLOGY OF THE CELL 12 (November 2001): 303A-303A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
12
Publish Date
2001
Start Page
303A
End Page
303A

Ankyrin-B COOH terminal domain is required for the segregation of calcium-release channels to spatially-privileged domains within the ER/SR membrane

Authors
Mohler, PJ; Gramolini, AO; Bennett, V
MLA Citation
Mohler, PJ, Gramolini, AO, and Bennett, V. "Ankyrin-B COOH terminal domain is required for the segregation of calcium-release channels to spatially-privileged domains within the ER/SR membrane." MOLECULAR BIOLOGY OF THE CELL 12 (November 2001): 451A-451A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
12
Publish Date
2001
Start Page
451A
End Page
451A

Neuronal migration and cell surface signaling: Doublecortin binds to a phospho-FIGQY-tyrosine motif conserved in the cytoplasmic domains of L1 family of cell adhesion molecules

Authors
Kizhatil, K; Wu, YX; Sen, A; Bennett, V
MLA Citation
Kizhatil, K, Wu, YX, Sen, A, and Bennett, V. "Neuronal migration and cell surface signaling: Doublecortin binds to a phospho-FIGQY-tyrosine motif conserved in the cytoplasmic domains of L1 family of cell adhesion molecules." MOLECULAR BIOLOGY OF THE CELL 12 (November 2001): 239A-239A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
12
Publish Date
2001
Start Page
239A
End Page
239A

Physiological evidence for an ankyrin-G dependent transcellular protein complex containing BetaIV spectrin, the voltage-gated sodium channel 1.6, and L1 CAMs at Purkinje neuron initial segments

Authors
Jenkins, SM; Bennett, V; Komada, M
MLA Citation
Jenkins, SM, Bennett, V, and Komada, M. "Physiological evidence for an ankyrin-G dependent transcellular protein complex containing BetaIV spectrin, the voltage-gated sodium channel 1.6, and L1 CAMs at Purkinje neuron initial segments." MOLECULAR BIOLOGY OF THE CELL 12 (November 2001): 184A-184A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
12
Publish Date
2001
Start Page
184A
End Page
184A

LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling.

This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.

Authors
Chen, L; Ong, B; Bennett, V
MLA Citation
Chen, L, Ong, B, and Bennett, V. "LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling." J Cell Biol 154.4 (August 20, 2001): 841-855.
PMID
11502758
Source
pubmed
Published In
The Journal of Cell Biology
Volume
154
Issue
4
Publish Date
2001
Start Page
841
End Page
855
DOI
10.1083/jcb.200009004

Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues.

The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

Authors
Bennett, V; Baines, AJ
MLA Citation
Bennett, V, and Baines, AJ. "Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues." Physiol Rev 81.3 (July 2001): 1353-1392. (Review)
PMID
11427698
Source
pubmed
Published In
Physiological reviews
Volume
81
Issue
3
Publish Date
2001
Start Page
1353
End Page
1392

Association of protein kinase C(lambda) with adducin in 3T3-L1 adipocytes.

There is evidence that the atypical protein kinases C (PKC(lambda), PKC(zeta)) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKC(lambda/zeta) from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKC(lambda) is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of alpha- and gamma-adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKC(lambda). Association of the adducins with PKC(lambda) was further indicated by the finding that the adducins coimmunoprecipitated proportionally with PKC(lambda) in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the adducins in a whole cell lysate or on the small portion of the adducins that coimmunoprecipitated with PKC(lambda). PKC(lambda) and the adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKC(lambda) to adducin may function to localize PKC(lambda) in 3T3-L1 adipocytes.

Authors
Laustsen, PG; Lane, WS; Bennett, V; Lienhard, GE
MLA Citation
Laustsen, PG, Lane, WS, Bennett, V, and Lienhard, GE. "Association of protein kinase C(lambda) with adducin in 3T3-L1 adipocytes." Biochim Biophys Acta 1539.1-2 (May 28, 2001): 163-172.
PMID
11389978
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1539
Issue
1-2
Publish Date
2001
Start Page
163
End Page
172

Ankyrins and cellular targeting of diverse membrane proteins to physiological sites.

Ankyrins are spectrin-binding proteins that associate via ANK repeats with a variety of ion channels/pumps, calcium release channels and cell adhesion molecules. Recent studies in mice indicate that ankyrins have a physiological role in restricting voltage-gated sodium channels and members of the L1 CAM family of cell adhesion molecules to excitable membranes in the central nervous system and in targeting calcium-release channels to the calcium homeostasis compartment of striated muscle.

Authors
Bennett, V; Chen, L
MLA Citation
Bennett, V, and Chen, L. "Ankyrins and cellular targeting of diverse membrane proteins to physiological sites." Curr Opin Cell Biol 13.1 (February 2001): 61-67. (Review)
PMID
11163135
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
13
Issue
1
Publish Date
2001
Start Page
61
End Page
67

FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.

Authors
Jenkins, SM; Kizhatil, K; Kramarcy, NR; Sen, A; Sealock, R; Bennett, V
MLA Citation
Jenkins, SM, Kizhatil, K, Kramarcy, NR, Sen, A, Sealock, R, and Bennett, V. "FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons." Journal of Cell Science 114.21 (2001): 3823-3835.
Source
scival
Published In
Journal of Cell Science
Volume
114
Issue
21
Publish Date
2001
Start Page
3823
End Page
3835

Partial ankyrin-B deficiency in heterozygous ankyrin-B (+/-) mice results in abnormal heart conduction and musculoskeletal defects

Authors
Bennett, V; Ali, M; Davis, L; Tuvia, S
MLA Citation
Bennett, V, Ali, M, Davis, L, and Tuvia, S. "Partial ankyrin-B deficiency in heterozygous ankyrin-B (+/-) mice results in abnormal heart conduction and musculoskeletal defects." MOLECULAR BIOLOGY OF THE CELL 11 (December 2000): 76A-76A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
11
Publish Date
2000
Start Page
76A
End Page
76A

LAD-1, the C-elegans L1CAM homologue, has essential cell adhesion roles in early embryogenesis, gonadal arm migration, and morphogenesis of multiple tissues

Authors
Chen, LS; Ong, B; Bennett, V
MLA Citation
Chen, LS, Ong, B, and Bennett, V. "LAD-1, the C-elegans L1CAM homologue, has essential cell adhesion roles in early embryogenesis, gonadal arm migration, and morphogenesis of multiple tissues." MOLECULAR BIOLOGY OF THE CELL 11 (December 2000): 272A-272A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
11
Publish Date
2000
Start Page
272A
End Page
272A

Ankyrin-B associated molecular complexes isolated from brain membranes and cytosol

Authors
Davis, JQ; Davis, LH; Bennett, V
MLA Citation
Davis, JQ, Davis, LH, and Bennett, V. "Ankyrin-B associated molecular complexes isolated from brain membranes and cytosol." MOLECULAR BIOLOGY OF THE CELL 11 (December 2000): 550A-550A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
11
Publish Date
2000
Start Page
550A
End Page
550A

FIGQY phosphorylation of L1 CAMs defines a novel signaling pathway in ankyrin-free domains of the PNS and migratory regions of the CNS

Authors
Jenkins, SM; Sen, A; Kramarcy, NR; Sealock, R; Bennett, V
MLA Citation
Jenkins, SM, Sen, A, Kramarcy, NR, Sealock, R, and Bennett, V. "FIGQY phosphorylation of L1 CAMs defines a novel signaling pathway in ankyrin-free domains of the PNS and migratory regions of the CNS." MOLECULAR BIOLOGY OF THE CELL 11 (December 2000): 553A-553A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
11
Publish Date
2000
Start Page
553A
End Page
553A

Doublecortin is a SH2/PTB independent phosphotyrosine specific adapter for L1 family cell adhesion molecules.

Authors
Kizhatil, K; Sen, A; Bennett, V
MLA Citation
Kizhatil, K, Sen, A, and Bennett, V. "Doublecortin is a SH2/PTB independent phosphotyrosine specific adapter for L1 family cell adhesion molecules." MOLECULAR BIOLOGY OF THE CELL 11 (December 2000): 57A-57A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
11
Publish Date
2000
Start Page
57A
End Page
57A

Taking a risk to save a life.

Authors
Bennett, V
MLA Citation
Bennett, V. "Taking a risk to save a life." Nursing 30.10 (October 2000): 96-.
PMID
11096982
Source
pubmed
Published In
Nursing
Volume
30
Issue
10
Publish Date
2000
Start Page
96

Adducin: structure, function and regulation.

Adducin is a ubiquitously expressed membrane-skeletal protein localized at spectrin-actin junctions that binds calmodulin and is an in vivo substrate for protein kinase C (PKC) and Rho-associated kinase. Adducin is a tetramer comprised of either alpha/beta or alpha/gamma heterodimers. Adducin subunits are related in sequence and all contain an N-terminal globular head domain, a neck domain and a C-terminal protease-sensitive tail domain. The tail domains of all adducin subunits end with a highly conserved 22-residue myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that has homology to MARCKS protein. Adducin caps the fast-growing ends of actin filaments and also preferentially recruits spectrin to the ends of filaments. Both the neck and the MARCKS-related domains are required for these activities. The neck domain self-associates to form oligomers. The MARCKS-related domain binds calmodulin and contains the major phosphorylation site for PKC. Calmodulin, gelsolin and phosphorylation by the kinase inhibit in vitro activities of adducin involving actin and spectrin. Recent observations suggest a role for adducin in cell motility, and as a target for regulation by Rho-dependent and Ca2+-dependent pathways. Prominent physiological sites of regulation of adducin include dendritic spines of hippocampal neurons, platelets and growth cones of axons.

Authors
Matsuoka, Y; Li, X; Bennett, V
MLA Citation
Matsuoka, Y, Li, X, and Bennett, V. "Adducin: structure, function and regulation." Cell Mol Life Sci 57.6 (June 2000): 884-895. (Review)
PMID
10950304
Source
pubmed
Published In
Cellular and Molecular Life Sciences
Volume
57
Issue
6
Publish Date
2000
Start Page
884
End Page
895
DOI
10.1007/PL00000731

alpha-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro.

G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Herein we report that alpha-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with alpha-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and alpha-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and alpha-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of alpha-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity.

Authors
Freeman, JL; Pitcher, JA; Li, X; Bennett, V; Lefkowitz, RJ
MLA Citation
Freeman, JL, Pitcher, JA, Li, X, Bennett, V, and Lefkowitz, RJ. "alpha-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro." FEBS Lett 473.3 (May 19, 2000): 280-284.
PMID
10818226
Source
pubmed
Published In
FEBS Letters
Volume
473
Issue
3
Publish Date
2000
Start Page
280
End Page
284

Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function.

The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.

Authors
Moorthy, S; Chen, L; Bennett, V
MLA Citation
Moorthy, S, Chen, L, and Bennett, V. "Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function." J Cell Biol 149.4 (May 15, 2000): 915-930.
PMID
10811831
Source
pubmed
Published In
The Journal of Cell Biology
Volume
149
Issue
4
Publish Date
2000
Start Page
915
End Page
930

Abnormal cardiac Na(+) channel properties and QT heart rate adaptation in neonatal ankyrin(B) knockout mice.

The cytoskeleton of the cardiomyocyte has been shown to modulate ion channel function. Cytoskeletal disruption in vitro alters Na(+) channel kinetics, producing a late Na(+) current that can prolong repolarization. This study describes the properties of the cardiac Na(+) channel and cardiac repolarization in neonatal mice lacking ankyrin(B), a cytoskeletal "adaptor" protein. Using whole-cell voltage clamp techniques, I(Na) density was lower in ankyrin(B)(-/-) ventricular myocytes than in wild-type (WT) myocytes (-307+/-26 versus -444+/-39 pA/pF, P<0.01). Ankyrin(B)(-/-) myocytes exhibited a hyperpolarizing shift in activation and inactivation kinetics compared with WT. Slower recovery from inactivation contributed to the negative shift in steady-state inactivation in ankyrin(B)(-/-). Single Na(+) channel mean open time was longer in ankyrin(B)(-/-) versus WT at test potentials (V(t)) of -40 mV (1.0+/-0.1 versus 0. 61+/-0.04 ms, P<0.05) and -50 mV (0.8+/-0.1 versus 0.39+/-0.05 ms, P<0.05). Ankyrin(B)(-/-) exhibited late single-channel openings at V(t) -40 and -50 mV, which were not seen in WT. Late I(Na) contributed to longer action potential durations measured at 90% repolarization (APD(90)) at 1 Hz stimulation in ankyrin(B)(-/-) compared with WT (354+/-26 versus 274+/-22 ms, P<0.05). From ECG recordings of neonatal mice, heart rates were slower in ankyrin(B)(-/-) than in WT (380+/-14 versus 434+/-13 bpm, P<0.01). Although the QT interval was similar in ankyrin(B)(-/-) and WT at physiological heart rates, QT-interval prolongation in response to heart rate deceleration was greater in ankyrin(B)(-/-). In conclusion, Na(+) channels in ankyrin(B)(-/-) display reduced I(Na) density and abnormal kinetics at the whole-cell and single-channel level that contribute to prolonged APD(90) and abnormal QT-rate adaptation.

Authors
Chauhan, VS; Tuvia, S; Buhusi, M; Bennett, V; Grant, AO
MLA Citation
Chauhan, VS, Tuvia, S, Buhusi, M, Bennett, V, and Grant, AO. "Abnormal cardiac Na(+) channel properties and QT heart rate adaptation in neonatal ankyrin(B) knockout mice." Circ Res 86.4 (March 3, 2000): 441-447.
PMID
10700449
Source
pubmed
Published In
Circulation Research
Volume
86
Issue
4
Publish Date
2000
Start Page
441
End Page
447

α-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro

G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Herein we report that α-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with α-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and α-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and α-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of α-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity. Copyright (C) 2000 Federation of European Biochemical Societies.

Authors
Freeman, JLR; Pitcher, JA; Li, X; Bennett, V; Lefkowitz, RJ
MLA Citation
Freeman, JLR, Pitcher, JA, Li, X, Bennett, V, and Lefkowitz, RJ. "α-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro." FEBS Letters 473.3 (2000): 280-284.
Source
scival
Published In
FEBS Letters
Volume
473
Issue
3
Publish Date
2000
Start Page
280
End Page
284
DOI
10.1016/S0014-5793(00)01543-X

A requirement for ankyrin binding to clathrin during coated pit budding.

Recent studies suggest that the mobility of clathrin-coated pits at the cell surface are restricted by an actin cytoskeleton and that there is an obligate reduction in the amount of spectrin on membranes during coated pit budding. The spectrin-actin cytoskeleton associates with membranes primarily through ankyrins, which interact with the cytoplasmic region of numerous integral membrane proteins. We now report that the fourth repeat domain (D4) of ankyrin(R) binds to the N-terminal domain of clathrin heavy chain with high affinity. Addition of peptides containing the D4 region inhibited clathrin-coated pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LDL). Ankyrin(R) peptides that contained repeat domains other than D4 had no effect on either in vitro budding or internalization of LDL. Finally, immunofluorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of clathrin-coated pits during endocytosis.

Authors
Michaely, P; Kamal, A; Anderson, RG; Bennett, V
MLA Citation
Michaely, P, Kamal, A, Anderson, RG, and Bennett, V. "A requirement for ankyrin binding to clathrin during coated pit budding." J Biol Chem 274.50 (December 10, 1999): 35908-35913.
PMID
10585476
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
50
Publish Date
1999
Start Page
35908
End Page
35913

Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins.

This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.

Authors
Tuvia, S; Buhusi, M; Davis, L; Reedy, M; Bennett, V
MLA Citation
Tuvia, S, Buhusi, M, Davis, L, Reedy, M, and Bennett, V. "Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins." J Cell Biol 147.5 (November 29, 1999): 995-1008.
PMID
10579720
Source
pubmed
Published In
The Journal of Cell Biology
Volume
147
Issue
5
Publish Date
1999
Start Page
995
End Page
1008

Human platelet adducin: A regulated actin filament barbed end-capping protein.

Authors
Barkalow, KL; Italiano, JE; Azim, AC; Matsuoka, Y; Bennett, V; Hartwig, JH
MLA Citation
Barkalow, KL, Italiano, JE, Azim, AC, Matsuoka, Y, Bennett, V, and Hartwig, JH. "Human platelet adducin: A regulated actin filament barbed end-capping protein." BLOOD 94.10 (November 15, 1999): 216A-216A.
Source
wos-lite
Published In
Blood
Volume
94
Issue
10
Publish Date
1999
Start Page
216A
End Page
216A

Abnormal Na channel properties and QT-heart rate adaptation in Ankyirn(B) knockout mice

Authors
Chauhan, VS; Tuvia, S; Buhusi, M; Bennett, V; Grant, AO
MLA Citation
Chauhan, VS, Tuvia, S, Buhusi, M, Bennett, V, and Grant, AO. "Abnormal Na channel properties and QT-heart rate adaptation in Ankyirn(B) knockout mice." CIRCULATION 100.18 (November 2, 1999): 351-351.
Source
wos-lite
Published In
Circulation
Volume
100
Issue
18
Publish Date
1999
Start Page
351
End Page
351

Potential role of ankyrins in endocytosis.

Authors
Michaely, P; Kamal, A; Anderson, RGW; Bennett, V
MLA Citation
Michaely, P, Kamal, A, Anderson, RGW, and Bennett, V. "Potential role of ankyrins in endocytosis." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 307A-307A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
307A
End Page
307A

Establishment of epithelial polarity in C-elegans does not require beta G spectrin

Authors
Chen, L; Moorthy, S; Otsuka, A; Bennett, V
MLA Citation
Chen, L, Moorthy, S, Otsuka, A, and Bennett, V. "Establishment of epithelial polarity in C-elegans does not require beta G spectrin." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 395A-395A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
395A
End Page
395A

ARM-1, a novel ankyrin-binding protein that may regulate Z disc spacing in C-elegans muscle

Authors
Chen, L; Davis, JQ; Hoffman, J; Bennett, V
MLA Citation
Chen, L, Davis, JQ, Hoffman, J, and Bennett, V. "ARM-1, a novel ankyrin-binding protein that may regulate Z disc spacing in C-elegans muscle." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 246A-246A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
246A
End Page
246A

Human platelet adducin: a regulated actin filament barbed end-capping protein.

Authors
Barkalow, K; Italiano, JE; Matsuoka, Y; Bennett, V; Hartwig, JH
MLA Citation
Barkalow, K, Italiano, JE, Matsuoka, Y, Bennett, V, and Hartwig, JH. "Human platelet adducin: a regulated actin filament barbed end-capping protein." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 29A-29A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
29A
End Page
29A

Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility.

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. Here we identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized alpha-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.

Authors
Fukata, Y; Oshiro, N; Kinoshita, N; Kawano, Y; Matsuoka, Y; Bennett, V; Matsuura, Y; Kaibuchi, K
MLA Citation
Fukata, Y, Oshiro, N, Kinoshita, N, Kawano, Y, Matsuoka, Y, Bennett, V, Matsuura, Y, and Kaibuchi, K. "Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility." J Cell Biol 145.2 (April 19, 1999): 347-361.
PMID
10209029
Source
pubmed
Published In
The Journal of Cell Biology
Volume
145
Issue
2
Publish Date
1999
Start Page
347
End Page
361

Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels.

440 kD ankyrin-B and 480/270 kD ankyrin-G are membrane skeletal proteins with closely related biochemical properties yet distinctive physiological roles in axons. These proteins associate with spectrin-actin networks and also bind to integral membrane proteins including the L1 CAM family of cell adhesion molecules and voltage-gated sodium channels. 440 kD ankyrin-B is expressed with L1 in premyelinated axon tracts, and is essential for survival of these axons, at least in the case of the optic nerve. 440 ankyrin-B may collaborate with L1 in transcellular structures that mediate axon fasciculation and mechanically stabilize axon bundles, although these proteins may also be involved in axon pathfinding. Ankyrin-B (-/-) mice exhibit loss of L1 from premyelinated axon tracts and a similar, although much more severe, phenotype to L1 (-/-) mice and humans with L1 mutations. Ankyrin-B and L1 thus are candidates to collaborate in the same structural pathway and defects in this pathway can lead to nervous system malformations and mental retardation. 480/270 kD ankyrin-G are highly concentrated along with the L1CAM family members neurofascin and NrCAM at nodes of Ranvier and axon initial segments. Voltage-gated sodium channels bind directly to ankyrins, and are likely to associate in a ternary complex containing neurofascin/NrCAM, and ankyrin-G. Mice with ankyrin-G expression abolished in the cerebellum exhibit loss of ability of Purkinje neurons to fire action potentials, as well as loss of restriction of neurofascin/NrCAM to axon initial segments. Ankyrin-G thus is a key component in assembly of functional components of the axon initial segment and possibly the node of Ranvier.

Authors
Bennett, V; Lambert, S
MLA Citation
Bennett, V, and Lambert, S. "Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels." J Neurocytol 28.4-5 (April 1999): 303-318. (Review)
PMID
10739573
Source
pubmed
Published In
Journal of neurocytology
Volume
28
Issue
4-5
Publish Date
1999
Start Page
303
End Page
318

Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels

440 kD ankyrin-B and 480/270 kD ankyrin(G) are membrane skeletal proteins with closely related biochemical properties yet distinctive physiological roles in axons. These proteins associate with spectrin-actin networks and also bind to integral membrane proteins including the L1 CAM family of cell adhesion molecules and voltage-gated sodium channels. 440 kD ankyrin-B is expressed with L1 in premyelinated axon tracts, and is essential for survival of these axons, at least in the case of the optic nerve. 440 ankyrin-B may collaborate with L1 in transcellular structures that mediate axon fasciculation and mechanically stabilize axon bundles, although these proteins may also be involved in axon pathfinding. Ankyrin-B (-/-) mice exhibit loss of L1 from premyelinated axon tracts and a similar, although much more severe, phenotype to L1 (-/-) mice and humans with L1 mutations. Ankyrin-B and L1 thus are candidates to collaborate in the same structural pathway and defects in this pathway can lead to nervous system malformations and mental retardation. 480/270 kD ankyrin(G) are highly concentrated along with the L1CAM family members neurofascin and NrCAM at nodes of Ranvier and axon initial segments. Voltage-gated sodium channels bind directly to ankyrins, and are likely to associate in a ternary complex containing neurofascin/NrCAM, and ankyrin(G). Mice with ankyrin(G) expression abolished in the cerebellum exhibit loss of ability of Purkinje neurons to fire action potentials, as well as loss of restriction of neurofascin/NrCAM to axon initial segments. Ankyrin(G) thus is a key component in assembly of functional components of the axon initial segment and possibly the node of Ranvier.

Authors
Bennett, V; Lambert, S
MLA Citation
Bennett, V, and Lambert, S. "Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels." Journal of Neurocytology 28.4-5 (1999): 303-318.
Source
scival
Published In
Journal of Neurocytology
Volume
28
Issue
4-5
Publish Date
1999
Start Page
303
End Page
318

Ankyrin(G) and the localization of sodium channels in the axonal membrane.

Authors
Zhou, D; Malen, PL; Walker, B; Carpenter, S; Boland, LM; Bennett, V; Lambert, S
MLA Citation
Zhou, D, Malen, PL, Walker, B, Carpenter, S, Boland, LM, Bennett, V, and Lambert, S. "Ankyrin(G) and the localization of sodium channels in the axonal membrane." JOURNAL OF NEUROCHEMISTRY 73 (1999): S6-S6.
Source
wos-lite
Published In
Journal of Neurochemistry
Volume
73
Publish Date
1999
Start Page
S6
End Page
S6

Nervous system defects of AnkyrinB (-/-) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons.

The L1 CAM family of cell adhesion molecules and the ankyrin family of spectrin-binding proteins are candidates to collaborate in transcellular complexes used in diverse contexts in nervous systems of vertebrates and invertebrates. This report presents evidence for functional coupling between L1 and 440-kD ankyrinB in premyelinated axons in the mouse nervous system. L1 and 440-kD ankyrinB are colocalized in premyelinated axon tracts in the developing nervous system and are both down-regulated after myelination. AnkyrinB (-/-) mice exhibit a phenotype similar to, but more severe, than L1 (-/-) mice and share features of human patients with L1 mutations. AnkyrinB (-/-) mice exhibit hypoplasia of the corpus callosum and pyramidal tracts, dilated ventricles, and extensive degeneration of the optic nerve, and they die by postnatal day 21. AnkyrinB (-/-) mice have reduced L1 in premyelinated axons of long fiber tracts, including the corpus callosum, fimbria, and internal capsule in the brain, and pyramidal tracts and lateral columns of the spinal cord. L1 was evident in the optic nerve at postnatal day 1 but disappeared by postnatal day 7 in mutant mice while NCAM was unchanged. Optic nerve axons of ankyrinB (-/-) mice become dilated with diameters up to eightfold greater than normal, and they degenerated by day 20. These findings provide the first evidence for a role of ankyrinB in the nervous system and support an interaction between 440-kD ankyrinB and L1 that is essential for maintenance of premyelinated axons in vivo.

Authors
Scotland, P; Zhou, D; Benveniste, H; Bennett, V
MLA Citation
Scotland, P, Zhou, D, Benveniste, H, and Bennett, V. "Nervous system defects of AnkyrinB (-/-) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons." J Cell Biol 143.5 (November 30, 1998): 1305-1315.
PMID
9832558
Source
pubmed
Published In
The Journal of Cell Biology
Volume
143
Issue
5
Publish Date
1998
Start Page
1305
End Page
1315

AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing.

Voltage-gated sodium channels (NaCh) are colocalized with isoforms of the membrane-skeletal protein ankyrinG at axon initial segments, nodes of Ranvier, and postsynaptic folds of the mammalian neuromuscular junction. The role of ankyrinG in directing NaCh localization to axon initial segments was evaluated by region-specific knockout of ankyrinG in the mouse cerebellum. Mutant mice exhibited a progressive ataxia beginning around postnatal day P16 and subsequent loss of Purkinje neurons. In mutant mouse cerebella, NaCh were absent from axon initial segments of granule cell neurons, and Purkinje cells showed deficiencies in their ability to initiate action potentials and support rapid, repetitive firing. Neurofascin, a member of the L1CAM family of ankyrin-binding cell adhesion molecules, also exhibited impaired localization to initial segments of Purkinje cell neurons. These results demonstrate that ankyrinG is essential for clustering NaCh and neurofascin at axon initial segments and is required for physiological levels of sodium channel activity.

Authors
Zhou, D; Lambert, S; Malen, PL; Carpenter, S; Boland, LM; Bennett, V
MLA Citation
Zhou, D, Lambert, S, Malen, PL, Carpenter, S, Boland, LM, and Bennett, V. "AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing." J Cell Biol 143.5 (November 30, 1998): 1295-1304.
PMID
9832557
Source
pubmed
Published In
The Journal of Cell Biology
Volume
143
Issue
5
Publish Date
1998
Start Page
1295
End Page
1304

Structural requirements for association of neurofascin with ankyrin.

This paper presents the first structural analysis of the cytoplasmic domain of neurofascin, which is highly conserved among the L1CAM family of cell adhesion molecules, and describes sequence requirements for neurofascin-ankyrin interactions in living cells. The cytoplasmic domain of neurofascin dimerizes in solution, has an asymmetric shape, and exhibits a reversible temperature-dependent beta-structure. Residues Ser56-Tyr81 are necessary for ankyrin binding but do not contribute to either dimerization or formation of structure. Transfected neurofascin recruits GFP-tagged 270-kDa ankyrinG to the plasma membrane of human embryo kidney 293 cells. Deletion mutants demonstrate that the sequence Ser56-Tyr81 contains the major ankyrin-recruiting activity of neurofascin. Mutations of the FIGQY tyrosine (Y81H/A/E) greatly impair neurofascin-ankyrin interactions. Mutation of human L1 at the equivalent tyrosine (Y1229H) is responsible for certain cases of mental retardation (Van Camp, G., Fransen, E., Vits, L., Raes, G., and Willems, P. J. (1996) Hum. Mutat. 8, 391). Mutations F77A and E73Q greatly impair ankyrin binding activity, whereas mutation D74N and a triple mutation of D57N/D58N/D62N result in less loss of ankyrin binding activity. These results provide evidence for a highly specific interaction between ankyrin and neurofascin and suggest that ankyrin association with L1 is required for L1 function in humans.

Authors
Zhang, X; Davis, JQ; Carpenter, S; Bennett, V
MLA Citation
Zhang, X, Davis, JQ, Carpenter, S, and Bennett, V. "Structural requirements for association of neurofascin with ankyrin." J Biol Chem 273.46 (November 13, 1998): 30785-30794.
PMID
9804856
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
46
Publish Date
1998
Start Page
30785
End Page
30794

A new adducin function: A molecular arbitrator between myosin and spectrin for access to cortical actin.

Authors
Matsuoka, Y; Li, X; Bennett, V
MLA Citation
Matsuoka, Y, Li, X, and Bennett, V. "A new adducin function: A molecular arbitrator between myosin and spectrin for access to cortical actin." MOLECULAR BIOLOGY OF THE CELL 9 (November 1998): 35A-35A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
9
Publish Date
1998
Start Page
35A
End Page
35A

Potential role for alpha-betaG-spectrin in C-elegans neuron function

Authors
Moorthy, S; Bennett, V
MLA Citation
Moorthy, S, and Bennett, V. "Potential role for alpha-betaG-spectrin in C-elegans neuron function." MOLECULAR BIOLOGY OF THE CELL 9 (November 1998): 411A-411A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
9
Publish Date
1998
Start Page
411A
End Page
411A

Ankyring is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing

Authors
Zhou, DX; Lambert, S; Malen, PL; Carpenter, S; Boland, LM; Bennett, V
MLA Citation
Zhou, DX, Lambert, S, Malen, PL, Carpenter, S, Boland, LM, and Bennett, V. "Ankyring is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing." MOLECULAR BIOLOGY OF THE CELL 9 (November 1998): 37A-37A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
9
Publish Date
1998
Start Page
37A
End Page
37A

Properties of the sodium channel in ankyrin-B knockout murine neonatal cardiomyocytes

Authors
Chauhan, VS; Tuvia, S; Bihushi, M; Bennett, V; Grant, AO
MLA Citation
Chauhan, VS, Tuvia, S, Bihushi, M, Bennett, V, and Grant, AO. "Properties of the sodium channel in ankyrin-B knockout murine neonatal cardiomyocytes." CIRCULATION 98.17 (October 27, 1998): 11-11.
Source
wos-lite
Published In
Circulation
Volume
98
Issue
17
Publish Date
1998
Start Page
11
End Page
11

Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains.

AnkyrinG (-/-) neurons fail to concentrate voltage-sensitive sodium channels and neurofascin at their axon proximal segments, suggesting that ankyrinG is a key component of a structural pathway involved in assembly of specialized membrane domains at axon proximal segments and possibly nodes of Ranvier (Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments by evaluation of localization of GFP-tagged ankyrinG constructs transfected into cultured dorsal root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin- GFP-tagged ankyrinG complexes in nonneuronal cells. A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding domain, contribute to restriction of ankyrinG to the axonal plasma membrane in dorsal root ganglion neurons. The ankyrinG-specific spectrin-binding and tail domains are capable of binding directly to sites on the plasma membrane of neuronal cell bodies and axon proximal segments, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrinG polypeptides, contributes to restriction of ankyrinG to axon proximal segments as well as limiting lateral diffusion of ankyrinG-neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrinG also contribute to limiting the lateral mobility of ankyrinG-neurofascin complexes. AnkyrinG thus functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains ability of ankyrinB and ankyrinG to sort to distinct sites in neurons and the fact that these ankyrins do not compensate for each other in ankyrin gene knockouts in mice.

Authors
Zhang, X; Bennett, V
MLA Citation
Zhang, X, and Bennett, V. "Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains." J Cell Biol 142.6 (September 21, 1998): 1571-1581.
PMID
9744885
Source
pubmed
Published In
The Journal of Cell Biology
Volume
142
Issue
6
Publish Date
1998
Start Page
1571
End Page
1581

Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons.

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.

Authors
Matsuoka, Y; Li, X; Bennett, V
MLA Citation
Matsuoka, Y, Li, X, and Bennett, V. "Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons." J Cell Biol 142.2 (July 27, 1998): 485-497.
PMID
9679146
Source
pubmed
Published In
The Journal of Cell Biology
Volume
142
Issue
2
Publish Date
1998
Start Page
485
End Page
497

Adducin preferentially recruits spectrin to the fast growing ends of actin filaments in a complex requiring the MARCKS-related domain and a newly defined oligomerization domain.

Adducin is a protein associated with spectrin and actin in membrane skeletons of erythrocytes and possibly other cells. Adducin has activities in in vitro assays of association with the sides of actin filaments, capping the fast growing ends of actin filaments, and recruiting spectrin to actin filaments. This study presents evidence that adducin exhibits a preference for the fast growing ends of actin filaments for recruiting spectrin to actin and for direct association with actin. beta-Adducin-(335-726) promoted recruitment of spectrin to gelsolin-sensitive sites at fast growing ends of actin filaments with half-maximal activity at 15 nM and to gelsolin-insensitive sites with half-maximal activity at 75 nM. beta-Adducin-(335-726) also exhibited a preference for actin filament ends in direct binding assays; the half-maximal concentration for binding of adducin to gelsolin-sensitive sites at filament ends was 60 nM, and the Kd for binding to lateral sites was 1.5 microM. The concentration of beta-adducin-(335-726) of 60 nM required for half-maximal binding to filament ends is in the same range as the concentration of 150 nM required for half-maximal actin capping activity. All interactions of adducin with actin require the myristoylated alanine-rich protein kinase C substrate-related domain as well as a newly defined oligomerization site localized in the neck domain of adducin. Surprisingly, the head domain of adducin is not required for spectrin-actin interactions, although it could play a role in forming tetramers. The relative activities of adducin imply that an important role of adducin in cells is to form a complex with the fast growing ends of actin filaments that recruits spectrin and prevents addition or loss of actin subunits.

Authors
Li, X; Matsuoka, Y; Bennett, V
MLA Citation
Li, X, Matsuoka, Y, and Bennett, V. "Adducin preferentially recruits spectrin to the fast growing ends of actin filaments in a complex requiring the MARCKS-related domain and a newly defined oligomerization domain." J Biol Chem 273.30 (July 24, 1998): 19329-19338.
PMID
9668123
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
30
Publish Date
1998
Start Page
19329
End Page
19338

Palmitoylation of neurofascin at a site in the membrane-spanning domain highly conserved among the L1 family of cell adhesion molecules.

This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.

Authors
Ren, Q; Bennett, V
MLA Citation
Ren, Q, and Bennett, V. "Palmitoylation of neurofascin at a site in the membrane-spanning domain highly conserved among the L1 family of cell adhesion molecules." J Neurochem 70.5 (May 1998): 1839-1849.
PMID
9572267
Source
pubmed
Published In
Journal of Neurochemistry
Volume
70
Issue
5
Publish Date
1998
Start Page
1839
End Page
1849

Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase.

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhanced the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.

Authors
Kimura, K; Fukata, Y; Matsuoka, Y; Bennett, V; Matsuura, Y; Okawa, K; Iwamatsu, A; Kaibuchi, K
MLA Citation
Kimura, K, Fukata, Y, Matsuoka, Y, Bennett, V, Matsuura, Y, Okawa, K, Iwamatsu, A, and Kaibuchi, K. "Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase." J Biol Chem 273.10 (March 6, 1998): 5542-5548.
PMID
9488679
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
10
Publish Date
1998
Start Page
5542
End Page
5548

The phosphorylation state of the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cell segregation.

Cell-cell recognition and patterning of cell contacts have a critical role in mediating reversible assembly of a variety of transcellular complexes in the nervous system. This study provides evidence for regulation of cell interactions through modulation of ankyrin binding to neurofascin, a member of the L1CAM family of nervous system cell adhesion molecules. The phosphorylation state of the conserved FIGQY tyrosine in the cytoplasmic domain of neurofascin regulates ankyrin binding and governs neurofascin-dependent cell aggregation as well as cell sorting when neurofascin is expressed in neuroblastoma cells. These findings suggest a general mechanism for the patterning of cell contact based on external signals that regulate tyrosine phosphorylation of L1CAM members and modulate their binding to ankyrin.

Authors
Tuvia, S; Garver, TD; Bennett, V
MLA Citation
Tuvia, S, Garver, TD, and Bennett, V. "The phosphorylation state of the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cell segregation." Proc Natl Acad Sci U S A 94.24 (November 25, 1997): 12957-12962.
PMID
9371782
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
24
Publish Date
1997
Start Page
12957
End Page
12962

Adducin preferentially recruits spectrin to fast-growing ends of actin filaments in a complex requiring the MARCKS related domain and an oligomerization domain.

Authors
Li, X; Matsuoka, Y; Bennett, V
MLA Citation
Li, X, Matsuoka, Y, and Bennett, V. "Adducin preferentially recruits spectrin to fast-growing ends of actin filaments in a complex requiring the MARCKS related domain and an oligomerization domain." MOLECULAR BIOLOGY OF THE CELL 8 (November 1997): 1591-1591.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
8
Publish Date
1997
Start Page
1591
End Page
1591

Phosphorylation of the MARSKS-related domain of adducin occurs in dendritic spines of hippocampal neurons, and modulates its actin-capping and spectrin-recruiting activities.

Authors
Matsuoka, Y; Li, X; Bennett, V
MLA Citation
Matsuoka, Y, Li, X, and Bennett, V. "Phosphorylation of the MARSKS-related domain of adducin occurs in dendritic spines of hippocampal neurons, and modulates its actin-capping and spectrin-recruiting activities." MOLECULAR BIOLOGY OF THE CELL 8 (November 1997): 1592-1592.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
8
Publish Date
1997
Start Page
1592
End Page
1592

Ankyrin(B) gene disruption in mice is neonatal lethal and abolishes L1 expression in the optic nerve.

Authors
Scotland, PB; Zhou, D; Bennett, V
MLA Citation
Scotland, PB, Zhou, D, and Bennett, V. "Ankyrin(B) gene disruption in mice is neonatal lethal and abolishes L1 expression in the optic nerve." MOLECULAR BIOLOGY OF THE CELL 8 (November 1997): 1019-1019.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
8
Publish Date
1997
Start Page
1019
End Page
1019

The spectrin-based membrane cytoskeleton in C. elegans.

Authors
Moorthy, S; Chen, LS; Bennett, V
MLA Citation
Moorthy, S, Chen, LS, and Bennett, V. "The spectrin-based membrane cytoskeleton in C. elegans." MOLECULAR BIOLOGY OF THE CELL 8 (November 1997): 1590-1590.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
8
Publish Date
1997
Start Page
1590
End Page
1590

The tyrosine phosphorylation state of neurofascin determines patterns of ankyrin-binding.

Authors
Tuvia, S; Garver, TD; Bennett, V
MLA Citation
Tuvia, S, Garver, TD, and Bennett, V. "The tyrosine phosphorylation state of neurofascin determines patterns of ankyrin-binding." MOLECULAR BIOLOGY OF THE CELL 8 (November 1997): 717-717.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
8
Publish Date
1997
Start Page
717
End Page
717

Morphogenesis of the node of Ranvier: co-clusters of ankyrin and ankyrin-binding integral proteins define early developmental intermediates.

AnkyrinG 480/270 kDa and three ankyrin-binding integral membrane proteins (neurofascin, NrCAM, and the voltage-dependent sodium channel) colocalize within a specialized domain of the spectrin-actin network found at axonal segments of nodes of Ranvier in myelinated axons. Before myelination in embryonic nerves, ankyrinG 480/270 kDa and the related ankyrin isoform ankyrinB 440 kDa are co-expressed along with NrCAM in an abundant, continuous distribution along the length of axons. This study has resolved intermediate stages in the developmental transition from a continuous distribution of ankyrinG 480/270 kDa in all axons to a highly polarized localization at the node of Ranvier in the developing rat sciatic nerve. The first detected event is formation of clusters containing the cell adhesion molecules neurofascin and NrCAM at sites independent of myelin-associated glycoprotein (MAG)-staining Schwann cell processes. Subsequent steps involve recruitment of ankyrinG 480/270 kDa and the voltage-dependent sodium channel to cluster sites containing cell adhesion molecules, and elaboration of MAG-staining Schwann cell processes adjacent to these cluster sites. Formation of the mature node of Ranvier results from the fusion of asynchronously formed pairs of clusters associated with MAG-positive Schwann cells flanking the site of presumed node formation. Studies with the hypomyelinating mutant mouse trembler demonstrate that the elaboration of compact myelin is not required for the formation of these clustered nodal intermediates. Clustering of neurofascin and NrCAM precedes redistribution of ankyrinG 480/270 kDa and the voltage-dependent sodium channel, suggesting that the adhesion molecules define the initial site for subsequent assembly of ankyrin and the voltage-dependent sodium channel.

Authors
Lambert, S; Davis, JQ; Bennett, V
MLA Citation
Lambert, S, Davis, JQ, and Bennett, V. "Morphogenesis of the node of Ranvier: co-clusters of ankyrin and ankyrin-binding integral proteins define early developmental intermediates." J Neurosci 17.18 (September 15, 1997): 7025-7036.
PMID
9278538
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
17
Issue
18
Publish Date
1997
Start Page
7025
End Page
7036

Clustering of voltage-sensitive sodium channels on axons is independent of direct Schwann cell contact in the dystrophic mouse.

The distribution of voltage-sensitive sodium channels on axons in the dorsal and ventral spinal roots of the dystrophic mouse 129/ReJ-Lama2dy was determined via immunocytochemistry. In these nerves there are regions in which Schwann cells fail to proliferate and myelinate axons in a normal manner, leaving bundles of closely packed large-diameter amyelinated axons. We have identified discrete and focal concentrations of sodium channel immunoreactivity on these axons by both confocal immunofluorescence and immunoelectron microscopy, using a peptide-derived polyclonal antibody. In addition, simultaneous labeling with an antibody recognizing neuronal-specific ankyrinG revealed a distinct colocalization with the sodium channels on both normal and amyelinated axons. The presence of patches of sodium channels along with their anchoring protein on amyelinated axons in the absence of intervening Schwann cells demonstrates that axons can form and maintain independently these initial aggregations. This confirms that direct contact between Schwann cell and axon is not required for the formation of sodium channel patches of nodal dimensions and density. Furthermore, this strongly suggests that local transfer of sodium channels from Schwann cells to axons is not required for this process.

Authors
Deerinck, TJ; Levinson, SR; Bennett, GV; Ellisman, MH
MLA Citation
Deerinck, TJ, Levinson, SR, Bennett, GV, and Ellisman, MH. "Clustering of voltage-sensitive sodium channels on axons is independent of direct Schwann cell contact in the dystrophic mouse." J Neurosci 17.13 (July 1, 1997): 5080-5088.
PMID
9185545
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
17
Issue
13
Publish Date
1997
Start Page
5080
End Page
5088

Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

Authors
Garver, TD; Ren, Q; Tuvia, S; Bennett, V
MLA Citation
Garver, TD, Ren, Q, Tuvia, S, and Bennett, V. "Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin." J Cell Biol 137.3 (May 5, 1997): 703-714.
PMID
9151675
Source
pubmed
Published In
The Journal of Cell Biology
Volume
137
Issue
3
Publish Date
1997
Start Page
703
End Page
714

Induction of sodium channel clustering by oligodendrocytes.

As oligodendrocytes wrap axons of the central nervous system (CNS) with insulating myelin sheaths, sodium channels that are initially continuously distributed along axons become segregated into regularly spaced gaps in the myelin called nodes of Ranvier. It is not known whether the regular spacing of nodes results from regularly spaced glial contacts or is instead intrinsically specified by the axonal cytoskeleton. Contact with Schwann cells induces clustering of sodium channels along the axons of peripheral neurons in vitro and in vivo. Similarly, it has been suggested that astrocyte contact induces clustering of sodium channels along CNS axons. Here we show that oligodendrocytes are necessary for clustering of sodium channels in vitro and in vivo. The induction, but not the maintenance, of sodium-channel clustering along the axons of highly purified rat retinal ganglion cells in culture depends on a protein secreted by oligodendrocytes. Surprisingly, the oligodendrocyte-induced clusters are regularly spaced at the predicted interval in the absence of glial-axonal contact. Mutant rats that are deficient in oligodendrocytes develop few axonal sodium channel clusters in vivo. These results demonstrate a crucial role for oligodendrocytes in inducing clustering of sodium channels.

Authors
Kaplan, MR; Meyer-Franke, A; Lambert, S; Bennett, V; Duncan, ID; Levinson, SR; Barres, BA
MLA Citation
Kaplan, MR, Meyer-Franke, A, Lambert, S, Bennett, V, Duncan, ID, Levinson, SR, and Barres, BA. "Induction of sodium channel clustering by oligodendrocytes." Nature 386.6626 (April 17, 1997): 724-728.
PMID
9109490
Source
pubmed
Published In
Nature
Volume
386
Issue
6626
Publish Date
1997
Start Page
724
End Page
728
DOI
10.1038/386724a0

Synthesis and secretion of von Willebrand factor and fibronectin in megakaryocytes at different phases of maturation.

Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.

Authors
Schick, PK; Walker, J; Profeta, B; Denisova, L; Bennett, V
MLA Citation
Schick, PK, Walker, J, Profeta, B, Denisova, L, and Bennett, V. "Synthesis and secretion of von Willebrand factor and fibronectin in megakaryocytes at different phases of maturation." Arterioscler Thromb Vasc Biol 17.4 (April 1997): 797-801.
PMID
9108796
Source
pubmed
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
17
Issue
4
Publish Date
1997
Start Page
797
End Page
801

Molecular architecture of the specialized axonal membrane at the node of Ranvier.

Authors
Bennett, V; Lambert, S; Davis, JQ; Zhang, X
MLA Citation
Bennett, V, Lambert, S, Davis, JQ, and Zhang, X. "Molecular architecture of the specialized axonal membrane at the node of Ranvier." Soc Gen Physiol Ser 52 (1997): 107-120. (Review)
PMID
9210224
Source
pubmed
Published In
Society of General Physiologists Series
Volume
52
Publish Date
1997
Start Page
107
End Page
120

Tales of the tenements: midwifery training in Glasgow.

Authors
Bennett, V
MLA Citation
Bennett, V. "Tales of the tenements: midwifery training in Glasgow." Int Hist Nurs J 3.2 (1997): 69-72.
PMID
11619813
Source
pubmed
Published In
International history of nursing journal : IHNJ
Volume
3
Issue
2
Publish Date
1997
Start Page
69
End Page
72

Modeling seasonal changes in intracellular freeze-tolerance of fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae)

Although seasonal changes in the freeze-tolerance of third-instar larvae of Eurosta solidaginis have been well documented for the whole organism, the nature of this cold-hardiness at the cellular level has not been examined. Seasonal changes in the survival of fat body cells from E. solidaginis larvae were assessed using fluorescent vital dyes after freezing at -10, -25 or -80 °C for 24 h both in vivo and in vitro. Cells frozen in vitro were frozen with glycerol, with sorbitol (both of which enhanced cell survival) or without cryoprotectants. Both cellular and organismal survival were low in August when larvae were not freeze-tolerant, then increased dramatically during September and October before leveling off from November to January. This observation for cells frozen without cryoprotectants indicates that the cells themselves have adapted. The single most important factor influencing cell survival, as determined by logistic regression modeling, was the time of larval collection, which reflects the level of cold-hardiness achieved by field acclimation. Cells frozen in vivo exhibited greater survival than did those frozen in vitro, even with the addition of cryoprotectants. Since no differences were observed between cells frozen with glycerol or sorbitol, the role of the multi-component cryoprotectant system present in E. solidaginis should be investigated.

Authors
Bennett, V; Lee, R
MLA Citation
Bennett, V, and Lee, R. "Modeling seasonal changes in intracellular freeze-tolerance of fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae)." J Exp Biol 200.Pt 1 (1997): 185-192.
PMID
9317602
Source
pubmed
Published In
The Journal of experimental biology
Volume
200
Issue
Pt 1
Publish Date
1997
Start Page
185
End Page
192

Molecular architecture of the specialized axonal membrane at the node of Ranvier.

Authors
Bennett, V; Lambert, S; Davis, JQ; Zhang, X
MLA Citation
Bennett, V, Lambert, S, Davis, JQ, and Zhang, X. "Molecular architecture of the specialized axonal membrane at the node of Ranvier." Society of General Physiologists series 52 (1997): 107-120.
Source
scival
Published In
Society of General Physiologists Series
Volume
52
Publish Date
1997
Start Page
107
End Page
120

Identification of O-linked N-acetylglucosamine modification of ankyrinG isoforms targeted to nodes of Ranvier.

AnkyrinGs of 270 and 480 kDa are localized at nodes of Ranvier and are candidates to couple the voltage-dependent sodium channel and neurofascin to the spectrin/actin network. This study presents evidence that these ankyrins contain O-linked GlcNAc residues and identifies as the site of glycosylation a serine-rich domain that distinguishes them from other ankyrin isoforms. The 480-kDa ankyrinG, extracted from brain membranes associated with wheat germ agglutinin-affinity columns, was [3H]galactose-labeled with UDP-[3H] galactose and galactosyltransferase, and cross-reacted with an antibody against O-GlcNAc monosaccharides. AnkyrinG-associated sugars are O-linked monosaccharides based on resistance to peptide-N-glycosidase F and analysis of saccharides released by beta-elimination. The serine-rich domain is the site of glycosylation based on wheat germ agglutinin binding activity of polypeptides produced by in vitro translation in reticulocyte lysates. Immunofluorescence revealed co-localization of ankyrinG and O-GlcNAc immunoreactivity at nodes of Ranvier. These observations suggest that ankyrin at the node of Ranvier is O-GlcNAc-glycosylated and are the first demonstration of a post-translational modification that is concentrated at the node of Ranvier and not in adjacent areas of myelinated axons.

Authors
Zhang, X; Bennett, V
MLA Citation
Zhang, X, and Bennett, V. "Identification of O-linked N-acetylglucosamine modification of ankyrinG isoforms targeted to nodes of Ranvier." J Biol Chem 271.49 (December 6, 1996): 31391-31398.
PMID
8940148
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
49
Publish Date
1996
Start Page
31391
End Page
31398

Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+/third FNIII domain-) and NrCAM at nodal axon segments.

Neurofascin, NrCAM, L1, and NgCAM are a family of Ig/FNIII cell adhesion molecules that share ankyrin-binding activity in their cytoplasmic domains, and are candidates to form membrane-spanning complexes with members of the ankyrin family of spectrin-binding proteins in a variety of cellular contexts in the nervous system. Specialized forms of ankyrin, 270 kD and/or 480 kD ankyrinG are components of the membrane undercoat of axons at the node of Ranvier. This paper focuses on definition of the isoforms of ankyrin-binding cell adhesion molecules localized with ankyrinG at the nodal axon segment. The exon usage of two major forms of neurofascin was determined by isolation of full-length cDNAs and used to prepare isoform-specific antibodies. An isoform of neurofascin containing a mucin-like domain and lacking the third FNIII domain was concentrated at axon initial segments and colocalized at nodes of Ranvier with ankyrinG and the voltage-dependent sodium channel. An alternative form of neurofascin lacking the mucin-like domain and containing the third FNIII domain was present in unmyelinated axons. The antibody initially raised against neurofascin was used to screen a rat brain cDNA expression library. In addition to neurofascin, this screen yielded a clone with 80% sequence identity to NrCAM from chicken. The sequences of two full-length cDNAs are presented. NrCAM is most closely related to neurofascin among the other members of the L1/neurofascin/NgCAM family, with over 70% identity between cytoplasmic domains. NrCAM, visualized with antibodies specific for the ecto-domain, also was found to be coexpressed with neurofascin at nodes of Ranvier and at axon initial segments. This is the first characterization of defined neuronal cell adhesion molecules localized to axonal membranes at the node of Ranvier of myelinated axons.

Authors
Davis, JQ; Lambert, S; Bennett, V
MLA Citation
Davis, JQ, Lambert, S, and Bennett, V. "Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+/third FNIII domain-) and NrCAM at nodal axon segments." J Cell Biol 135.5 (December 1996): 1355-1367.
PMID
8947556
Source
pubmed
Published In
The Journal of Cell Biology
Volume
135
Issue
5
Publish Date
1996
Start Page
1355
End Page
1367

Identification of O-GlcNAc glycosylation of isoforms of ankyrin targeted to nodes of Ranvier.

Authors
Zhang, X; Bennett, V
MLA Citation
Zhang, X, and Bennett, V. "Identification of O-GlcNAc glycosylation of isoforms of ankyrin targeted to nodes of Ranvier." MOLECULAR BIOLOGY OF THE CELL 7 (December 1996): 3208-3208.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
7
Publish Date
1996
Start Page
3208
End Page
3208

Association of ankyrin with the neuronal cell adhesion molecule neurofascin: Regulation by both tyrosine phosphorylation and alternative exon usage.

Authors
Garver, TD; Davis, JQ; Bennett, V
MLA Citation
Garver, TD, Davis, JQ, and Bennett, V. "Association of ankyrin with the neuronal cell adhesion molecule neurofascin: Regulation by both tyrosine phosphorylation and alternative exon usage." MOLECULAR BIOLOGY OF THE CELL 7 (December 1996): 2233-2233.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
7
Publish Date
1996
Start Page
2233
End Page
2233

Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C.

Adducin promotes association of spectrin with actin and caps the fast growing end of actin filaments. Adducin contains N-terminal core, neck, and C-terminal tail domains, is a substrate for protein kinases A (PKA) and C (PKC), and binds to Ca2+/calmodulin. Ser-726 and Ser-713 in the C-terminal MARCKS-related domains of alpha- and beta-adducin, respectively, were identified as the major phosphorylation sites common for PKA and PKC. PKA, in addition, phosphorylated alpha-adducin at Ser-408, -436, and -481 in the neck domain. Phosphorylation by PKA, but not PKC, reduced the affinity of adducin for spectrin-F-actin complexes as well as the activity of adducin in promoting binding of spectrin to F-actin. The myristoylated alanine-rich protein kinase C substrate-related domain of beta-adducin was identified as the dominant Ca2+-dependent calmodulin-binding site. Calmodulin-binding was inhibited by phosphorylation of beta-adducin and of a MARCKS-related domain peptide by PKA and PKC. Calmodulin in turn inhibited the rate, but not the extent, of phosphorylation of beta-adducin, but not alpha-adducin, by PKA and that of each subunit by PKC. These findings suggest a complex reciprocal relationship between regulation of adducin function by calmodulin binding and phosphorylation by PKA and PKC.

Authors
Matsuoka, Y; Hughes, CA; Bennett, V
MLA Citation
Matsuoka, Y, Hughes, CA, and Bennett, V. "Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C." J Biol Chem 271.41 (October 11, 1996): 25157-25166.
PMID
8810272
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
41
Publish Date
1996
Start Page
25157
End Page
25166

Ankyrin clustering in the coordinate recruitment of voltage-dependent Na channels and adhesion molecules during morphogenesis of the node of Ranvier

Authors
Lambert, RS; Davis, JQ; Bennett, V
MLA Citation
Lambert, RS, Davis, JQ, and Bennett, V. "Ankyrin clustering in the coordinate recruitment of voltage-dependent Na channels and adhesion molecules during morphogenesis of the node of Ranvier." JOURNAL OF GENERAL PHYSIOLOGY 108.2 (August 1996): 10-10.
Source
wos-lite
Published In
The Journal of General Physiology
Volume
108
Issue
2
Publish Date
1996
Start Page
10
End Page
10

Genes for the neuronal immunoglobulin domain cell adhesion molecules neurofascin and Nr-CAM map to mouse chromosomes 1 and 12 and homologous human chromosomes.

Authors
Burmeister, M; Ren, Q; Makris, GJ; Samson, D; Bennett, V
MLA Citation
Burmeister, M, Ren, Q, Makris, GJ, Samson, D, and Bennett, V. "Genes for the neuronal immunoglobulin domain cell adhesion molecules neurofascin and Nr-CAM map to mouse chromosomes 1 and 12 and homologous human chromosomes." Mamm Genome 7.7 (July 1996): 558-559.
PMID
8672144
Source
pubmed
Published In
Mammalian Genome
Volume
7
Issue
7
Publish Date
1996
Start Page
558
End Page
559

Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes.

Adducin is an actin-binding protein that has been proposed to function as a regulated assembly factor for the spectrin/actin network. This study has addressed the question of the subunit and domains of spectrin required for formation of spectrin/adducin/actin complexes in in vitro assays. Quantitative evidence is presented that the beta-spectrin N-terminal domain plus the first two alpha-helical domains are required for optimal participation of spectrin in spectrin/adducin/actin complexes. The alpha subunit exhibited no detectable activity either alone or following association with beta-spectrin. The critical domains of beta-spectrin involved in complex formation were determined using recombinant proteins expressed in bacteria. The N-terminal domain (residues 1-313) of beta-spectrin associated with F-actin with a Kd of 26 microM, and promoted adducin binding to F-actin with half-maximal activation at 110 nM. Addition of the first alpha-helical domain (residues 1-422) lowered the Kdfor F-actin by 4-fold to 6 microM, but also reduced the capacity by 3-fold and had no effect on interaction with adducin. Further addition of the second alpha-helical domain (residues 1-528) did not alter binding to F-actin but resulted in a 2-fold increased activity in promoting adducin binding with half-maximal activation at 50 nM. Addition of up to eight additional alpha-helical domains (residues 1-1388) resulted in no further change in F-actin binding or association with adducin. These results demonstrate an unanticipated role of the first repeat of beta-spectrin in actin binding activity and of the second repeat in association with adducin/actin, and imply the possibility of an extended contact between adducin, spectrin, and actin involving several actin subunits.

Authors
Li, X; Bennett, V
MLA Citation
Li, X, and Bennett, V. "Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes." J Biol Chem 271.26 (June 28, 1996): 15695-15702.
PMID
8663089
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
26
Publish Date
1996
Start Page
15695
End Page
15702

On duty. Interview by Antonia Owen.

Authors
Bennett, V
MLA Citation
Bennett, V. "On duty. Interview by Antonia Owen." Nurs Times 92.17 (April 24, 1996): 189-. (Interview)
PMID
8710600
Source
pubmed
Published In
Nursing times
Volume
92
Issue
17
Publish Date
1996
Start Page
189

A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments.

Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.

Authors
Kuhlman, PA; Hughes, CA; Bennett, V; Fowler, VM
MLA Citation
Kuhlman, PA, Hughes, CA, Bennett, V, and Fowler, VM. "A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments." J Biol Chem 271.14 (April 5, 1996): 7986-7991.
PMID
8626479
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
14
Publish Date
1996
Start Page
7986
End Page
7991

Fibronectin isoforms in megakaryocytes.

Our studies have shown that megakaryocytes (MK) can synthesize fibronectin (FN) and alternatively spliced fibronectin, FN EIIIB. FN EIIIB is primarily present in embryonic, proliferating and migrating cells, and thought to be important for cell maturation. MK, but not nonmegakaryocytic bone marrow cells, contain FN EIIIB and thus, MK and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. Thrombin can induce the secretion of general FN, but does not cause the secretion of FN EIIIB into the medium. Analysis of immunostained cells by confocal microscopy revealed that both general FN and FN EIIIB accumulated on the MK surface following thrombin treatment. Thus, FN EIIIB can be released only to be bound to the MK surface. The expression of FN EIIIB on the MK surface may have a unique role in MK migration and maturation.

Authors
Schick, PK; Wojensk, CM; Bennett, V; Denisova, L
MLA Citation
Schick, PK, Wojensk, CM, Bennett, V, and Denisova, L. "Fibronectin isoforms in megakaryocytes." Stem Cells 14 Suppl 1 (1996): 212-219.
PMID
11012224
Source
pubmed
Published In
Stem Cells
Volume
14 Suppl 1
Publish Date
1996
Start Page
212
End Page
219
DOI
10.1002/stem.5530140728

Chapter 7 Axonal Ankyrins and Ankyrin-Binding Proteins: Potential Participants in Lateral Membrane Domains and Transcellular Connections at the Node of Ranvier

Authors
Lambert, S; Bennett, V
MLA Citation
Lambert, S, and Bennett, V. "Chapter 7 Axonal Ankyrins and Ankyrin-Binding Proteins: Potential Participants in Lateral Membrane Domains and Transcellular Connections at the Node of Ranvier." Current Topics in Membranes and Transport 43.C (1996): 129-145.
Source
scival
Published In
Current Topics in Membranes and Transport
Volume
43
Issue
C
Publish Date
1996
Start Page
129
End Page
145
DOI
10.1016/S0070-2161(08)60387-1

Axonal ankyrins and ankyrin-binding proteins: Potential participants in lateral membrane domains and transcellular connections at the node of Ranvier

Authors
Lambert, S; Bennett, V
MLA Citation
Lambert, S, and Bennett, V. "Axonal ankyrins and ankyrin-binding proteins: Potential participants in lateral membrane domains and transcellular connections at the node of Ranvier." MEMBRANE PROTEIN-CYTOSKELETON INTERACTIONS 43 (1996): 129-145.
Source
wos-lite
Published In
MEMBRANE PROTEIN-CYTOSKELETON INTERACTIONS
Volume
43
Publish Date
1996
Start Page
129
End Page
145

Mechanism for binding site diversity on ankyrin. Comparison of binding sites on ankyrin for neurofascin and the Cl-/HCO3- anion exchanger.

Ankyrins are a family of spectrin-binding proteins that associate with at least seven distinct membrane proteins, including ion transporters and cell adhesion molecules. The membrane-binding domain of ankyrin is comprised of a tandem array of 24 ANK repeats organized into four 6-repeat folding domains. Tandem arrays of ANK repeats have been proposed to mediate protein interactions in a variety of proteins including factors involved in the regulation of transcription and the cell cycle. This report provides several new insights into the versatility of ANK repeats of ankyrin in protein recognition, using neurofascin and the Cl-/HCO3- anion exchanger as model ligands and ankyrinR as the prototypic ankyrin. Different combinations of ANK repeat domains from this ankyrin form two distinct, high affinity binding sites for neurofascin. One site requires both repeat domains 3 and 4. The other site involves both repeat domains 2 and 3, although domain 2 has significant activity alone. The sites appear to be independent with Kd values of 3 and 14 nM, respectively. Both the Cl-/HCO3- anion exchanger and neurofascin can interact simultaneously with repeat domains 3 and 4, because neurofascin is unable to displace binding of the anion exchanger cytoplasmic domain to domains 3 and 4, despite having a 3-5-fold higher affinity. These results demonstrate two levels of diversity in the binding sites on ankyrin: one resulting from different combinations of ANK repeat domains and another from different determinants within the same combination of repeat domains. One consequence of this diversity is that ankyrin can accommodate two neurofascin molecules as well as the anion exchanger through interactions mediated by ANK repeats. The ability of ankyrin to simultaneously associate with multiple types of membrane proteins is an unanticipated finding with implications for the assembly of integral membrane proteins into specialized regions of the plasma membrane.

Authors
Michaely, P; Bennett, V
MLA Citation
Michaely, P, and Bennett, V. "Mechanism for binding site diversity on ankyrin. Comparison of binding sites on ankyrin for neurofascin and the Cl-/HCO3- anion exchanger." J Biol Chem 270.52 (December 29, 1995): 31298-31302.
PMID
8537399
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
52
Publish Date
1995
Start Page
31298
End Page
31302

THE 4TH REPEAT DOMAIN OF ANKYRIN ASSOCIATES WITH THE N-TERMINAL DOMAIN OF CLATHRIN WITH HIGH-AFFINITY

Authors
MICHAELY, P; BENNETT, V
MLA Citation
MICHAELY, P, and BENNETT, V. "THE 4TH REPEAT DOMAIN OF ANKYRIN ASSOCIATES WITH THE N-TERMINAL DOMAIN OF CLATHRIN WITH HIGH-AFFINITY." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 2396-2396.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
2396
End Page
2396

IDENTIFICATION OF THE SPECTRIN SUBUNIT AND DOMAINS REQUIRED FOR FORMATION OF ADDUCIN/SPECTRIN/ACTIN COMPLEXES

Authors
LI, XL; BENNETT, V
MLA Citation
LI, XL, and BENNETT, V. "IDENTIFICATION OF THE SPECTRIN SUBUNIT AND DOMAINS REQUIRED FOR FORMATION OF ADDUCIN/SPECTRIN/ACTIN COMPLEXES." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 1564-1564.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
1564
End Page
1564

SPECTRIN/ACTIN ASSEMBLY ACTIVIN OF ADDUCIN IS REGULATED BY PHOSPHORYLATION OF THE MARCKS-RELATED DOMAIN BY PROTEIN-KINASE-C

Authors
MATSUOKA, Y; HUGHES, CA; BENNETT, V
MLA Citation
MATSUOKA, Y, HUGHES, CA, and BENNETT, V. "SPECTRIN/ACTIN ASSEMBLY ACTIVIN OF ADDUCIN IS REGULATED BY PHOSPHORYLATION OF THE MARCKS-RELATED DOMAIN BY PROTEIN-KINASE-C." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 1563-1563.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
1563
End Page
1563

NEUROFASCIN - CORRELATION OF MOLECULAR SPLICING WITH TARGETING TO NODES OF RANVIER AND AXON INITIAL SEGMENTS

Authors
DAVIS, J; ZHANG, F; REN, Q; BENNETT, V
MLA Citation
DAVIS, J, ZHANG, F, REN, Q, and BENNETT, V. "NEUROFASCIN - CORRELATION OF MOLECULAR SPLICING WITH TARGETING TO NODES OF RANVIER AND AXON INITIAL SEGMENTS." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 1027-1027.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
1027
End Page
1027

ANKYRIN CLUSTERING IN THE COORDINATE RECRUITMENT OF ION CHANNELS AND ADHESION MOLECULES DURING MORPHOGENESIS OF THE NODE OF RANVIER

Authors
LAMBERT, S; MICHAELY, P; DAVIS, JQ; BENNETT, V
MLA Citation
LAMBERT, S, MICHAELY, P, DAVIS, JQ, and BENNETT, V. "ANKYRIN CLUSTERING IN THE COORDINATE RECRUITMENT OF ION CHANNELS AND ADHESION MOLECULES DURING MORPHOGENESIS OF THE NODE OF RANVIER." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 568-568.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
568
End Page
568

PURIFICATION OF A CALMODULIN-SENSITIVE SPECTRIN-BINDING MEMBRANE GLYCOPROTEIN FROM BRAIN

Authors
DAVIS, L; BENNETT, V
MLA Citation
DAVIS, L, and BENNETT, V. "PURIFICATION OF A CALMODULIN-SENSITIVE SPECTRIN-BINDING MEMBRANE GLYCOPROTEIN FROM BRAIN." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 1566-1566.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
1566
End Page
1566

The ANK repeats of erythrocyte ankyrin form two distinct but cooperative binding sites for the erythrocyte anion exchanger.

The 24 ANK repeats of the membrane-binding domain of ankyrin form four folded subdomains of six ANK repeats each. These four repeat subdomains mediate interactions with at least seven different families of membrane proteins. In the erythrocyte, the main membrane target of ankyrin is the Cl-/HCO3- anion exchanger. This report presents the first evidence that ankyrin contains two separate binding sites for anion exchanger dimers. One site utilizes repeat subdomain two (repeats 7-12) while the other requires both repeat subdomains three and four (repeats 13-24). The two sites are positively coupled with a Hill coefficient of 1.4. Since the anion exchanger exists as a dimer in the membrane, the presence of two binding sites on ankyrin allows ankyrin to interact with four anion exchangers simultaneously. These findings provide a direct demonstration of the versatility of ANK repeats in protein recognition, and have important implications for the organization of ankyrin-linked integral membrane proteins in erythrocytes as well as other cells.

Authors
Michaely, P; Bennett, V
MLA Citation
Michaely, P, and Bennett, V. "The ANK repeats of erythrocyte ankyrin form two distinct but cooperative binding sites for the erythrocyte anion exchanger." J Biol Chem 270.37 (September 15, 1995): 22050-22057.
PMID
7665627
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
37
Publish Date
1995
Start Page
22050
End Page
22057

Adducin: a physical model with implications for function in assembly of spectrin-actin complexes.

Adducin binds to spectrin-actin complexes, promotes association of spectrin with actin, and is subject to regulation by calmodulin as well as protein kinases A and C. Adducin is a heteromer comprised of homologous alpha and beta-subunits with an NH2-terminal protease-resistant head domain, connected by a neck region to a COOH-terminal hydrophilic, protease-sensitive region. This study provides evidence that adducin in solution is a mixture of heterodimers and tetramers. CD spectroscopy of COOH-terminal domains of alpha- and beta-adducin bacterial recombinants provides direct evidence for an unstructured random coil configuration. Cross-linking, proteolysis, and blot-binding experiments suggest a model for the adducin tetramer in which four head domains contact one another to form a globular core with extended interacting alpha- and beta-adducin tails. The site for binding to spectrin-actin complexes on adducin was identified as the COOH-terminal tail of both the alpha- and beta-adducin subunits. The capacity of native adducin to recruit spectrin to actin filaments is similar to that of adducin tail domains. Thus, adducin tail domains alone are sufficient to interact with F-actin and a single spectrin and to recruit additional spectrin molecules to the ternary complex.

Authors
Hughes, CA; Bennett, V
MLA Citation
Hughes, CA, and Bennett, V. "Adducin: a physical model with implications for function in assembly of spectrin-actin complexes." J Biol Chem 270.32 (August 11, 1995): 18990-18996.
PMID
7642559
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
32
Publish Date
1995
Start Page
18990
End Page
18996

Assignment of the human beta-adducin gene (ADD2) to 2p13-p14 by in situ hybridization.

Authors
Gilligan, DM; Lieman, J; Bennett, V
MLA Citation
Gilligan, DM, Lieman, J, and Bennett, V. "Assignment of the human beta-adducin gene (ADD2) to 2p13-p14 by in situ hybridization." Genomics 28.3 (August 10, 1995): 610-612.
PMID
7490111
Source
pubmed
Published In
Genomics
Volume
28
Issue
3
Publish Date
1995
Start Page
610
End Page
612
DOI
10.1006/geno.1995.1205

Chromosomal localization of the ankyrinG gene (ANK3/Ank3) to human 10q21 and mouse 10.

The ankyrin3 gene encodes a novel form of ankyrin, AnkyrinG, expressed in multiple tissues but characteristically present at the axonal initial segment and nodes of Ranvier of neurons in the central and peripheral nervous systems. We have localized ANK3 to human Chromosome 10q21 by fluorescence in situ hybridization. The position of the murine homologue was determined by intersubspecific backcross analysis, mapping Ank3 to mouse Chromosome 10, between microsatellite marker D10Mit31 and the Bcr gene. This interval is known to comprise a region syntenic to human 10q. The localization of ANK3 is a preliminary step in identifying neurological disorders potentially associated with the gene.

Authors
Kapfhamer, D; Miller, DE; Lambert, S; Bennett, V; Glover, TW; Burmeister, M
MLA Citation
Kapfhamer, D, Miller, DE, Lambert, S, Bennett, V, Glover, TW, and Burmeister, M. "Chromosomal localization of the ankyrinG gene (ANK3/Ank3) to human 10q21 and mouse 10." Genomics 27.1 (May 1, 1995): 189-191.
PMID
7665168
Source
pubmed
Published In
Genomics
Volume
27
Issue
1
Publish Date
1995
Start Page
189
End Page
191
DOI
10.1006/geno.1995.1023

Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia.

OBJECTIVE: To evaluate the recovery rate of spermatozoa from the epididymis using a percutaneous aspiration technique and to examine the fertilization rate after intracytoplasmic sperm injection. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London. SUBJECTS: Twenty patients with obstructive azoospermia who each had an attempt at IVF. The sperm used for intracytoplasmic sperm injection was retrieved by percutaneous epididymal sperm aspiration in 16 patients. In one patient, microepididymal sperm aspiration was performed in addition because the quality of the sperm obtained by percutaneous epididymal sperm aspiration was not considered suitable for microinjection. In the remaining three patients, neither percutaneous epididymal sperm aspiration nor microepididymal sperm aspiration resulted in the recovery of sperm, which was obtained by testicular biopsy in one of them. INTERVENTION: Assisted fertilization with intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Normal fertilization and pregnancy rates. RESULTS: A total of 179 eggs were collected and 157 subsequently were microinjected. Normal fertilization occurred in 22 oocytes (14%) and the total number of embryos cleaved was 30. Twelve patients underwent ET in which three conceived (pregnancy rate 25% per transfer). The implantation rate was 10% and failed fertilization occurred in four cycles. CONCLUSION: Percutaneous epididymal sperm aspiration can be used successfully to recover sperm in men with obstructive azoospermia for use in assisted fertilization IVF cycles. The technique is simple, effective, and less traumatic compared with an open microsurgical operation.

Authors
Craft, I; Tsirigotis, M; Bennett, V; Taranissi, M; Khalifa, Y; Hogewind, G; Nicholson, N
MLA Citation
Craft, I, Tsirigotis, M, Bennett, V, Taranissi, M, Khalifa, Y, Hogewind, G, and Nicholson, N. "Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia." Fertil Steril 63.5 (May 1995): 1038-1042.
PMID
7720914
Source
pubmed
Published In
Fertility and Sterility
Volume
63
Issue
5
Publish Date
1995
Start Page
1038
End Page
1042

Late intracytoplasmic sperm injection in unexpected failed fertilization in vitro: diagnostic or therapeutic?

OBJECTIVE: To evaluate fertilization potential of 24-hour-old unfertilized oocytes using intracytoplasmic sperm injection and the pregnancy potential of resultant embryos. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London, United Kingdom. PATIENTS: Fifteen patients with a history of infertility who underwent treatment with IVF and showed failure of fertilization on the day after oocyte retrieval. INTERVENTION: Assisted fertilization with intracytoplasmic sperm injection was carried out at 24 hours after oocyte retrieval. RESULTS: A total of 121 metaphase II oocytes were subjected to intracytoplasmic sperm injection. Of these, 9 were damaged (7%), 2 were polyploidic (2%), and 58 showed normal fertilization (48%). Of the latter, 47 cleaved normally (81%). Forty embryos were transferred and three were cryopreserved. One patient conceived (7%) but in this case only one of three embryos transferred was from intracytoplasmic sperm injection. CONCLUSION: Late (24 hours) intracytoplasmic sperm injection can give good fertilization and cleavage rates but the potential of the generated embryos to achieve pregnancy seems to be low.

Authors
Tsirigotis, M; Nicholson, N; Taranissi, M; Bennett, V; Pelekanos, M; Craft, I
MLA Citation
Tsirigotis, M, Nicholson, N, Taranissi, M, Bennett, V, Pelekanos, M, and Craft, I. "Late intracytoplasmic sperm injection in unexpected failed fertilization in vitro: diagnostic or therapeutic?." Fertil Steril 63.4 (April 1995): 816-819.
PMID
7890068
Source
pubmed
Published In
Fertility and Sterility
Volume
63
Issue
4
Publish Date
1995
Start Page
816
End Page
819

Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells.

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G-spectrin, although alpha-spectrin was still present. Beta G-spectrin-deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin-associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)-ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.

Authors
Hu, RJ; Moorthy, S; Bennett, V
MLA Citation
Hu, RJ, Moorthy, S, and Bennett, V. "Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells." J Cell Biol 128.6 (March 1995): 1069-1080.
PMID
7896872
Source
pubmed
Published In
The Journal of Cell Biology
Volume
128
Issue
6
Publish Date
1995
Start Page
1069
End Page
1080

Of mice and men: the mice were right.

Authors
Bennett, V
MLA Citation
Bennett, V. "Of mice and men: the mice were right." J Clin Invest 95.3 (March 1995): 921-922.
PMID
7883989
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
95
Issue
3
Publish Date
1995
Start Page
921
End Page
922
DOI
10.1172/JCI117797

AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier.

We have characterized a new ankyrin gene, expressed in brain and other tissues, that is subject to extensive tissue-specific alternative mRNA processing. The full-length polypeptide has a molecular mass of 480 kDa and includes a predicted globular head domain, with membrane- and spectrin-binding activities, as well as an extended "tail" domain. We term this gene ankyrinG based on its giant size and general expression. Two brain-specific isoforms of 480 kDa and 270 kDa were identified that contain a unique stretch of sequence highly enriched in serine and threonine residues immediately following the globular head domain. Antibodies against the serine-rich domain and spectrin-binding domain revealed labeling of nodes of Ranvier and axonal initial segments. Ankyrin-binding proteins also known to be localized in these specialized membrane domains include the voltage-dependent sodium channel, the sodium/potassium ATPase, sodium/calcium exchanger, and members of the neurofascin/L1 family of cell adhesion molecules. The neural-specific ankyrinG polypeptides are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to nodes of Ranvier and axonal initial segments.

Authors
Kordeli, E; Lambert, S; Bennett, V
MLA Citation
Kordeli, E, Lambert, S, and Bennett, V. "AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier." J Biol Chem 270.5 (February 3, 1995): 2352-2359.
PMID
7836469
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
5
Publish Date
1995
Start Page
2352
End Page
2359

Ankyrin binding activity shared by the neurofascin/L1/NrCAM family of nervous system cell adhesion molecules.

Neurofascin, L1, NrCAM, NgCAM, and neuroglian are membrane-spanning cell adhesion molecules with conserved cytoplasmic domains that are believed to play important roles in development of the nervous system. This report presents biochemical evidence that the cytoplasmic domains of these molecules associate directly with ankyrins, a family of spectrin-binding proteins located on the cytoplasmic surface of specialized plasma membrane domains. Rat neurofascin and NrCAM together comprise over 0.5% of the membrane protein in adult brain tissue. Linkage of these ankyrin-binding cell adhesion molecules to spectrin-based structures may provide a major class of membrane-cytoskeletal connections in adult brain as well as earlier stages of development.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "Ankyrin binding activity shared by the neurofascin/L1/NrCAM family of nervous system cell adhesion molecules." J Biol Chem 269.44 (November 4, 1994): 27163-27166.
PMID
7961622
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
44
Publish Date
1994
Start Page
27163
End Page
27166

Experience with subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) on unfertilized aged human oocytes.

OBJECTIVE: The aim of this study was to assess the fertilizability of unfertilized aged human oocytes from failed in vitro fertilization (IVF) cycles using SUZI and ICSI. METHODS: A total of 363 oocytes which showed no fertilization after conventional IVF was subjected to assisted fertilization using SUZI or ICSI. The microinjected oocytes which were derived from 72 patients undergoing their first IVF treatment had an intact polar body and no signs of degeneration. SUZI was carried out in 265 oocytes and ICSI in the remaining 98. RESULTS: Significantly more oocytes were damaged after ICSI (9 vs 0.3%, P < 0.01). Normal fertilization rates were higher at 24 hr in both groups and occurred more frequently after ICSI, although the difference did not reach statistical significance. Abnormal fertilization occurred significantly more often after SUZI at 48 hr (P < 0.005), but not at 24 hr. Cleavage rates were significantly higher after ICSI (94.4 vs 57.1%, P < 0.025) at 24 hr, but this was not observed at 48 hr, although the ICSI group still showed better cleavage rates (33.3 vs 19.1%). There was no difference in embryo quality in either group. CONCLUSIONS: Our results indicate that micromanipulation rather than reinsemination should be carried out on unfertilized human oocytes from failed IVF attempts. Both techniques can be used to achieve fertilization which occurs more often after ICSI. However, the trauma from the former technique on the microinjected oocytes may impair the potential of the generated embryos to achieve pregnancy compared to SUZI. Prospective randomized trials are necessary to address the problem.

Authors
Tsirigotis, M; Bennett, V; Nicholson, N; Khalifa, Y; Hogewind, G; Yazdani, N; Craft, I
MLA Citation
Tsirigotis, M, Bennett, V, Nicholson, N, Khalifa, Y, Hogewind, G, Yazdani, N, and Craft, I. "Experience with subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) on unfertilized aged human oocytes." J Assist Reprod Genet 11.8 (September 1994): 389-394.
PMID
7606150
Source
pubmed
Published In
Journal of Assisted Reproduction and Genetics
Volume
11
Issue
8
Publish Date
1994
Start Page
389
End Page
394

Identification of two regions of beta G spectrin that bind to distinct sites in brain membranes.

This study analyzed the complex interactions of intact spectrin with bovine brain membranes by evaluating membrane associations of defined regions of beta G spectrin, the subunit responsible for high affinity membrane binding. Two regions of beta G spectrin were expressed in bacteria and demonstrated to contain fully functional binding site(s) for a subset of spectrin-binding sites in brain membranes depleted of peripheral proteins. One region, located near the NH2 terminus, was comprised of 106-residue repeats and required repeats 2-7 for full activity. The other binding domain was located at the COOH terminus, which is the most variable between beta G and beta R spectrins, is distinct from the 106-residue repeats, and contains a pleckstrin homology domain. NH2-terminal beta spectrin polypeptides interacted with a membrane site(s) that recognized both brain and erythrocyte isoforms of spectrin, was inhibited by calcium/calmodulin, and was not blocked by the COOH-terminal polypeptide. The COOH-terminal region associated with a membrane site(s) that was specific for brain spectrin, was not inhibited by calcium/calmodulin, and was not blocked by the NH2-terminal polypeptide. These observations demonstrate membrane association of spectrin with at least two independent sites, which differ with regard to regulation by calcium/calmodulin and in selectivity for spectrin isoforms.

Authors
Davis, LH; Bennett, V
MLA Citation
Davis, LH, and Bennett, V. "Identification of two regions of beta G spectrin that bind to distinct sites in brain membranes." J Biol Chem 269.6 (February 11, 1994): 4409-4416.
PMID
8308011
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
6
Publish Date
1994
Start Page
4409
End Page
4416

440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons.

440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha-helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.

Authors
Chan, W; Kordeli, E; Bennett, V
MLA Citation
Chan, W, Kordeli, E, and Bennett, V. "440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons." J Cell Biol 123.6 Pt 1 (December 1993): 1463-1473.
PMID
8253844
Source
pubmed
Published In
The Journal of Cell Biology
Volume
123
Issue
6 Pt 1
Publish Date
1993
Start Page
1463
End Page
1473

The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats.

Ankyrin repeats are a 33-amino acid motif present in a number of proteins of diverse functions including transcription factors, cell differentiation molecules, and structural proteins. This motif has been shown to mediate protein interactions in the case of ankyrin as well as several other repeat-bearing proteins. In ankyrin, 24 tandemly arrayed repeats are arranged to form a globular, membrane-binding domain. This report provides evidence that the repeats in this domain fold into four independently folded subdomains of six repeats each. Limited proteolytic digestions of defined regions of the membrane-binding domain identified protease-sensitive sites, which divided this domain into subdomains of approximately six repeats each. Hydrodynamic measurements and circular dichroism spectroscopy of expressed subdomains confirmed that these six-repeat regions exist as folded, globular structures. The requirement of a complete set of six repeats for proper folding was determined using a series of protein constructs, which sequentially deleted repeats from the last subdomain. Deletion of even one repeat resulted in a 40% loss of alpha-helicity. Deletions removing three or more repeats abolished the helical signal completely. The spherical shapes of the intact domain and of the subdomains (inferred from hydrodynamic values) suggest that the four subdomains are organized in either a tetrahedral or square planar configuration. Two six-repeat subdomains were found to be required for high affinity association with the anion exchanger, suggesting that at least some of the protein interactions mediated by ankyrin repeats involve multiple subdomains.

Authors
Michaely, P; Bennett, V
MLA Citation
Michaely, P, and Bennett, V. "The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats." J Biol Chem 268.30 (October 25, 1993): 22703-22709.
PMID
8226780
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
30
Publish Date
1993
Start Page
22703
End Page
22709

Fertilising ability of testicular spermatozoa.

Authors
Craft, I; Bennett, V; Nicholson, N
MLA Citation
Craft, I, Bennett, V, and Nicholson, N. "Fertilising ability of testicular spermatozoa." Lancet 342.8875 (October 2, 1993): 864-. (Letter)
PMID
8104288
Source
pubmed
Published In
The Lancet
Volume
342
Issue
8875
Publish Date
1993
Start Page
864

Postmitotic expression of ankyrinR and beta R-spectrin in discrete neuronal populations of the rat brain.

Isoforms of ankyrin (ankyrinR) are expressed in both the erythrocyte and the brain. Four cDNAs representing regulatory domains of ankyrinR expressed in the rat spleen and brain were cloned and sequenced. These different cDNAs were found to result from tissue-specific alternative mRNA processing of the ankyrinR regulatory domain. One of the isolated cDNAs was used to develop an antibody to brain isoforms of ankyrinR, and this antibody was used to study the localization of ankyrinR in the rat brain. The protein was found to be widely expressed in neurons of the metencephalon but limited to a discrete subset of neurons in the rat forebrain. In the thalamus and areas of the basal ganglia, these neurons were grouped in defined nuclei, whereas in the cortex, hippocampus, and caudate putamen they appeared as isolated cells distributed randomly throughout these structures. A similar study using an antibody raised against erythrocyte spectrin (beta R) showed a comparable localization to that of ankyrinR. Both proteins were expressed late in the developing rat brain, as part of the maturation stage of neural development. These data suggest a specific role for these erythrocyte structural proteins in the postmitotic development of a subset of neurons in the rat brain.

Authors
Lambert, S; Bennett, V
MLA Citation
Lambert, S, and Bennett, V. "Postmitotic expression of ankyrinR and beta R-spectrin in discrete neuronal populations of the rat brain." J Neurosci 13.9 (September 1993): 3725-3735.
PMID
8366343
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
13
Issue
9
Publish Date
1993
Start Page
3725
End Page
3735

The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin.

Na+-Ca2+ exchange is the major pathway of Ca2+ efflux during excitation-contraction coupling in cardiac muscle. The Na+-Ca2+ exchanger is present in cardiac transverse tubules with an apparent high density (Frank, J.S., Mottino, G., Reid, D., Molday, R. S., and Philipson, K.D. (1992) J. Cell Biol. 117, 337-345). The mechanism for this localization is unknown but may involve interactions with the cytoskeleton. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with the cytoskeletal protein ankyrin. On immunoblots of isolated canine cardiac sarcolemma, an antibody raised against purified rabbit red blood cell-ankyrin (RBC-ankyrin) recognized a 220-kDa protein, which is the same size as RBC-ankyrin. Alkaline extraction of sarcolemma removed this protein. The Na+-Ca2+ exchange protein, purified from recombinant baculovirus-infected insect cells, bound 125I-labeled-RBC-ankyrin with a KD of 42 +/- 3 nm. 125I-RBC-ankyrin was co-precipitated by antibodies to the Na+-Ca2+ exchanger after preincubation with solubilized cardiac sarcolemma. Myocardial ankyrin could be localized to both surface and T-tubular sarcolemma by immunofluorescence techniques. These results demonstrate that the cardiac Na+-Ca2+ exchanger binds ankyrin with high affinity. This interaction may be important for localizing the Na+-Ca2+ exchanger to specific domains of the sarcolemma.

Authors
Li, ZP; Burke, EP; Frank, JS; Bennett, V; Philipson, KD
MLA Citation
Li, ZP, Burke, EP, Frank, JS, Bennett, V, and Philipson, KD. "The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin." J Biol Chem 268.16 (June 5, 1993): 11489-11491.
PMID
8505285
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
16
Publish Date
1993
Start Page
11489
End Page
11491

Degradation of spectrin and ankyrin in the ischemic rat kidney.

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.

Authors
Doctor, RB; Bennett, V; Mandel, LJ
MLA Citation
Doctor, RB, Bennett, V, and Mandel, LJ. "Degradation of spectrin and ankyrin in the ischemic rat kidney." Am J Physiol 264.4 Pt 1 (April 1993): C1003-C1013.
PMID
8386446
Source
pubmed
Published In
The American journal of physiology
Volume
264
Issue
4 Pt 1
Publish Date
1993
Start Page
C1003
End Page
C1013

Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.

Authors
Davis, JQ; McLaughlin, T; Bennett, V
MLA Citation
Davis, JQ, McLaughlin, T, and Bennett, V. "Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain." J Cell Biol 121.1 (April 1993): 121-133.
PMID
8458865
Source
pubmed
Published In
The Journal of Cell Biology
Volume
121
Issue
1
Publish Date
1993
Start Page
121
End Page
133

DEGRADATION OF SPECTRIN AND ANKYRIN IN THE ISCHEMIC RAT-KIDNEY

Authors
DOCTOR, RB; BENNETT, V; MANDEL, LJ
MLA Citation
DOCTOR, RB, BENNETT, V, and MANDEL, LJ. "DEGRADATION OF SPECTRIN AND ANKYRIN IN THE ISCHEMIC RAT-KIDNEY." AMERICAN JOURNAL OF PHYSIOLOGY 264.4 (April 1993): C1003-C1013.
Source
wos-lite
Published In
The American journal of physiology
Volume
264
Issue
4
Publish Date
1993
Start Page
C1003
End Page
C1013

THE CARDIAC NA+-CA-2+ EXCHANGER BINDS TO THE CYTOSKELETAL PROTEIN ANKYRIN

Authors
LI, Z; BURKE, EP; FRANK, JS; BENNETT, V; PHILIPSON, KD
MLA Citation
LI, Z, BURKE, EP, FRANK, JS, BENNETT, V, and PHILIPSON, KD. "THE CARDIAC NA+-CA-2+ EXCHANGER BINDS TO THE CYTOSKELETAL PROTEIN ANKYRIN." BIOPHYSICAL JOURNAL 64.2 (February 1993): A400-A400.
Source
wos-lite
Published In
Biophysical Journal
Volume
64
Issue
2
Publish Date
1993
Start Page
A400
End Page
A400

ANKYRIN-BINDING PROTEINS RELATED TO NEURAL CELL-ADHESION MOLECULES - CANDIDATES TO PROVIDE TRANSMEMBRANE AND INTERCELLULAR CONNECTIONS IN ADULT BRAIN

Authors
BENNETT, V; DAVIS, JQ
MLA Citation
BENNETT, V, and DAVIS, JQ. "ANKYRIN-BINDING PROTEINS RELATED TO NEURAL CELL-ADHESION MOLECULES - CANDIDATES TO PROVIDE TRANSMEMBRANE AND INTERCELLULAR CONNECTIONS IN ADULT BRAIN." JOURNAL OF CELLULAR BIOCHEMISTRY (January 26, 1993): 264-264.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1993
Start Page
264
End Page
264

From anemia to cerebellar dysfunction. A review of the ankyrin gene family.

The focus of this review is on the ankyrin gene family, key elements in the interaction of the spectrin-based membrane skeleton with the plasma membrane in a variety of tissues and multicellular organisms. The structure/function relationships of ankyrin molecules are reviewed, illustrating how these proteins are uniquely suited to serve as adaptors between the membrane skeleton and a number of integral membrane proteins. Advances in the understanding of ankyrin biology in the brain are discussed and used to show how ankyrins may be involved in the establishment and/or maintenance of specialized plasma membrane domains. Finally, recent research in hematological and neurological disorders are reviewed, suggesting that ankyrins have a role in the development of human disease.

Authors
Lambert, S; Bennett, V
MLA Citation
Lambert, S, and Bennett, V. "From anemia to cerebellar dysfunction. A review of the ankyrin gene family." Eur J Biochem 211.1-2 (January 15, 1993): 1-6. (Review)
PMID
8425519
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
211
Issue
1-2
Publish Date
1993
Start Page
1
End Page
6

The junctional complex of the membrane skeleton.

Authors
Gilligan, DM; Bennett, V
MLA Citation
Gilligan, DM, and Bennett, V. "The junctional complex of the membrane skeleton." Semin Hematol 30.1 (January 1993): 74-83. (Review)
PMID
8434261
Source
pubmed
Published In
Seminars in Hematology
Volume
30
Issue
1
Publish Date
1993
Start Page
74
End Page
83

Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain." J Cell Sci Suppl 17 (1993): 109-117.
PMID
8144685
Source
pubmed
Published In
Journal of cell science. Supplement
Volume
17
Publish Date
1993
Start Page
109
End Page
117

The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane.

Authors
Bennett, V; Gilligan, DM
MLA Citation
Bennett, V, and Gilligan, DM. "The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane." Annu Rev Cell Biol 9 (1993): 27-66. (Review)
PMID
8280463
Source
pubmed
Published In
Annual Review of Cell Biology
Volume
9
Publish Date
1993
Start Page
27
End Page
66
DOI
10.1146/annurev.cb.09.110193.000331

440-kD ankyrinB: Structure of the major developmentally regulated domain and selective localization in unmyelinated axons

440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible α-helix or β sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination. © The Rockefeller University Press.

Authors
Chan, W; Kordeli, E; Bennett, V
MLA Citation
Chan, W, Kordeli, E, and Bennett, V. "440-kD ankyrinB: Structure of the major developmentally regulated domain and selective localization in unmyelinated axons." Journal of Cell Biology 123.6 PART 1 (1993): 1463-1473.
Source
scival
Published In
Journal of Cell Biology
Volume
123
Issue
6 PART 1
Publish Date
1993
Start Page
1463
End Page
1473

Degradation of spectrin and ankyrin in the ischemic rat kidney

This study investigates ischemia-induced degradation of the spectrin- based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na+-K+-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.

Authors
Doctor, RB; Bennett, V; Mandel, LJ
MLA Citation
Doctor, RB, Bennett, V, and Mandel, LJ. "Degradation of spectrin and ankyrin in the ischemic rat kidney." American Journal of Physiology - Cell Physiology 264.4 33-4 (1993): C1003-C1013.
Source
scival
Published In
American Journal of Physiology - Cell Physiology
Volume
264
Issue
4 33-4
Publish Date
1993
Start Page
C1003
End Page
C1013

The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin

Na+-Ca2+ exchange is the major pathway of Ca2+ efflux during excitation-contraction coupling in cardiac muscle. The Na+-Ca2+ exchanger is present in cardiac transverse tubules with an apparent high density (Frank, J. S., Mottino, G., Reid, D., Molday, R. S., and Philipson, K. D. (1992) J. Cell Biol. 117, 337-345). The mechanism for this localization is unknown but may involve interactions with the cytoskeleton. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with the cytoskeletal protein ankyrin. On immunoblots of isolated canine cardiac sarcolemma, an antibody raised against purified rabbit red blood cell-ankyrin (RBC-ankyrin) recognized a 220-kDa protein, which is the same size as RBC-ankyrin. Alkaline extraction of sarcolemma removed this protein. The Na+-Ca2+ exchange protein, purified from recombinant baculovirus-infected insect cells, bound 125I-labeled-RBC-ankyrin with a KD of 42 ± 3 nM. 125I-RBC-ankyrin was co-precipitated by antibodies to the Na+-Ca2+ exchanger after preincubation with solubilized cardiac sarcolemma. Myocardial ankyrin could be localized to both surface and T-tubular sarcolemma by immunofluorescence techniques. These results demonstrate that the cardiac Na+-Ca2+ exchanger binds ankyrin with high affinity. This interaction may be important for localizing the Na+-Ca2+ exchanger to specific domains of the sarcolemma.

Authors
Li, Z; Burke, EP; Frank, JS; Bennett, V; Philipson, KD
MLA Citation
Li, Z, Burke, EP, Frank, JS, Bennett, V, and Philipson, KD. "The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin." Journal of Biological Chemistry 268.16 (1993): 11489-11491.
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
16
Publish Date
1993
Start Page
11489
End Page
11491

Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator.

This study of two forms of ankyrin (protein 2.1 and 2.2) from human erythrocytes has revealed a role for alternate exon usage at the level of regulation of protein interactions. The smaller form of ankyrin (protein 2.2), which lacks a portion of the regulatory domain due to alternative splicing of pre-mRNA, exhibits increased affinity for the cytoplasmic domain of the anion exchanger, spectrin, and tubulin. Direct evidence that at least one of these associations is modulated by the alternatively spliced segment of the regulatory domain is provided by experiments utilizing a polypeptide that is comprised of residues 1513-1674 corresponding to the portion of the regulatory domain missing from protein 2.2. Addition of this regulatory domain polypeptide to binding assays reversed the increase in affinity of protein 2.2 for the anion exchanger. The inhibitory activity of the regulatory domain polypeptide in these assays is accompanied by a direct interaction with a site that is available on the smaller form of ankyrin and is distinct from the binding site for the anion exchanger. These results support the idea that the alternatively spliced segment within the regulatory domain of erythrocyte ankyrin performs a repressor function and acts through an allosteric mechanism involving interaction(s) at a site separate from the binding site for the anion exchanger.

Authors
Davis, LH; Davis, JQ; Bennett, V
MLA Citation
Davis, LH, Davis, JQ, and Bennett, V. "Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator." J Biol Chem 267.26 (September 15, 1992): 18966-18972.
PMID
1388161
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
26
Publish Date
1992
Start Page
18966
End Page
18972

Characterization of human brain cDNA encoding the general isoform of beta-spectrin.

The complete primary structure of the general form of human beta-spectrin (beta G) has been deduced from cDNAs isolated from human brain. beta G-Spectrin is encoded by a gene located on human chromosome 2. beta G-Spectrin and erythrocyte beta-spectrin (beta R) share identical domain organization, with sequence identity of 60% and sequence similarity of 77%. beta-Spectrins have closely related N-terminal domains implicated in binding to actin, and 17 copies of a 106-residue repeat motif with consensus residues that are highly conserved between beta-spectrins as well as alpha-spectrins. C-terminal domains of beta G and the 270-kDa beta R-spectrins are candidate regions to associate with alpha-spectrin, and exhibit 75% similarity. beta G- and beta R-spectrins exhibit different patterns of expression in tissues and follow different developmental programs in those tissues where they are co-expressed. beta G-Spectrin is present in all tissues examined except for erythrocytes, while beta R-spectrin could be detected only in erythrocytes, brain, and heart. beta G- and beta R-Spectrins are both expressed in brain, but beta R appeared later in post-natal development and was highly enriched in cerebellum in contrast to the broad regional distribution of beta G-spectrin. beta-Spectrins are likely to perform related but distinct functions, with beta G in a general, constitutive role and beta R-spectrin involved in more specialized activities of differentiated cells.

Authors
Hu, RJ; Watanabe, M; Bennett, V
MLA Citation
Hu, RJ, Watanabe, M, and Bennett, V. "Characterization of human brain cDNA encoding the general isoform of beta-spectrin." J Biol Chem 267.26 (September 15, 1992): 18715-18722.
PMID
1527002
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
26
Publish Date
1992
Start Page
18715
End Page
18722

OLIGOMERIC STRUCTURE AND SUBUNIT ASSOCIATION IN ERYTHROCYTE ADDUCIN

Authors
HUGHES, CA; GILLIGAN, DM; BENNETT, V
MLA Citation
HUGHES, CA, GILLIGAN, DM, and BENNETT, V. "OLIGOMERIC STRUCTURE AND SUBUNIT ASSOCIATION IN ERYTHROCYTE ADDUCIN." MOLECULAR BIOLOGY OF THE CELL 3 (September 1992): A366-A366.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
3
Publish Date
1992
Start Page
A366
End Page
A366

DIFFERENTIAL-EFFECTS OF ISCHEMIA ON SPECTRIN AND ANKYRIN IN RAT-BRAIN, HEART AND KIDNEY

Authors
DOCTOR, RB; BENNETT, V; MANDEL, LJ
MLA Citation
DOCTOR, RB, BENNETT, V, and MANDEL, LJ. "DIFFERENTIAL-EFFECTS OF ISCHEMIA ON SPECTRIN AND ANKYRIN IN RAT-BRAIN, HEART AND KIDNEY." MOLECULAR BIOLOGY OF THE CELL 3 (September 1992): A269-A269.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
3
Publish Date
1992
Start Page
A269
End Page
A269

ANK-CAMS - A FAMILY OF ANKYRIN-BINDING PROTEINS RELATED TO L1/NEUROGLIAN EXPRESSED IN POSTNATAL BRAIN

Authors
DAVIS, JQ; MCLAUGHLIN, T; BENNETT, V
MLA Citation
DAVIS, JQ, MCLAUGHLIN, T, and BENNETT, V. "ANK-CAMS - A FAMILY OF ANKYRIN-BINDING PROTEINS RELATED TO L1/NEUROGLIAN EXPRESSED IN POSTNATAL BRAIN." MOLECULAR BIOLOGY OF THE CELL 3 (September 1992): A265-A265.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
3
Publish Date
1992
Start Page
A265
End Page
A265

ANK REPEATS OF RBC ANKYRIN FOLD COOPERATIVELY IN SPECIFIC ORDERED-GROUPS OF 6 REPEATS

Authors
MICHAELY, P; BENNETT, V
MLA Citation
MICHAELY, P, and BENNETT, V. "ANK REPEATS OF RBC ANKYRIN FOLD COOPERATIVELY IN SPECIFIC ORDERED-GROUPS OF 6 REPEATS." MOLECULAR BIOLOGY OF THE CELL 3 (September 1992): A264-A264.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
3
Publish Date
1992
Start Page
A264
End Page
A264

Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm.

Authors
Bennett, V
MLA Citation
Bennett, V. "Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm." J Biol Chem 267.13 (May 5, 1992): 8703-8706. (Review)
PMID
1533619
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
13
Publish Date
1992
Start Page
8703
End Page
8706

ADAPTERS BETWEEN DIVERSE PLASMA-MEMBRANE PROTEINS AND THE CYTOPLASM

Authors
BENNETT, V
MLA Citation
BENNETT, V. "ADAPTERS BETWEEN DIVERSE PLASMA-MEMBRANE PROTEINS AND THE CYTOPLASM." JOURNAL OF BIOLOGICAL CHEMISTRY 267.13 (May 5, 1992): 8703-8706.
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
267
Issue
13
Publish Date
1992
Start Page
8703
End Page
8706

The ANK repeat: A ubiquitous motif involved in macromolecular recognition

Many proteins rely on stable, noncovalent interactions with other macromolecules to perform their function. The identification of a repeated sequence motif, the ANK repeat, in diverse proteins whose common function involves binding to other proteins indicates one way nature may achieve a wide range of protein-protein interactions. In this article, we describe evidence that these ANK repeats are involved in the specific recognition of proteins and possibly DNA, and present a model for the folding of the motif.

Authors
Michaely, P; Bennett, V
MLA Citation
Michaely, P, and Bennett, V. "The ANK repeat: A ubiquitous motif involved in macromolecular recognition." Trends in Cell Biology 2.5 (1992): 127-129.
PMID
14731966
Source
scival
Published In
Trends in Cell Biology
Volume
2
Issue
5
Publish Date
1992
Start Page
127
End Page
129

A new 440-kD isoform is the major ankyrin in neonatal rat brain.

This report describes initial characterization of a 440-kD isoform of brain ankyrin (ankyrinB) representing an alternatively spliced mRNA product of the gene encoding the major isoform of ankyrin in adult human brain (Otto, E., M. Kunimoto, T. McLaughlin, V. Bennett, J. Cell Biology. 114:241-253). Northern and immunoblot analyses indicate that 440-kD ankyrinB includes the spectrin and membrane-binding domains as well as a regulatory domain of the major 220-kD isoform. 440-kD ankyrinB contains, in addition, a sequence of a predicted size of 220 kD which is inserted between the regulatory domain and spectrin/membrane-binding domains. 440-kD ankyrinB has properties expected of a peripherally associated membrane-skeletal protein: it is exclusively present in the particulate fraction of brain homogenates, is extracted with NaOH, and remains associated with Triton-X-100-resistant structures. Expression of 440-kD ankyrinB in rat brain began at birth before other ankyrins could be detected, peaked 10 d after birth, and then decreased progressively to 30% of the maximum in adults. Expression of the 220-kD ankyrinB and ankyrinR (erythroid ankyrin) began approximately 10 d after the 440-kD isoform, increased rapidly between 10 and 15 d after birth, and finally achieved their maximal levels in adults. 440-kD ankyrinB is present in approximately equivalent amounts in all regions of neonatal brain while in adult brain it is present in highest levels in cerebellum and lowest in brain stem. 440-kD ankyrinB was localized by immunofluorescence in regions of neonatal and adult brain containing primarily dendrites and unmyelinated axons. 440-kD ankyrinB thus may play a specialized role in neuronal processes.

Authors
Kunimoto, M; Otto, E; Bennett, V
MLA Citation
Kunimoto, M, Otto, E, and Bennett, V. "A new 440-kD isoform is the major ankyrin in neonatal rat brain." J Cell Biol 115.5 (December 1991): 1319-1331.
PMID
1835461
Source
pubmed
Published In
The Journal of Cell Biology
Volume
115
Issue
5
Publish Date
1991
Start Page
1319
End Page
1331

Diversity of ankyrins in the brain.

Authors
Bennett, V; Otto, E; Kunimoto, M; Kordeli, E; Lambert, S
MLA Citation
Bennett, V, Otto, E, Kunimoto, M, Kordeli, E, and Lambert, S. "Diversity of ankyrins in the brain." Biochem Soc Trans 19.4 (November 1991): 1034-1039. (Review)
PMID
1838986
Source
pubmed
Published In
Biochemical Society transactions
Volume
19
Issue
4
Publish Date
1991
Start Page
1034
End Page
1039

Primary structure and domain organization of human alpha and beta adducin.

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.

Authors
Joshi, R; Gilligan, DM; Otto, E; McLaughlin, T; Bennett, V
MLA Citation
Joshi, R, Gilligan, DM, Otto, E, McLaughlin, T, and Bennett, V. "Primary structure and domain organization of human alpha and beta adducin." J Cell Biol 115.3 (November 1991): 665-675.
PMID
1840603
Source
pubmed
Published In
The Journal of Cell Biology
Volume
115
Issue
3
Publish Date
1991
Start Page
665
End Page
675

Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization.

Adducin is a calmodulin-binding protein involved in the assembly of the erythrocyte membrane skeleton. To investigate the expression of adducin during human erythropoiesis, we performed immunofluorescence studies on smears of cultured human erythroblasts. Adducin immunoreactivity was found in the early stages of erythropoiesis. Proerythroblasts were the first erythroid precursor cells positive for adducin. The adducin signal was very similar to the signal of erythroid beta-spectrin in that both proteins lined the membrane of erythroid precursor cells. Cell fractionation experiments were performed to further analyze the intracellular distribution of adducin in erythroid cells. In erythroblasts, about 60% of total cellular adducin appeared in the Triton-soluble fraction. In reticulocytes, the Triton-soluble fraction decreased to 30% of total reticulocyte adducin. Erythrocytes had no detectable amount of adducin in the Triton-soluble pool. Instead, adducin was quantitatively bound to the Triton-insoluble erythrocyte cytoskeleton. Our results suggest that adducin is expressed early in the development of the erythrocyte membrane skeleton, whereas stable assembly onto the membrane skeleton does not occur before the final stages of mammalian erythropoiesis.

Authors
Nehls, V; Drenckhahn, D; Joshi, R; Bennett, V
MLA Citation
Nehls, V, Drenckhahn, D, Joshi, R, and Bennett, V. "Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization." Blood 78.7 (October 1, 1991): 1692-1696.
PMID
1912559
Source
pubmed
Published In
Blood
Volume
78
Issue
7
Publish Date
1991
Start Page
1692
End Page
1696

In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s).

This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.

Authors
Hu, RJ; Bennett, V
MLA Citation
Hu, RJ, and Bennett, V. "In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s)." J Biol Chem 266.27 (September 25, 1991): 18200-18205.
PMID
1833394
Source
pubmed
Published In
The Journal of biological chemistry
Volume
266
Issue
27
Publish Date
1991
Start Page
18200
End Page
18205

Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice.

Isoforms of ankyrin (ankyrinsR) immunologically related to erythrocyte ankyrin (ankyrinRo) are associated with distinct neuronal plasma membrane domains of functional importance, such as cell bodies and dendrites, axonal hillock and initial segments, and nodes of Ranvier. AnkyrinRo is expressed in brain, and accounts for at least one of the ankyrinR isoforms. Another ankyrin isoform of brain, ankyrinB, is encoded by a distinct gene and is immunologically distinct from ankyrinsR. Mutant mice with normoblastosis (nb/nb) constitute the first described genetic model of ankyrin deficiency: they display a severe hemolytic anemia due to a significantly reduced expression of the ankyrinRo gene in reticulocytes as well as brain (Peters L. L., C. S. Birkenmeier, R. T. Bronson, R. A. White, S. E. Lux, E. Otto, V. Bennett, A. Higgins, and J. E. Barker. 1991. J. Cell Biol. 114:1233-1241). In the present report, we distinguish between ankyrinRo and other ankyrinR isoforms using immunoblot analysis and immunofluorescence localization of ankyrinsR throughout the nervous system (forebrain, cerebellum, brain stem, spinal cord, and sciatic nerve) of nb/nb and normal mice. This is the first immunocytochemical characterization of the neurological component of the nb mutation and shows the following. (a) The isoform of ankyrin at the nodes of Ranvier and initial axonal segments is present in the nb/nb mice and does not cross-react with an ankyrinRo-specific antibody; this isoform, therefore, is distinct from both ankyrin isoforms identified in brain, ankyrinRo and ankyrinB, and is probably the product of a distinct gene and a unique component of the specialized membrane skeleton associated with nodes of Ranvier. (b) AnkyrinRo missing from nb/nb mice is selectively associated with neuronal cell bodies and dendrites, excluded from myelinated axons, and displays a selective pattern of expression in the nervous system whereby expression is almost ubiquitous in neurons of the cerebellum (Purkinje and granule cells) and spinal cord, and restricted to a very minor subset of neurons in hippocampus and neocortex of forebrain.

Authors
Kordeli, E; Bennett, V
MLA Citation
Kordeli, E, and Bennett, V. "Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice." J Cell Biol 114.6 (September 1991): 1243-1259.
PMID
1832678
Source
pubmed
Published In
The Journal of Cell Biology
Volume
114
Issue
6
Publish Date
1991
Start Page
1243
End Page
1259

Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice.

Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear.

Authors
Peters, LL; Birkenmeier, CS; Bronson, RT; White, RA; Lux, SE; Otto, E; Bennett, V; Higgins, A; Barker, JE
MLA Citation
Peters, LL, Birkenmeier, CS, Bronson, RT, White, RA, Lux, SE, Otto, E, Bennett, V, Higgins, A, and Barker, JE. "Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice." J Cell Biol 114.6 (September 1991): 1233-1241.
PMID
1716634
Source
pubmed
Published In
The Journal of Cell Biology
Volume
114
Issue
6
Publish Date
1991
Start Page
1233
End Page
1241

Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes.

The mouse oocyte expresses a polypeptide of Mr 120,000 that cross-reacts with an antibody to the brain membrane skeletal protein adducin. Immunofluorescence localization showed a bright chromosomal staining reaction in metaphase I and metaphase II oocytes. Following in vitro fertilization the maternal chromosomes lost their immunoreactivity during pronuclear development. The fertilizing sperm chromatin and male pronucleus did not show any detectable staining reaction. Bright chromosomal fluorescence was again observed in the first mitotic metaphase when both maternal and paternal chromosomes gave a positive staining reaction. In contrast to the immunoreactivity of the maternal meiotic chromosomes, the meiotic chromosomes of male germ line cells failed to exhibit any detectable staining reaction and this difference was confirmed by immunolabeling of oocyte and spermatocyte karyotypes. Mitotic chromosomes in preimplantation embryos, fetal liver, adult intestinal epithelium, and MDCK cells also failed to show any detectable labeling reaction. The results suggest that expression of the immunoreactive chromosomal adducin may be a unique feature of oogenesis.

Authors
Pinto-Correia, C; Goldstein, EG; Bennett, V; Sobel, JS
MLA Citation
Pinto-Correia, C, Goldstein, EG, Bennett, V, and Sobel, JS. "Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes." Dev Biol 146.2 (August 1991): 301-311.
PMID
1864459
Source
pubmed
Published In
Developmental Biology
Volume
146
Issue
2
Publish Date
1991
Start Page
301
End Page
311

Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes.

Ankyrins are a family of membrane-associated proteins that can be divided into two immunologically distinct groups: (a) erythrocyte-related isoforms (ankyrinR) that have polarized distributions in particular cell types; and (b) brain-related isoforms (ankyrinB) that display a broader distribution. In this paper, we report the isolation and sequences of cDNAs related to two ankyrinB isoforms, human brain ankyrin 1 and 2, and show that these isoforms are produced from alternatively spliced mRNAs of a single gene. Human brain ankyrin 1 and 2 share a common NH2-terminus that is similar to human erythrocyte ankyrins, with the most striking conservation occurring between areas composed of a repeated 33-amino acid motif and between areas corresponding to the central portion of the spectrin-binding domain. In contrast, COOH-terminal sequences of brain ankyrin 1 and 2 are distinct from one another and from human erythrocyte ankyrins, and thus are candidates to mediate protein interactions that distinguish these isoforms. The brain ankyrin 2 cDNA sequence includes a stop codon and encodes a polypeptide with a predicted molecular mass of 202 kD, which is similar to the Mr of the major form of ankyrin in adult bovine brain membranes. Moreover, an antibody raised against the conserved NH2-terminal domain of brain ankyrin cross-reacts with a single Mr = 220 kD polypeptide in adult human brain. These results strongly suggest that the amino acid sequence of brain ankyrin 2 determined in this report represents the complete coding sequence of the major form of ankyrin in adult human brain. In contrast, the brain ankyrin 1 cDNAs encode only part of a larger isoform. An immunoreactive polypeptide of Mr = 440 kD, which is evident in brain tissue of young rats, is a candidate to be encoded by brain ankyrin 1 mRNA. The COOH-terminal portion of brain ankyrin 1 includes 15 contiguous copies of a novel 12-amino acid repeat. Analysis of DNA from a panel of human/rodent cell hybrids linked this human brain ankyrin gene to chromosome 4. This result, coupled with previous reports assigning the human erythrocyte ankyrin gene to chromosome 8, demonstrates that human brain and erythrocyte ankyrins are encoded by distinct members of a multigene family.

Authors
Otto, E; Kunimoto, M; McLaughlin, T; Bennett, V
MLA Citation
Otto, E, Kunimoto, M, McLaughlin, T, and Bennett, V. "Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes." J Cell Biol 114.2 (July 1991): 241-253.
PMID
1830053
Source
pubmed
Published In
The Journal of Cell Biology
Volume
114
Issue
2
Publish Date
1991
Start Page
241
End Page
253

Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion exchanger.

Erythrocyte ankyrin contains an 89-kDa domain (residues 2-827) comprised almost entirely of 22 tandem repeats of 33 amino acids which are responsible for the high affinity interaction of ankyrin with the anion exchanger (Davis, L., and Bennett, V. (1990) J. Biol. Chem. 265, 10589-10596). The question of whether the repeats are equivalent with respect to binding to the anion exchanger was addressed using defined regions of erythrocyte and brain ankyrins expressed in bacteria. The conclusion is that the repeats are not interchangeable and that the 44 residues from 722 to 765 are essential for high affinity binding between erythrocyte ankyrin and the anion exchanger. Residues 348-765 were active whereas a polypeptide of the same size (residues 305-721) but missing the 44 residues was not active. The difference between the active and inactive polypeptides was not caused by the degree of folding based on circular dichroism spectra. The 44 residues from 722 to 765 were not sufficient for binding since deletions of residues from 348 to 568 resulted in a 10-fold loss of activity. However, the role of residues 348-568 may be at the level of folding rather than a direct contact since the deleted sequences were not active in the absence of 722-765 and since circular dichroism spectra revealed significant loss of structure in the smaller polypeptides. Further evidence that the 33-residue repeats are not equivalent in ability to bind to the anion exchanger is that a region of human brain ankyrin containing 18 33-residue repeats with 67% overall sequence identity to erythrocyte ankyrin was 8-fold less active than a region of erythrocyte ankyrin containing only 12 repeats. The fact that the anion exchanger binds to certain repeats suggests that the other 33-amino acid repeats could interact with proteins distinct from the anion exchanger and provide ankyrin with the potential for considerable diversity in association with membrane proteins as well as cytoplasmic proteins. Tubulin was identified as one example of a protein that can interact with ankyrin repeats that are not recognized by the anion exchanger.

Authors
Davis, LH; Otto, E; Bennett, V
MLA Citation
Davis, LH, Otto, E, and Bennett, V. "Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion exchanger." J Biol Chem 266.17 (June 15, 1991): 11163-11169.
PMID
1828247
Source
pubmed
Published In
The Journal of biological chemistry
Volume
266
Issue
17
Publish Date
1991
Start Page
11163
End Page
11169

The spectrin skeleton: from red cells to brain.

Authors
Bennett, V; Lambert, S
MLA Citation
Bennett, V, and Lambert, S. "The spectrin skeleton: from red cells to brain." J Clin Invest 87.5 (May 1991): 1483-1489. (Review)
PMID
1850755
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
87
Issue
5
Publish Date
1991
Start Page
1483
End Page
1489
DOI
10.1172/JCI115157

Ankyrins: A family of proteins that link diverse membrane proteins to the spectrin skeleton

Authors
Bennett, V; Otto, E; Davis, J; Davis, L; Kordeli, E
MLA Citation
Bennett, V, Otto, E, Davis, J, Davis, L, and Kordeli, E. "Ankyrins: A family of proteins that link diverse membrane proteins to the spectrin skeleton." Current Topics in Membranes 38.C (1991): 65-77.
Source
scival
Published In
Current Topis in Membranes
Volume
38
Issue
C
Publish Date
1991
Start Page
65
End Page
77
DOI
10.1016/S0070-2161(08)60782-0

Primary structure and domain organization of human alpha and beta adducin

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.

Authors
Joshi, R; Gilligan, DM; Otto, E; McLaughlin, T; Bennett, V
MLA Citation
Joshi, R, Gilligan, DM, Otto, E, McLaughlin, T, and Bennett, V. "Primary structure and domain organization of human alpha and beta adducin." Journal of Cell Biology 115.3 (1991): 665-675.
Source
scival
Published In
Journal of Cell Biology
Volume
115
Issue
3
Publish Date
1991
Start Page
665
End Page
675
DOI
10.1083/jcb.115.3.665

The anion exchanger and Na+K(+)-ATPase interact with distinct sites on ankyrin in in vitro assays.

This report demonstrates that the high affinity binding of ankyrin to two well characterized ankyrin-binding proteins, the erythrocyte anion exchanger and kidney Na+K(+)-ATPase, requires interaction of these proteins with unique sites on the ankyrin molecule. Binding of 125I-labeled erythrocyte ankyrin and ankyrin proteolytic domains was measured to the anion exchanger and Na+K(+)-ATPase incorporated into phosphatidylcholine liposomes. 125I-Labeled ankyrin associated with both anion exchanger and Na+K(+)-ATPase liposomes with a high affinity (KD ranging from 10 to 25 nM), and a capacity approaching 1 mol of ankyrin/2 mol of ATPase and 1 mol of ankyrin/8 mol of anion exchanger. The 43 kDa cytoplasmic domain of the erythrocyte anion exchanger inhibited binding of ankyrin to both the anion exchanger and Na+K(+)-ATPase liposomes with a 50% reduction at approximately 90 nM for both proteins. Further binding experiments using proteolytic domains derived from ankyrin demonstrated the following differences between the anion exchanger and Na+K(+)-ATPase in interactions with ankyrin: 1) 125I-Labeled Na+K(+)-ATPase associated with both the 89-kDa domain as well as the spectrin binding domain of ankyrin, while the anion exchanger only associated with the 89-kDa domain. 2) The 125I-labeled 89-kDa domain of ankyrin associated with Na+K(+)-ATPase liposomes with at least a 20-fold lower affinity compared with intact ankyrin while this domain associated with the anion exchanger with a 2-3-fold increase in affinity compared with intact ankyrin. 3) The 125I-labeled spectrin-binding domain of ankyrin associated with the Na+K(+)-ATPase liposomes to at least an 8-fold greater extent than to anion exchanger liposomes. The data are consistent with an independent acquisition of high affinity ankyrin binding activity for the anion exchanger and Na+K(+)-ATPase proteins through a convergent evolutionary process.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "The anion exchanger and Na+K(+)-ATPase interact with distinct sites on ankyrin in in vitro assays." J Biol Chem 265.28 (October 5, 1990): 17252-17256.
PMID
2170369
Source
pubmed
Published In
The Journal of biological chemistry
Volume
265
Issue
28
Publish Date
1990
Start Page
17252
End Page
17256

Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm.

Authors
Bennett, V
MLA Citation
Bennett, V. "Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm." Physiol Rev 70.4 (October 1990): 1029-1065. (Review)
PMID
2271059
Source
pubmed
Published In
Physiological reviews
Volume
70
Issue
4
Publish Date
1990
Start Page
1029
End Page
1065

The prevalence and characteristics of congenital pigmented lesions in newborn babies in Oxford.

Melanocytic naevi (MN) are recognised risk factors for malignant melanoma but the epidemiology of MN is poorly understood. Some MN are present at birth and the study of congenital lesions is an important first step toward understanding the development of MN in early life. In this study, the prevalence and characteristics of congenital pigmented lesions were documented in 1012 White caucasian newborn babies at a maternity hospital in Oxford. Only 12 babies (1.2%) were found to have pigmented skin marks and each of these had only one lesion and no other abnormalities. MN were identified with certainty in only five babies (0.5% of the population; 95% CI, 0.06%-0.93%) thus confirming the relatively low prevalence of this lesion. Four of the MN were noted to be 'small' (less than 15 mm diameter) and all five were less than or equal to 20 mm in diameter. In other respects, the MN displayed a diversity of features. Of the other lesions there were two Mongolian blue spots, one melanocytic pustule, one 'probable' MN and three unusual skin marks where the diagnosis was uncertain but considered unlikely to be MN. During the course of the study, examinations were also carried out incidentally on 39 non-White babies. Twenty-two of these were noted to have Mongolian spots (57%) and three had other pigmented lesions (8%).

Authors
Goss, BD; Forman, D; Ansell, PE; Bennett, V; Swerdlow, AJ; Burge, S; Ryan, TJ
MLA Citation
Goss, BD, Forman, D, Ansell, PE, Bennett, V, Swerdlow, AJ, Burge, S, and Ryan, TJ. "The prevalence and characteristics of congenital pigmented lesions in newborn babies in Oxford." Paediatr Perinat Epidemiol 4.4 (October 1990): 448-457.
PMID
2267186
Source
pubmed
Published In
Paediatric & Perinatal Epidemiology
Volume
4
Issue
4
Publish Date
1990
Start Page
448
End Page
457

Mapping the domain structure of human erythrocyte adducin.

Adducin is a 200-kDa heterodimeric protein associated with the erythrocyte membrane skeleton which binds to Ca2+/calmodulin, promotes binding of spectrin to actin, and is a substrate for protein kinases C and A. Adducin polypeptides can be structurally and functionally divided into two distinct regions. The amino-terminal 39-kDa domain of each subunit is more basic and resistant to proteases than the C-terminal 60-64-kDa domain, which is very sensitive to proteolytic degradation. Two-dimensional peptide map analysis revealed that the 39-kDa protease-resistant domains represent a portion of adducin which is highly conserved between the alpha and beta subunits whereas the protease-sensitive regions are different in each subunit. Comparison of the structural and functional properties of purified 39-kDa domains with intact adducin showed that the 39-kDa domains were not phosphorylated by protein kinases C or A and did not bind to Ca2+/calmodulin or interact with spectrin and actin. This suggests that the protease-sensitive domains may perform the various functions of adducin since these activities were all lacking from the protease-resistant domains. It is also possible that the conserved and variable domains are both required for one or more activities of adducin or that the 39-kDa domains play a role in maintaining the oligomeric state of adducin necessary for interaction of the variable domains with spectrin-actin complexes.

Authors
Joshi, R; Bennett, V
MLA Citation
Joshi, R, and Bennett, V. "Mapping the domain structure of human erythrocyte adducin." J Biol Chem 265.22 (August 5, 1990): 13130-13136.
PMID
2376589
Source
pubmed
Published In
The Journal of biological chemistry
Volume
265
Issue
22
Publish Date
1990
Start Page
13130
End Page
13136

Stimulation of mutations suppressing the loss of replication control by small alcohols.

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.

Authors
Hayes, S; Hayes, C; Duncan, D; Bennett, V; Blushke, J
MLA Citation
Hayes, S, Hayes, C, Duncan, D, Bennett, V, and Blushke, J. "Stimulation of mutations suppressing the loss of replication control by small alcohols." Mutat Res 231.2 (August 1990): 151-163.
PMID
2143557
Source
pubmed
Published In
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume
231
Issue
2
Publish Date
1990
Start Page
151
End Page
163

Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin.

This report describes initial characterization of the binding sites of ankyrin for spectrin and the anion exchanger using defined subfragments isolated from purified ankyrin domains. The spectrin-binding domain of ankyrin is comprised of two subdomains: an acidic, proline-rich region (pI = 4) involving the amino-terminal 80 residues from 828 to 908 and a basic region (pI = 8.8) that extends from 898 to 1386. The amino-terminal 70 amino acids of the spectrin-binding domain are critical for association with spectrin, since a subfragment missing this region is only 5% as active as the intact domain in displacing binding of spectrin to inside-out membrane vesicles, while deletion of the first 38 residues of the acidic domain results in a 10-fold reduction in activity. The anion exchanger-binding site is confined to an 89-kDa domain that was isolated and characterized as a globular molecule with approximately 30% alpha-helical configuration. A subfragment of the 89-kDa domain extending from residues 403 to 779 (or possibly 740) retains ability to associate with the anion exchanger. The 89-kDa domain is comprised of a series of tandem repeats of 33 amino acids that extend from residues 35 to 778 (Lux, S., John, K., and Bennett, V. (1990) Nature 344, 36-42). The activity of residues 403-779 demonstrates that the 33-amino acid repeats of the 89-kDa domain are responsible for association between ankyrin and the anion exchanger. The 33-amino acid repeating sequence of ankyrin represents an ancient motif also found in proteins of Drosophila, yeast, and Caenor habditis elegans. The finding that the 33-amino acid repeating sequence is involved in interaction with the anion exchanger implies that this motif may perform a role in molecular recognition in diverse proteins.

Authors
Davis, LH; Bennett, V
MLA Citation
Davis, LH, and Bennett, V. "Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin." J Biol Chem 265.18 (June 25, 1990): 10589-10596.
PMID
2141335
Source
pubmed
Published In
The Journal of biological chemistry
Volume
265
Issue
18
Publish Date
1990
Start Page
10589
End Page
10596

Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8.

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.

Authors
Lux, SE; Tse, WT; Menninger, JC; John, KM; Harris, P; Shalev, O; Chilcote, RR; Marchesi, SL; Watkins, PC; Bennett, V
MLA Citation
Lux, SE, Tse, WT, Menninger, JC, John, KM, Harris, P, Shalev, O, Chilcote, RR, Marchesi, SL, Watkins, PC, and Bennett, V. "Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8." Nature 345.6277 (June 21, 1990): 736-739.
PMID
2141669
Source
pubmed
Published In
Nature
Volume
345
Issue
6277
Publish Date
1990
Start Page
736
End Page
739
DOI
10.1038/345736a0

An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves.

Two variants of ankyrin have been distinguished in rat brain tissue using antibodies: a broadly distributed isoform (ankyrinB) that represents the major form of ankyrin in brain and another isoform with a restricted distribution (ankyrinR) that shares epitopes with erythrocyte ankyrin. The ankyrinR isoform was localized by immunofluorescence in cryosections of rat spinal cord gray matter and myelinated central and peripheral nerves to: (a) perikarya and initial axonal segments of neuron cells, (b) nodes of Ranvier of myelinated nerve with no detectable labeling in other areas of the myelinated axons, and (c) the axolemma of unmyelinated axons. Immunogold EM on ultrathin cryosections of myelinated nerve showed that ankyrinR was localized on the cytoplasmic face of the axolemma and was restricted to the nodal and, in some cases, paranodal area. The major isoform of ankyrin in brain (ankyrinB) displayed a broad distribution on glial and neuronal cells of the gray matter and a mainly glial distribution in central myelinated axons with no significant labeling on the axolemma. These results show that (a) ankyrin isoforms display a differential distribution on glial and neuronal cells of the nervous tissue; (b) an isoform of ankyrin codistributes with the voltage-dependent sodium channel in both myelinated and unmyelinated nerve fibers. Ankyrin interacts in vitro with the voltage-dependent sodium channel (Srinivasan, Y., L. Elmer, J. Davis, V. Bennett, and K. Angelides. 1988. Nature (Lond.). 333:177-180). A specific interaction of an isoform of ankyrin with the sodium channel thus may play an important role in the morphogenesis and/or maintenance of the node of Ranvier.

Authors
Kordeli, E; Davis, J; Trapp, B; Bennett, V
MLA Citation
Kordeli, E, Davis, J, Trapp, B, and Bennett, V. "An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves." J Cell Biol 110.4 (April 1990): 1341-1352.
PMID
2139035
Source
pubmed
Published In
The Journal of Cell Biology
Volume
110
Issue
4
Publish Date
1990
Start Page
1341
End Page
1352

Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins.

Analysis of complementary DNA for human erythroid ankyrin indicates that the mature protein contains 1,880 amino acids comprising an N-terminal domain binding integral membrane proteins and tubulin, a central domain binding spectrin and vimentin, and an acidic C-terminal 'regulatory' domain containing an alternatively spliced sequence missing from ankyrin variant 2.2. The N-terminal domain is almost entirely composed of 22 tandem 33-amino-acid repeats. Similar repeats are found in yeast and invertebrate proteins involved in cell-cycle control and tissue differentiation.

Authors
Lux, SE; John, KM; Bennett, V
MLA Citation
Lux, SE, John, KM, and Bennett, V. "Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins." Nature 344.6261 (March 1, 1990): 36-42.
PMID
2137557
Source
pubmed
Published In
Nature
Volume
344
Issue
6261
Publish Date
1990
Start Page
36
End Page
42
DOI
10.1038/344036a0

Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm.

The spectrin skeleton of non-erythroid cells is likely to interact with a variety of integral membrane proteins and participate both in stable linkages as well as dynamic structures capable of rapid disassembly and assembly. The basis for diversity of roles for spectrin includes multiple, functionally distinct isoforms of spectrin, ankyrin and other associated proteins, regulation of protein interactions through phosphorylation and calcium/calmodulin, as well as differential expression of accessory proteins that determine the organization and localization of spectrin in cells. Spectrin is highly conserved from Drosophila to man and is likely to be involved in fundamental aspects of membrane structure requiring long range order and organization. Spectrin is a candidate to interact with many integral membrane proteins in roles basic to metazoan cells which must associate into tissues. Organization of cells into tissues requires loss of cell motility, formation of specialized membrane domains and assembly of cell junctions, which are all activities potentially involving spectrin. Future challenges lie in devising direct experiments to evaluate the functions of spectrin in cells and tissues.

Authors
Bennett, V
MLA Citation
Bennett, V. "Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm." Curr Opin Cell Biol 2.1 (February 1990): 51-56. (Review)
PMID
2183842
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
2
Issue
1
Publish Date
1990
Start Page
51
End Page
56

MAPPING THE PROTEIN-BINDING SITES OF ERYTHROCYTE ANKYRIN FOR BAND-3, SPECTRIN, AND THE NA/K ATPASE

Authors
BENNETT, V; DAVIS, L; DAVIS, J
MLA Citation
BENNETT, V, DAVIS, L, and DAVIS, J. "MAPPING THE PROTEIN-BINDING SITES OF ERYTHROCYTE ANKYRIN FOR BAND-3, SPECTRIN, AND THE NA/K ATPASE." BIOPHYSICAL JOURNAL 57.2 (February 1990): A27-A27.
Source
wos-lite
Published In
Biophysical Journal
Volume
57
Issue
2
Publish Date
1990
Start Page
A27
End Page
A27

REVERSIBLE ASSOCIATION OF SPECTRIN WITH BRAIN MEMBRANE-PROTEINS AND IMPLICATIONS OF A DYNAMIC SPECTRIN-BASED MEMBRANE SKELETON

Authors
STEINER, JP; BENNETT, V
MLA Citation
STEINER, JP, and BENNETT, V. "REVERSIBLE ASSOCIATION OF SPECTRIN WITH BRAIN MEMBRANE-PROTEINS AND IMPLICATIONS OF A DYNAMIC SPECTRIN-BASED MEMBRANE SKELETON." 1990.
Source
wos-lite
Published In
CELLULAR AND MOLECULAR BIOLOGY OF NORMAL AND ABNORMAL ERYTHROID MEMBRANES
Volume
118
Publish Date
1990
Start Page
1
End Page
26

Adducin: Ca++-dependent association with sites of cell-cell contact.

Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.

Authors
Kaiser, HW; O'Keefe, E; Bennett, V
MLA Citation
Kaiser, HW, O'Keefe, E, and Bennett, V. "Adducin: Ca++-dependent association with sites of cell-cell contact." J Cell Biol 109.2 (August 1989): 557-569.
PMID
2503523
Source
pubmed
Published In
The Journal of Cell Biology
Volume
109
Issue
2
Publish Date
1989
Start Page
557
End Page
569

Mapping the ankyrin-binding site of the human erythrocyte anion exchanger.

This report describes initial efforts to map the ankyrin-binding site of the cytoplasmic domain of the human erythrocyte anion exchanger. The conclusions are that this site is likely to involve a fairly extended sequence in the midregion of the cytoplasmic domain and requires interactions that are not provided by isolated peptides. The region of the sequence involving residues 174-186 is likely to participate in the ankyrin-binding site based on several experiments. Limited tryptic cleavage in the midregion of the cytoplasmic domain (residues 174 and/or 181) nearly abolished the ability of the cytoplasmic domain to inhibit binding of ankyrin to the anion exchanger. Ankyrin protected the cytoplasmic domain from tryptic digestion. Finally, peptide-specific antibodies against the sequence encompassing the site(s) of tryptic cleavage (residues 174-186) blocked binding of ankyrin to the anion exchanger. However, the sequence comprising the tryptic site is not sufficient for high affinity binding of ankyrin. A 39-amino acid peptide (residues 161-200) that includes the tryptic cleavage site(s) was inactive in inhibiting binding of ankyrin to the anion exchanger. Further evidence for a complex ankyrin-binding site is that peptide-specific antibodies against two different, noncontiguous regions (residues 118-162 and 174-186) both inhibited binding of ankyrin to the anion exchanger and were only 10-20% as effective as antibody against the entire cytoplasmic domain. Finally, the ankyrin-binding site of the anion exchanger did not renature following sodium dodecyl sulfate electrophoresis and transfer to nitrocellulose paper even though spectrin did recover ability to bind ankyrin under the same conditions. Thus, the ankyrin-binding site is not defined by a short continuous sequence. A simple consensus sequence for ankyrin-binding regions in other proteins is not likely.

Authors
Davis, L; Lux, SE; Bennett, V
MLA Citation
Davis, L, Lux, SE, and Bennett, V. "Mapping the ankyrin-binding site of the human erythrocyte anion exchanger." J Biol Chem 264.16 (June 5, 1989): 9665-9672.
PMID
2470759
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
16
Publish Date
1989
Start Page
9665
End Page
9672

Desmoplakin I and desmoplakin II. Purification and characterization.

Desmoplakins I and II (DP1 and DP2), major cytoskeletal structural proteins concentrated in desmosomes, have been purified in milligram quantities from keratomed pig tongue epithelium. DP1 and DP2 extracted from purified desmosomes in 4 M urea were chromatographed on DEAE-cellulose and remained soluble after removal of urea during subsequent chromatography. The two proteins differed by only about 15% in molecular weight (Mr = 285,000 for DP1 and 225,000 for DP2 on sodium dodecyl sulfate-polyacrylamide gels) were found to have similar Svedberg constants, 6.7 S (DP1) and 6.4 S (DP2); nevertheless, separation was readily achieved by gel filtration, since DP1 has a Stokes radius (Rs) of 164 nm, but DP2 has a Rs = 90 nm. Calculated molecular mass was 462,000 daltons for DP1 and 242,000 daltons for DP2, suggesting that DP1 may be a dimer in solution and DP2 a monomer. Cross-linking by disuccinimidyl suberate of 125I-labeled DP1 or DP2 at nanomolar concentrations confirmed that DP1 is a dimer by doubling of its apparent Mr on sodium dodecyl sulfate gels and indicated that DP2, which failed to become cross-linked, is a monomer. DP1 in the presence of 8 M urea could not be cross-linked, indicating that urea dissociated the dimers. Calculated frictional ratios (f/f0 = 3 for DP1 and 2 for DP2) indicate that both proteins are highly asymmetric. Rotary shadowing of DP1 demonstrated flexible dumbbell-like extended shapes with a maximal length of about 180 nm with a central rod and coiled or folded end domains. DP2 showed variable extended shapes of maximal length of 78-93 nm. The increased length and Rs of desmoplakin I is probably accounted for by formation of tail-to-tail dimers. Two-dimensional peptide maps and amino acid analysis showed very similar profiles for the two proteins. Purified keratin filaments failed to bind DP1 or DP2, and prekeratins polymerized in vitro and sedimented failed to remove desmoplakins, suggesting that desmoplakins do not bind keratins directly. These studies provide a basis for functional and detailed structural studies with purified native desmosomal proteins.

Authors
O'Keefe, EJ; Erickson, HP; Bennett, V
MLA Citation
O'Keefe, EJ, Erickson, HP, and Bennett, V. "Desmoplakin I and desmoplakin II. Purification and characterization." J Biol Chem 264.14 (May 15, 1989): 8310-8318.
PMID
2470743
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
14
Publish Date
1989
Start Page
8310
End Page
8318

Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes.

This report presents evidence for diversity in membrane binding sites between three forms of ankyrin: brain ankyrin, erythrocyte ankyrin, and a variant of erythrocyte ankyrin (protein 2.2) present in circulating human erythrocytes that is missing a regulatory domain. These ankyrins were compared with respect to binding to kidney microsomes and exhibited the following behavior. 1) Brain and erythrocyte ankyrin each bind to distinct sites. 2) Protein 2.2 is an activated ankyrin that binds to all of the sites accessible to both brain and erythrocyte ankyrin and, in addition, associates with its own specialized sites. 3) The specificity of these membrane sites for various ankyrins is not absolute but reflects 2.5-10-fold differences in relative affinities. Further evidence that binding sites of different ankyrins share some common features is that the cytoplasmic domain of the erythrocyte anion transporter associates with all three ankyrins and displaces binding of the ankyrin variants to kidney membranes. The differences between erythrocyte and brain ankyrins in association with kidney membranes are likely to have physiological relevance to kidney because immunologically related isoforms of ankyrin are expressed in this tissue: erythroid ankyrin which is restricted to the basolateral domains of two cell types and a brain-related ankyrin expressed in all cells and present on apical as well as basolateral membrane surfaces. An unanticipated observation was the discovery of a membrane-associated ankyrin protease in kidney that is specific for erythrocyte ankyrin and may selectively activate the erythroid isoform of ankyrin. The variety of binding sites within this group of ankyrin proteins supports the idea that ankyrins are capable of linking a number of different membrane proteins to the spectrin-actin skeleton.

Authors
Davis, J; Davis, L; Bennett, V
MLA Citation
Davis, J, Davis, L, and Bennett, V. "Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes." J Biol Chem 264.11 (April 15, 1989): 6417-6426.
PMID
2522931
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
11
Publish Date
1989
Start Page
6417
End Page
6426

DESMOPLAKIN-I AND DESMOPLAKIN-II - PURIFICATION AND CHARACTERIZATION

Authors
OKEEFE, EJ; ERICKSON, HP; BENNETT, V
MLA Citation
OKEEFE, EJ, ERICKSON, HP, and BENNETT, V. "DESMOPLAKIN-I AND DESMOPLAKIN-II - PURIFICATION AND CHARACTERIZATION." CLINICAL RESEARCH 37.2 (April 1989): A638-A638.
Source
wos-lite
Published In
Clinical Research
Volume
37
Issue
2
Publish Date
1989
Start Page
A638
End Page
A638

STRUCTURE OF HUMAN-ERYTHROCYTE ANKYRIN - HOMOLOGY TO PROTEINS INVOLVED IN DIFFERENTIATION AND CELL-CYCLE REGULATION

Authors
LUX, SE; JOHN, KM; BENNETT, V
MLA Citation
LUX, SE, JOHN, KM, and BENNETT, V. "STRUCTURE OF HUMAN-ERYTHROCYTE ANKYRIN - HOMOLOGY TO PROTEINS INVOLVED IN DIFFERENTIATION AND CELL-CYCLE REGULATION." CLINICAL RESEARCH 37.2 (April 1989): A547-A547.
Source
wos-lite
Published In
Clinical Research
Volume
37
Issue
2
Publish Date
1989
Start Page
A547
End Page
A547

DESMOPLAKIN-I AND DESMOPLAKIN-II - PURIFICATION AND CHARACTERIZATION

Authors
OKEEFE, EJ; ERICKSON, HP; BENNETT, V
MLA Citation
OKEEFE, EJ, ERICKSON, HP, and BENNETT, V. "DESMOPLAKIN-I AND DESMOPLAKIN-II - PURIFICATION AND CHARACTERIZATION." JOURNAL OF INVESTIGATIVE DERMATOLOGY 92.3 (March 1989): 494-494.
Source
wos-lite
Published In
Journal of Investigative Dermatology
Volume
92
Issue
3
Publish Date
1989
Start Page
494
End Page
494

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes.

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.

Authors
Steiner, JP; Walke, HT; Bennett, V
MLA Citation
Steiner, JP, Walke, HT, and Bennett, V. "Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes." J Biol Chem 264.5 (February 15, 1989): 2783-2791.
PMID
2492524
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
5
Publish Date
1989
Start Page
2783
End Page
2791

The spectrin-actin junction of erythrocyte membrane skeletons.

High-resolution electron microscopy of erythrocyte membrane skeletons has provided striking images of a regular lattice-like organization with five or six spectrin molecules attached to short actin filaments to form a sheet of five- and six-sided polygons. Visualization of the membrane skeletons has focused attention on the (spectrin)5,6-actin oligomers, which form the vertices of the polygons, as basic structural units of the lattice. Membrane skeletons and isolated junctional complexes contain four proteins that are stable components of this structure in the following ratios: 1 mol of spectrin dimer, 2-3 mol of actin, 1 mol of protein 4.1 and 0.1-0.5 mol of protein 4.9 (numbers refer to mobility on SDS gels). Additional proteins have been identified that are candidates to interact with the junction, based on in vitro assays, although they have not yet been localized to this structure and include: tropomyosin, tropomyosin-binding protein and adducin. The spectrin-actin complex with its associated proteins has a key structural role in mediating cross-linking of spectrin into the network of the membrane skeleton, and is a potential site for regulation of membrane properties. The purpose of this article is to review properties of known and potential constituent proteins of the spectrin-actin junction, regulation of their interactions, the role of junction proteins in erythrocyte membrane dysfunction, and to consider aspects of assembly of the junctions.

Authors
Bennett, V
MLA Citation
Bennett, V. "The spectrin-actin junction of erythrocyte membrane skeletons." Biochim Biophys Acta 988.1 (January 18, 1989): 107-121. (Review)
PMID
2642392
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
988
Issue
1
Publish Date
1989
Start Page
107
End Page
121

Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin.

Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.

Authors
Steiner, JP; Bennett, V
MLA Citation
Steiner, JP, and Bennett, V. "Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin." J Biol Chem 263.28 (October 5, 1988): 14417-14425.
PMID
2971657
Source
pubmed
Published In
The Journal of biological chemistry
Volume
263
Issue
28
Publish Date
1988
Start Page
14417
End Page
14425

Ankyrin and spectrin associate with voltage-dependent sodium channels in brain.

The segregation of voltage-dependent sodium channels to specialized regions of the neuron is crucial for propagation of an action potential. Studies of their lateral mobility indicate that sodium channels are freely mobile on the neuronal cell body but are immobile at the axon hillock, presynaptic terminal and at focal points along the axon. To elucidate the mechanisms that regulate sodium channel topography and mobility, we searched for specific proteins from the brain that associate with sodium channels. Here we show that sodium channels labelled with 3H-saxitoxin (STX) are precipitated in the presence of exogenous brain ankyrin by anti-ankyrin antibodies and that 125I-labelled ankyrin binds with high affinity to sodium channels reconstituted into lipid vesicles. The cytoplasmic domain of the erythrocyte anion transporter competes for the latter interaction. Neither the neuronal GABA (gamma-aminobutyric acid) receptor channel complex nor the dihydropyridine (DHP) receptor bind brain ankyrin. The results indicate that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeleton and may help to maintain axolemmal membrane heterogeneity and control sodium channel mobility.

Authors
Srinivasan, Y; Elmer, L; Davis, J; Bennett, V; Angelides, K
MLA Citation
Srinivasan, Y, Elmer, L, Davis, J, Bennett, V, and Angelides, K. "Ankyrin and spectrin associate with voltage-dependent sodium channels in brain." Nature 333.6169 (May 12, 1988): 177-180.
PMID
2452986
Source
pubmed
Published In
Nature
Volume
333
Issue
6169
Publish Date
1988
Start Page
177
End Page
180
DOI
10.1038/333177a0

Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction.

Erythrocyte adducin is a membrane skeletal protein that binds to calmodulin, is a major substrate for protein kinase C, and associates preferentially with spectrin-actin complexes. Erythrocyte adducin also promotes association of spectrin with actin, and this activity is inhibited by calmodulin. This study describes the isolation and characterization of a brain peripheral membrane protein closely related to erythrocyte adducin. Brain and erythrocyte adducin have at least 50% antigenic sites in common, each contains a protease-resistant core of Mr = 48,000-48,500, and both proteins are comprised of two partially homologous polypeptides of Mr = 103,000 and 97,000 (erythrocytes) and Mr = 104,000 and 107,000-110,000 (brain). Brain and erythrocyte adducin associate preferentially with spectrin-actin complexes as compared to spectrin or actin alone, and both proteins also promote binding of spectrin to actin. Brain adducin binds calmodulin in a calcium-dependent manner, although the Kd of 1.3 microM is weaker by 5-6-fold than the Kd of erythrocyte adducin for calmodulin. Brain adducin is a substrate for protein kinase C in vitro and can accept up to 2 mol of phosphate/mol of protein. Adducin provides a potential mechanism in cells for mediating site-directed assembly of additional spectrin molecules and possibly other proteins at the spectrin-actin junction. Brain tissue contains 12 pmol of adducin/mg of membrane protein, which is the most of any tissue examined other than erythrocytes, which have 50 pmol/mg. The presence of high amounts of adducin in brain suggests some role for this protein in specialized activities of nerve cells.

Authors
Bennett, V; Gardner, K; Steiner, JP
MLA Citation
Bennett, V, Gardner, K, and Steiner, JP. "Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction." J Biol Chem 263.12 (April 25, 1988): 5860-5869.
PMID
2451672
Source
pubmed
Published In
The Journal of biological chemistry
Volume
263
Issue
12
Publish Date
1988
Start Page
5860
End Page
5869

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes.

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.

Authors
Atkinson, CT; Aikawa, M; Perry, G; Fujino, T; Bennett, V; Davidson, EA; Howard, RJ
MLA Citation
Atkinson, CT, Aikawa, M, Perry, G, Fujino, T, Bennett, V, Davidson, EA, and Howard, RJ. "Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes." Eur J Cell Biol 45.2 (February 1988): 192-199.
PMID
2966734
Source
pubmed
Published In
European Journal of Cell Biology
Volume
45
Issue
2
Publish Date
1988
Start Page
192
End Page
199

Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes.

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.

Authors
Virshup, DM; Bennett, V
MLA Citation
Virshup, DM, and Bennett, V. "Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes." J Cell Biol 106.1 (January 1988): 39-50.
PMID
2892842
Source
pubmed
Published In
The Journal of Cell Biology
Volume
106
Issue
1
Publish Date
1988
Start Page
39
End Page
50

The spectrin-based membrane skeleton: extensions of the current paradigm.

Authors
Bennett, V; Davis, J; Gardner, K; Steiner, JP
MLA Citation
Bennett, V, Davis, J, Gardner, K, and Steiner, JP. "The spectrin-based membrane skeleton: extensions of the current paradigm." Soc Gen Physiol Ser 43 (1988): 101-109. (Review)
PMID
3077538
Source
pubmed
Published In
Society of General Physiologists Series
Volume
43
Publish Date
1988
Start Page
101
End Page
109

Diversity in protein associations of the spectrin-based membrane skeleton of nonerythroid cells

The purpose of this review is to summarize recent progress in understanding interactions of spectrin with membranes from brain and other tissues. Spectrin has at least two choices in linkages with the membrane, one through ankyrin, which in turn is associated with integral membrane proteins, and another linkage directly with integral membrane sites identified recently in brain membranes. Some of the integral membrane protein sites in brain bind preferentially with one spectrin isoform, while some can interact with both erythroid and the general isoform of spectrin. Ankyrin also has different isoforms, and these exhibit specificity in binding to spectrin isoforms and associate with distinct integral membrane proteins. The membrane binding sites for ankyrin include several integral membrane proteins, which are differentially expressed in different cells: the anion exchanger of intercalated cells of mammalian kidney, the sodium/potassium ATPase of kidney, and the voltage-dependent sodium channel of neurons. Ankyrin is present in many other cell types and it is likely that additional ankyrin-binding proteins will be identified. Each of the proteins that now are candidates for ankyrin binding proteins are ion channels or transporters and are localized in specialized cellular domains. The polarized localization of the ankyrin-associated membrane proteins is an essential aspect of their function at a physiological level. Spectrin and ankyrin thus exhibit an unsuspected diversity in protein linkages and have the potential for cell domain-specific interactions with a variety of membrane proteins. © 1988 Springer-Verlag.

Authors
Bennett, V; Steiner, J; Davis, J
MLA Citation
Bennett, V, Steiner, J, and Davis, J. "Diversity in protein associations of the spectrin-based membrane skeleton of nonerythroid cells." Protoplasma 145.2-3 (1988): 89-94.
Source
scival
Published In
Protoplasma
Volume
145
Issue
2-3
Publish Date
1988
Start Page
89
End Page
94
DOI
10.1007/BF01349343

REGULATION OF THE SPECTRIN-BASED MEMBRANE SKELETON

Authors
BENNETT, V
MLA Citation
BENNETT, V. "REGULATION OF THE SPECTRIN-BASED MEMBRANE SKELETON." JOURNAL OF GENERAL PHYSIOLOGY 90.6 (December 1987): A3-A3.
Source
wos-lite
Published In
The Journal of General Physiology
Volume
90
Issue
6
Publish Date
1987
Start Page
A3
End Page
A3

Regulatory domains of erythrocyte ankyrin.

This report provides evidence for regulatory domains of erythrocyte ankyrin that modulate associations of this protein with the anion transporter and spectrin. Two domains have been identified that are located at opposite ends of the polypeptide chain. One domain (Mr = 20,000), which is released by calpain, is primarily involved in regulation of the association of ankyrin with the anion transporter. The Mr = 195,000 fragment remaining after calpain cleavage binds to ankyrin-depleted inside-out vesicles with a 8-fold reaction in affinity, although with a 2-fold increase in number of high affinity sites. Cleavage of ankyrin by calpain induces a reduction in the frictional ratio from 1.55 to 1.33 suggesting either that the calpain-sensitive domain is present as a tail extending from a globular domain, or that upon cleavage ankyrin undergoes a major change in conformation. The other proposed regulatory domain is missing in protein 2.2, a form of ankyrin present in human erythrocytes that has a molecular weight about 29,000 smaller than ankyrin. Protein 2.2 is distinct from the calpain fragment based on peptide maps and reaction with domain-specific antibodies. The activity of the domain deleted from protein 2.2 has been inferred by comparison of ankyrin and protein 2.2, with the assumption that differences between these proteins are due to the missing domain. Protein 2.2 is an activated form of ankyrin that has a 3-fold higher affinity for spectrin and binds to twice the number of high affinity anion transporter sites. These observations suggested that removal of terminal domains of ankyrin may have a physiological role in modulation of ankyrin activity.

Authors
Hall, TG; Bennett, V
MLA Citation
Hall, TG, and Bennett, V. "Regulatory domains of erythrocyte ankyrin." J Biol Chem 262.22 (August 5, 1987): 10537-10545.
PMID
3038887
Source
pubmed
Published In
The Journal of biological chemistry
Volume
262
Issue
22
Publish Date
1987
Start Page
10537
End Page
10545

Modulation of spectrin-actin assembly by erythrocyte adducin.

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.

Authors
Gardner, K; Bennett, V
MLA Citation
Gardner, K, and Bennett, V. "Modulation of spectrin-actin assembly by erythrocyte adducin." Nature 328.6128 (July 23, 1987): 359-362.
PMID
3600811
Source
pubmed
Published In
Nature
Volume
328
Issue
6128
Publish Date
1987
Start Page
359
End Page
362
DOI
10.1038/328359a0

Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors.

In the present study we have examined several types of nucleated cells with respect to the occurrence and subcellular distribution of ankyrin. In red blood cells ankyrin links and integral membrane protein, the anion channel (band 3), to the subplasmalemmal cytoskeleton which is comprised largely of spectrin and actin. Since nucleated cells also contain spectrin and other constituents of the erythrocyte membrane skeleton it is possible that in nonerythroid cells ankyrin is also important for connecting membrane proteins to the cytoskeleton. We show here that membrane fractions of rat brain and various types of rat epithelial cells contain analogs of ankyrin at Mr 210,000 and 190,000 that are immunologically related to human erythrocyte ankyrin. In transporting epithelial cells, such as epithelia of the intestine, pancreas, prostate or kidney (various species) the analogs of ankyrin are confined to the basolateral plasma membrane and are absent from the apical membrane. In neurons of the central and peripheral nervous system and in photoreceptors of the retina, ankyrin was found restricted to the membrane of the cell body and axons and was not detected by immunostaining along the afferent processes (dendrites, photoreceptor inner and outer segments). Linkage of integral membrane proteins via ankyrin to the spectrin-based membrane cytoskeleton may provide a molecular basis for restricting the lateral mobility of certain membrane proteins and localizing them in a nonrandom or polarized fashion at specialized domains of the plasma membrane.

Authors
Drenckhahn, D; Bennett, V
MLA Citation
Drenckhahn, D, and Bennett, V. "Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors." Eur J Cell Biol 43.3 (June 1987): 479-486.
PMID
2957198
Source
pubmed
Published In
European Journal of Cell Biology
Volume
43
Issue
3
Publish Date
1987
Start Page
479
End Page
486

Synapsin I: a regulated synaptic vesicle organizing protein.

Synapsin is a protein initially discovered and characterized as a target for cyclic AMP and Ca/calmodulin-regulated protein kinases that is concentrated in nerve endings and is localized on the surface of small synaptic vesicles. Synapsin shares antigenic sites and some local regions of homology with erythrocyte protein 4.1, although these proteins in general are quite different in sequence. Protein 4.1 and synapsin share several local regions of homology with erythrocyte spectrin alpha subunit. Protein 4.1 and synapsin may be related to each other through a common relationship with spectrin. Synapsin binds to synaptic vesicles and membrane sites with a Kd of 0.01-0.02 microM and associates with a Kd of 0.5-4 microM to spectrin, microtubules and neurofilaments in in vitro assays. Synapsin interconnects synaptic vesicles to membranes, and this activity is inhibited by phosphorylation with Ca/calmodulin-dependent protein kinase. Synapsin may have a role in neurons as a structural protein capable of interconnecting small synaptic vesicles with a number of proteins, including spectrin, microtubules, neurofilaments, and membrane sites. A physiological function of synapsin could be as a vesicle-organizing protein that mediates calcium-regulated association of vesicles with cytoskeletal proteins during axonal transport and attaches vesicles to active zones in nerve endings.

Authors
Steiner, JP; Gardner, K; Baines, A; Bennett, V
MLA Citation
Steiner, JP, Gardner, K, Baines, A, and Bennett, V. "Synapsin I: a regulated synaptic vesicle organizing protein." Brain Res Bull 18.6 (June 1987): 777-785. (Review)
PMID
3113674
Source
pubmed
Published In
Brain Research Bulletin
Volume
18
Issue
6
Publish Date
1987
Start Page
777
End Page
785

Nearest neighbor analysis for brain synapsin I. Evidence from in vitro reassociation assays for association with membrane protein(s) and the Mr = 68,000 neurofilament subunit.

Synapsin I, a major neuron-specific substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinases, associates in in vitro assays with brain integral membrane protein site(s) distinct from secretory vesicles and with the neurofilament Mr = 68,000 subunit. The membrane sites for synapsin involve protein(s) and are likely to have physiological relevance since the binding of 125I-labeled synapsin is abolished by digestion with chymotrypsin, is displaced by unlabeled synapsin, is of high affinity (KD = 10 nM), and has a capacity (42 pmol/mg membrane protein) that is comparable to the amount of synapsin in brain, optimal binding occurs at physiological pH (6.8-7.2) and salt concentrations (50 mM), and synapsin binding to membranes is inhibited by phosphorylation with Ca2+/calmodulin-dependent protein kinase. The brain membrane protein sites for synapsin are not due to synaptic vesicles, since synaptic vesicles do not sediment under the conditions of the binding assay. Association between synapsin and the Mr = 68,000 neurofilament subunit has also been demonstrated. The binding of synapsin with the neurofilament subunit is specific since this binding interaction is saturable, with a 1:1 stoichiometry, the binding involves only certain proteolytically derived domains of synapsin, and is therefore not a simple electrostatic interaction between the basic domains of synapsin and the acidic regions in the neurofilament subunit, and Ca2+/calmodulin-dependent phosphorylation of synapsin inhibits this interaction. Synapsin promotes cross-linking of synaptic vesicles to brain membranes, and these complexes are reduced by phosphorylation of synapsin. This interconnecting function of synapsin may be a general characteristic of synapsin binding, with a membrane (synaptic vesicle or nonsecretory vesicle)-bound synapsin associating with microtubules, neurofilaments, or spectrin.

Authors
Steiner, JP; Ling, E; Bennett, V
MLA Citation
Steiner, JP, Ling, E, and Bennett, V. "Nearest neighbor analysis for brain synapsin I. Evidence from in vitro reassociation assays for association with membrane protein(s) and the Mr = 68,000 neurofilament subunit." J Biol Chem 262.2 (January 15, 1987): 905-914.
PMID
3100521
Source
pubmed
Published In
The Journal of biological chemistry
Volume
262
Issue
2
Publish Date
1987
Start Page
905
End Page
914

Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s).

An assay has been developed to measure association of brain ankyrin with protein site(s) in brain membranes that are independent of spectrin and tubulin, behave as integral membrane proteins, and appear to be similar in several respects to the erythrocyte anion channel. Brain membranes were depleted of ankyrin, spectrin, and other peripheral membrane proteins by a brief incubation in 0.1 M sodium hydroxide. Binding of ankyrin to these membranes fulfilled experimentally testable criteria for a specific protein-protein association. Binding was optimal at physiological values for ionic strength and pH, was of high affinity (Kd = 20-60 nM), and the capacity of 25 pmol/mg of brain membrane protein is in the same range as the number of spectrin tetramers (30 pmol/mg). The membrane-binding site(s) for brain ankyrin are likely to be related in some way to the cytoplasmic domain of the erythrocyte anion channel since binding was inhibited by the anion channel domain and by erythrocyte ankyrin. The binding site(s) for brain ankyrin were released from the membrane by limited proteolysis as active water-soluble fragments capable of inhibiting binding of ankyrin to membranes. Ankyrin-binding fragments of Mr = 40,000 and 68,000 were selectively bound to an erythrocyte ankyrin affinity column. The fragment of Mr = 40,000 is close to the size of the cytoplasmic domain of the erythrocyte anion channel. It is likely based on these results that membrane attachment proteins for ankyrin are present in brain and other tissues and that these membrane proteins have domains homologous at least in conformation to the ankyrin-binding site of the erythrocyte anion channel.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s)." J Biol Chem 261.34 (December 5, 1986): 16198-16206.
PMID
2946682
Source
pubmed
Published In
The Journal of biological chemistry
Volume
261
Issue
34
Publish Date
1986
Start Page
16198
End Page
16206

Selective externalization of an ATP-binding protein structurally related to the clathrin-uncoating ATPase/heat shock protein in vesicles containing terminal transferrin receptors during reticulocyte maturation.

Transferrin receptors are lost from reticulocytes in vesicles that are released during the final stage of erythrocyte maturation (Pan, B. T., and Johnstone, R. M. (1983) Cell 33, 967-977). Transferrin receptor-containing vesicles have a major protein component present in a 1:1 ratio with the receptor that migrates on sodium dodecyl sulfate gels as two polypeptides of Mr = 71,000 and 72,000. The Mr = 71,000/72,000 doublet is indistinguishable from the clathrin-uncoating ATPase/heat shock protein based on cross-reaction with affinity-purified antibody against the uncoating protein, by comparison of peptide maps of the Mr = 72,000 and 71,000 polypeptides and the uncoating protein, and by selective binding of these polypeptides to ATP-agarose. This finding suggests a possible activity of proteins related to the uncoating/heat shock protein family in the disposal of aged membrane proteins by a pathway independent of lysosomes.

Authors
Davis, JQ; Dansereau, D; Johnstone, RM; Bennett, V
MLA Citation
Davis, JQ, Dansereau, D, Johnstone, RM, and Bennett, V. "Selective externalization of an ATP-binding protein structurally related to the clathrin-uncoating ATPase/heat shock protein in vesicles containing terminal transferrin receptors during reticulocyte maturation." J Biol Chem 261.33 (November 25, 1986): 15368-15371.
PMID
3536900
Source
pubmed
Published In
The Journal of biological chemistry
Volume
261
Issue
33
Publish Date
1986
Start Page
15368
End Page
15371

Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes.

A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.

Authors
Ling, E; Gardner, K; Bennett, V
MLA Citation
Ling, E, Gardner, K, and Bennett, V. "Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes." J Biol Chem 261.30 (October 25, 1986): 13875-13878.
PMID
3771508
Source
pubmed
Published In
The Journal of biological chemistry
Volume
261
Issue
30
Publish Date
1986
Start Page
13875
End Page
13878

A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification.

A new protein that binds calmodulin has been identified and purified to greater than 95% homogeneity from the Triton X-100-insoluble residue of human erythrocyte ghost membranes (cytoskeletons) by DEAE chromatography and preparative rate zonal sucrose gradient sedimentation. This ghost calmodulin-binding protein is an alpha/beta heterodimer with subunits of Mr = 103,000 (alpha) and 97,000 (beta). The protein exhibits a Stokes radius of 6.9 nm and a sedimentation coefficient of 6.8 S, corresponding to a molecular weight of 197,000. Moreover, the protein is cross-linked by Cu2+/phenanthroline to a dimer of Mr = 200,000. The Mr = 97,000 beta subunit was identified as the calmodulin-binding site by photoaffinity labeling with 125I-azidocalmodulin. A 230 nM affinity for calmodulin was estimated by displacement of two different concentrations of the 125I-azidocalmodulin with unmodified calmodulin and subsequent Dixon plot analysis. This calmodulin-binding protein is present in erythrocytes at 30,000 copies/cell and is associated exclusively with the membrane. It is tightly bound to a site on red cell cytoskeletons and is totally solubilized in the low ionic strength extract derived from red cell ghost membranes. Visualization of this calmodulin-binding protein in the electron microscope by rotary shadowing, negative staining, and unidirectional shadowing indicates that it is a flattened circular molecule with a 12.4-nm diameter and a 5.4-nm height. Affinity-purified antibodies against the calmodulin-binding protein identify a cross-reacting Mr = 100,000 polypeptide(s) in brain membranes.

Authors
Gardner, K; Bennett, V
MLA Citation
Gardner, K, and Bennett, V. "A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification." J Biol Chem 261.3 (January 25, 1986): 1339-1348.
PMID
3511042
Source
pubmed
Published In
The Journal of biological chemistry
Volume
261
Issue
3
Publish Date
1986
Start Page
1339
End Page
1348

Synapsin I is a microtubule-bundling protein.

Synapsin I, a synaptic vesicle protein, is thought to be involved in the regulation of neurotransmission through its phosphorylation by the cyclic AMP-dependent and Ca2+/calmodulin-dependent protein kinases which become activated upon depolarization of nerve endings. However, despite its recent characterization as a spectrin-binding protein immunologically related to erythrocyte protein 4.1, other interactions of synapsin I with structural proteins remain unknown. We report here that synapsin I can co-cycle with microtubules through three cycles of warm polymerization and cold depolymerization. Synapsin I binds saturably to microtubules stabilized by taxol, with an estimated dissociation constant (Kd) of 4.5 microM and a stoichiometry of 1.2 mol of synapsin binding sites per mol tubulin dimer. Synapsin I also increases the turbidity of tubulin solutions at 37 degrees C, but without causing detectable alterations in the critical concentration required for polymerization. Mixtures of synapsin I and tubulin observed by negative stain electron microscopy contain bundles of microtubules, accounting for the effect of synapsin I on tubulin turbidity. Synapsin I is thus a candidate to mediate or regulate the interaction of synaptic vesicles with microtubules.

Authors
Baines, AJ; Bennett, V
MLA Citation
Baines, AJ, and Bennett, V. "Synapsin I is a microtubule-bundling protein." Nature 319.6049 (January 9, 1986): 145-147.
PMID
2417124
Source
pubmed
Published In
Nature
Volume
319
Issue
6049
Publish Date
1986
Start Page
145
End Page
147
DOI
10.1038/319145a0

Purification of brain analogs of red blood cell membrane skeletal proteins: ankyrin, protein 4.1 (synapsin), spectrin, and spectrin subunits.

Authors
Bennett, V; Baines, AJ; Davis, J
MLA Citation
Bennett, V, Baines, AJ, and Davis, J. "Purification of brain analogs of red blood cell membrane skeletal proteins: ankyrin, protein 4.1 (synapsin), spectrin, and spectrin subunits." Methods Enzymol 134 (1986): 55-69.
PMID
2950299
Source
pubmed
Published In
Methods in Enzymology
Volume
134
Publish Date
1986
Start Page
55
End Page
69

[7] Purification of brain analogs of red blood cell membrane skeletal proteins: Ankyrin, protein 4.1 (synapsin), spectrin, and spectrin subunits

Authors
Bennett, V; Baines, AJ; Davis, J
MLA Citation
Bennett, V, Baines, AJ, and Davis, J. "[7] Purification of brain analogs of red blood cell membrane skeletal proteins: Ankyrin, protein 4.1 (synapsin), spectrin, and spectrin subunits." Methods in Enzymology 134.C (1986): 55-69.
Source
scival
Published In
Methods in Enzymology
Volume
134
Issue
C
Publish Date
1986
Start Page
55
End Page
69
DOI
10.1016/0076-6879(86)34075-8

Colocalization of band 3 with ankyrin and spectrin at the basal membrane of intercalated cells in the rat kidney.

An immunoreactive form of the anion channel protein of erythrocytes, band 3, has been identified in the rat kidney. It is found in the intercalated cells of the distal tubule and collecting ducts. Immunostaining specific for band 3 is confined to the basolateral plasma membrane of these cells, where this protein probably mediates the transport of bicarbonate across the tubular wall. Double-immunolabeling studies demonstrate that band 3 is colocalized with immunoreactive forms of ankyrin and spectrin along the basolateral plasma membrane. The polarized distribution of band 3 may be the result of the association of its cytoplasmic domain with ankyrin, which in turn links band 3 to spectrin and the cytoskeleton. These observations help to explain how the collecting ducts of the kidney can direct the transport of bicarbonate ions, thus maintaining the acid-base balance.

Authors
Drenckhahn, D; Schlüter, K; Allen, DP; Bennett, V
MLA Citation
Drenckhahn, D, Schlüter, K, Allen, DP, and Bennett, V. "Colocalization of band 3 with ankyrin and spectrin at the basal membrane of intercalated cells in the rat kidney." Science 230.4731 (December 13, 1985): 1287-1289.
PMID
2933809
Source
pubmed
Published In
Science
Volume
230
Issue
4731
Publish Date
1985
Start Page
1287
End Page
1289

Human erythrocyte clathrin and clathrin-uncoating protein.

Clathrin, a Mr = 72,000 clathrin-associated protein, and myosin were purified in milligram quantities from the same erythrocyte hemolysate fraction. Erythrocyte clathrin closely resembled brain clathrin in several respects: (a) both are triskelions as visualized by electron microscopy with arms 40 nm in length with globular ends and a flexible hinge region in the middle of each arm, and these triskelions assemble into polyhedral "cages" at appropriate pH and ionic strength; (b) both molecules contain heavy chains of Mr = 170,000 that are indistinguishable by two-dimensional maps of 125I-labeled peptides; and (c) both molecules contain light chains of Mr approximately 40,000 in a 1:1 molar ratio with the heavy chain. Erythrocyte clathrin is not identical to brain clathrin since antibody raised against the erythrocyte protein reacts better with erythrocyte clathrin than with brain clathrin and since brain clathrin contains two light chains resolved on sodium dodecyl sulfate gels while the light chain of erythrocyte clathrin migrates as a single band. The erythrocyte Mr = 72,000 clathrin-associated protein is closely related to a protein in brain that mediates ATP-dependent disassembly of clathrin from coated vesicles and binds tightly to clathrin triskelions (Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733). The erythrocyte and brain proteins have identical Mr on sodium dodecyl sulfate gels and identical maps of 125I-labeled peptides, share antigenic sites, and bind tightly to ATP immobilized on agarose. Clathrin and the uncoating protein are not restricted to reticulocytes since equivalent amounts of these proteins are present in whole erythrocyte populations and reticulocyte-depleted erythrocytes. Clathrin is present at 6,000 triskelions/cells, while the uncoating protein is in substantial excess at 250,000 copies/cell.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "Human erythrocyte clathrin and clathrin-uncoating protein." J Biol Chem 260.27 (November 25, 1985): 14850-14856.
PMID
4055802
Source
pubmed
Published In
The Journal of biological chemistry
Volume
260
Issue
27
Publish Date
1985
Start Page
14850
End Page
14856

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1.

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.

Authors
Baines, AJ; Bennett, V
MLA Citation
Baines, AJ, and Bennett, V. "Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1." Nature 315.6018 (May 30, 1985): 410-413.
PMID
3923367
Source
pubmed
Published In
Nature
Volume
315
Issue
6018
Publish Date
1985
Start Page
410
End Page
413

Partial deficiency of erythrocyte spectrin in hereditary spherocytosis.

Hereditary spherocytosis (HS) is a common, clinically heterogeneous haemolytic anaemia in which the primary erythrocyte defect is believed to be some abnormality in the spectrin-actin membrane skeleton, leading to loss of surface membrane. Recessively inherited spectrin deficiency with extreme erythrocyte fragility and spherocytosis has been identified in certain mutant mice and two severely anaemic humans. Although suspected, deficiency of spectrin has not been demonstrated in less severe forms of human HS. We not report the quantitation of erythrocytes spectrin by radioimmunoassay. We found that normal erythrocytes contained 240,000 copies of spectrin heterodimer, whereas erythrocytes from 14 patients with a variety of types of HS were all partially deficient in spectrin (range 74,000-200,000 copies), the magnitude of the deficiency correlating with the severity of the disease. Spectrin deficiency of varying degrees is common in HS and probably represents the principal structural defect leading to loss of surface membrane.

Authors
Agre, P; Casella, JF; Zinkham, WH; McMillan, C; Bennett, V
MLA Citation
Agre, P, Casella, JF, Zinkham, WH, McMillan, C, and Bennett, V. "Partial deficiency of erythrocyte spectrin in hereditary spherocytosis." Nature 314.6009 (March 28, 1985): 380-383.
PMID
3982506
Source
pubmed
Published In
Nature
Volume
314
Issue
6009
Publish Date
1985
Start Page
380
End Page
383

Plasmodium falciparum malaria: band 3 as a possible receptor during invasion of human erythrocytes.

Human erythrocyte band 3, a major membrane-spanning protein, was purified and incorporated into liposomes. These liposomes, at nanomolar concentrations of protein, inhibited invasion of human erythrocytes in vitro by the malaria parasite Plasmodium falciparum. Liposomes containing human band 3 were ten times more effective in inhibiting invasion than those with pig band 3 and six times more effective than liposomes containing human erythrocyte glycophorin. Liposomes alone or liposomes containing erythrocyte glycolipids did not inhibit invasion. These results suggest that band 3 participates in the invasion process in a step involving a specific, high-affinity interaction between band 3 and some component of the parasite.

Authors
Okoye, VC; Bennett, V
MLA Citation
Okoye, VC, and Bennett, V. "Plasmodium falciparum malaria: band 3 as a possible receptor during invasion of human erythrocytes." Science 227.4683 (January 11, 1985): 169-171.
PMID
3880920
Source
pubmed
Published In
Science
Volume
227
Issue
4683
Publish Date
1985
Start Page
169
End Page
171

Human erythrocyte myosin: identification and purification.

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.

Authors
Fowler, VM; Davis, JQ; Bennett, V
MLA Citation
Fowler, VM, Davis, JQ, and Bennett, V. "Human erythrocyte myosin: identification and purification." J Cell Biol 100.1 (January 1985): 47-55.
PMID
3880759
Source
pubmed
Published In
The Journal of Cell Biology
Volume
100
Issue
1
Publish Date
1985
Start Page
47
End Page
55

The membrane skeleton of human erythrocytes and its implications for more complex cells.

Authors
Bennett, V
MLA Citation
Bennett, V. "The membrane skeleton of human erythrocytes and its implications for more complex cells." Annu Rev Biochem 54 (1985): 273-304. (Review)
PMID
3161450
Source
pubmed
Published In
Annual Review of Biochemistry
Volume
54
Publish Date
1985
Start Page
273
End Page
304
DOI
10.1146/annurev.bi.54.070185.001421

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes.

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.

Authors
Bennett, V; Baines, AJ; Davis, JQ
MLA Citation
Bennett, V, Baines, AJ, and Davis, JQ. "Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes." J Cell Biochem 29.2 (1985): 157-169.
PMID
2933418
Source
pubmed
Published In
Journal of Cellular Biochemistry
Volume
29
Issue
2
Publish Date
1985
Start Page
157
End Page
169
DOI
10.1002/jcb.240290210

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel.

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel." J Biol Chem 259.21 (November 10, 1984): 13550-13559.
PMID
6092380
Source
pubmed
Published In
The Journal of biological chemistry
Volume
259
Issue
21
Publish Date
1984
Start Page
13550
End Page
13559

Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes.

Two new vascular smooth muscle relaxants, bepridil and cetiedil, were found to possess specific CaM-inhibitory properties which resembled those of trifluoperazine. Trifluoperazine, bepridil, and cetiedil inhibited Ca2+-dependent 125I-CaM binding to erythrocyte membranes and CaM activation of membrane Ca2+-ATPase with IC50 values of approximately 12, approximately 17, and approximately 40 microM, respectively. This does not appear to be the result of a nonspecific hydrophobic interaction since inhibition was not observed with micromolar concentrations of many other hydrophobic agents. The predominant inhibition of binding and Ca2+-ATPase activation was competitive with respect to CaM. Bepridil and cetiedil bind directly to CaM since these drugs displaced [3H]trifluoperazine from sites on CaM. Inhibition of Ca2+-ATPase and binding by the drugs was not due to interference with the catalytic activity of this enzyme since: (a) neither inhibition of CaM-independent basal Ca2+-ATPase activity nor inhibition of proteolytically-activated Ca2+-ATPase activities were produced by these agents, and (b) no drug-induced inhibition of CaM binding was detected when membranes were preincubated with these agents but washed prior to addition of 125I-CaM. Thus, bepridil and cetiedil competitively inhibit Ca2+-dependent interactions of CaM with erythrocyte membranes, most likely by a direct interaction between these drugs and CaM. The principal clinical actions of these drugs may be explained by their interactions with CaM or CaM-related proteins leading to reduced activation of Ca2+-regulated enzymes in certain other tissues, such as myosin light chain kinase in vascular smooth muscle.

Authors
Agre, P; Virshup, D; Bennett, V
MLA Citation
Agre, P, Virshup, D, and Bennett, V. "Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes." J Clin Invest 74.3 (September 1984): 812-820.
PMID
6088585
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
74
Issue
3
Publish Date
1984
Start Page
812
End Page
820
DOI
10.1172/JCI111497

Erythrocyte membrane tropomyosin. Purification and properties.

Two polypeptides of Mr approximately 29,000 and 27,000 have been identified in human erythrocyte membranes that cross-react specifically with affinity purified antibodies to chicken gizzard tropomyosin. The cross-reacting polypeptides are quantitatively retained on the membrane after cell lysis if millimolar concentrations of magnesium are included in the lysis and wash buffers, indicating that they are membrane-bound proteins under physiological conditions. Milligram quantities of these immunoreactive polypeptides have been purified to greater than 95% purity from a low salt extract of membranes by DEAE-chromatography, precipitation at pH 4.4, and heating to 85 degrees C to denature contaminants. Physical similarities of the erythrocyte protein to other tropomyosins include (a) amino acid composition (b) anomalous migration of the Mr approximately 29,000 and 27,000 polypeptides on sodium dodecyl sulfate-gels in the presence of 6 M urea to apparent Mr approximately 43,000 and 38,000, respectively (c) arrangement of chains as dimers of Mr approximately 60,000 based on cross-linking studies and calculation of molecular weight from hydrodynamic values (Rs = 5.9 nm, sedimentation coefficient = 2.5 S; partial specific volume = 0.72 cm3/g) and (d) highly asymmetric shape, based on a frictional ratio of 2.07. Binding of erythrocyte tropomyosin to muscle F-actin saturates at one tropomyosin molecule (Mr approximately 60,000) to 6-7 actin monomers and is highly cooperative with a Hill coefficient of about 2.8, similar to muscle tropomyosins. Binding also exhibits a high degree of cooperativity as a function of the magnesium concentration with a transition between no binding and complete binding between 1 and 2 mM MgCl2. Increasing the magnesium concentration from 2 to 10 mM increases the apparent affinity of tropomyosin for actin from approximately 2.6 X 10(6) M-1 to approximately 2.7 X 10(7) M-1 without effect on the Hill coefficient. The tropomyosin polypeptides comprise about 1% of the erythrocyte membrane protein and are present in a ratio of one Mr approximately 60,000 tropomyosin molecule to 7-8 actin monomers, an amount almost sufficient to coat all of the F-actin on the membrane. These data are consistent with the association of two tropomyosin molecules with each of the short actin filaments (12-17 monomers long) thought to exist in the erythrocyte membrane cytoskeleton. The erythrocyte tropomyosin could function to mechanically stabilize these actin filaments as well as play a role in regulating the interaction of spectrin with actin.(ABSTRACT TRUNCATED AT 400 WORDS)

Authors
Fowler, VM; Bennett, V
MLA Citation
Fowler, VM, and Bennett, V. "Erythrocyte membrane tropomyosin. Purification and properties." J Biol Chem 259.9 (May 10, 1984): 5978-5989.
PMID
6715382
Source
pubmed
Published In
The Journal of biological chemistry
Volume
259
Issue
9
Publish Date
1984
Start Page
5978
End Page
5989

Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain.

Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.

Authors
Davis, JQ; Bennett, V
MLA Citation
Davis, JQ, and Bennett, V. "Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain." J Biol Chem 259.3 (February 10, 1984): 1874-1881.
PMID
6229540
Source
pubmed
Published In
The Journal of biological chemistry
Volume
259
Issue
3
Publish Date
1984
Start Page
1874
End Page
1881

The work of a health visitor in a residential family unit.

Authors
Bennett, V
MLA Citation
Bennett, V. "The work of a health visitor in a residential family unit." Health Visit 57.2 (February 1984): 49-50.
PMID
6561184
Source
pubmed
Published In
Health Visitor
Volume
57
Issue
2
Publish Date
1984
Start Page
49
End Page
50

Tropomyosin: a new component of the erythrocyte membrane skeleton.

Authors
Fowler, VM; Bennett, V
MLA Citation
Fowler, VM, and Bennett, V. "Tropomyosin: a new component of the erythrocyte membrane skeleton." Prog Clin Biol Res 159 (1984): 57-71.
PMID
6473466
Source
pubmed
Published In
Progress in Clinical and Biological Research
Volume
159
Publish Date
1984
Start Page
57
End Page
71

Proteins closely related to spectrin and ankyrin are general components of cell membranes.

Membrane-associated analogues of erythrocyte spectrin and ankyrin have been detected in nonerythroid cells by crossreaction with antibodies. Brain spectrin and a spectrin-binding domain of brain ankyrin have been purified and demonstrated to have all known functional activities of their erythrocyte counterparts. The nonerythroid forms of spectrin and ankyrin may have functions such as providing support for the lipid bilayer, mediating some types of actin-membrane interactions, and integrating membrane-spanning proteins with cytoplasmic structural proteins.

Authors
Bennett, V; Davis, JQ
MLA Citation
Bennett, V, and Davis, JQ. "Proteins closely related to spectrin and ankyrin are general components of cell membranes." Prog Clin Biol Res 165 (1984): 457-472.
PMID
6239291
Source
pubmed
Published In
Progress in Clinical and Biological Research
Volume
165
Publish Date
1984
Start Page
457
End Page
472

Brain ankyrin: Purification of a 72,000 M spectrin-binding domain

Authors
Bennett, V; Davis, JQ
MLA Citation
Bennett, V, and Davis, JQ. "Brain ankyrin: Purification of a 72,000 M spectrin-binding domain." Federation Proceedings 43.7 (1984): no.-2525.
Source
scival
Published In
Federation Proceedings
Volume
43
Issue
7
Publish Date
1984
Start Page
no.
End Page
2525

Polychlorinated biphenyl toxicity in vitamin E or selenium deficient rats

Authors
Gee, DL; Johnston, T; Bennett, V
MLA Citation
Gee, DL, Johnston, T, and Bennett, V. "Polychlorinated biphenyl toxicity in vitamin E or selenium deficient rats." Federation Proceedings 43.3 (1984): no.-1187.
Source
scival
Published In
Federation Proceedings
Volume
43
Issue
3
Publish Date
1984
Start Page
no.
End Page
1187

Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits.

Brain spectrin tetramer was purified from pig brain membranes in milligram quantities. The tetramer had subunits of Mr = 265,000 (alpha) and Mr = 260,000 (beta), Rs = 21.4 nM, S20,w = 11 S, V = 0.725 ml/g, frictional ratio of 2.9, and calculated molecular weight of 9.7 x 10(5). The subunits were isolated in greater than 95% purity by chromatography on hydroxylapatite in 7 M urea, as described for erythrocyte spectrin (Calvert, R., Bennett, P., Gratzer, W. (1980) Eur. J. Biochem. 107, 355-361). Peptide maps of the subunits revealed few if any common peptides. The subunits were visualized by rotary shadowing as single-stranded flexible rods 100 nm in length with no homodimers by lateral or end association. When the subunits were renatured together, double-stranded tetramers 200 nm in length were formed, as well as higher oligomers. These results indicate that spectrin tetramers are formed by laterally associated alpha,beta dimers attached by head-to-head linkage of each alpha chain with a beta chain. The reassembled subunits regained the ability to increase the low shear viscosity of actin, although isolated alpha or beta subunits were inactive. Hybrid molecules were formed with brain alpha and pig erythrocyte spectrin beta subunit and were visualized as double-stranded rods 100 nm long with no tetramers. 125I-labeled brain alpha chain that was hybridized with erythrocyte beta subunit acquired the ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain alpha chain bound only to beta subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate gels. Affinity-purified antibodies against brain spectrin cross-reacted with both subunits of erythrocyte spectrin and with polypeptides of a similar molecular weight to brain spectrin in membranes of all tissues examined. Cross-reacting polypeptides were localized exclusively in plasma membranes of subcellular fractions from liver. These studies provide strong additional evidence that brain and mammalian erythrocyte spectrin belong to a closely related family of proteins with conserved functions in both subunits.

Authors
Davis, J; Bennett, V
MLA Citation
Davis, J, and Bennett, V. "Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits." J Biol Chem 258.12 (June 25, 1983): 7757-7766.
PMID
6863263
Source
pubmed
Published In
The Journal of biological chemistry
Volume
258
Issue
12
Publish Date
1983
Start Page
7757
End Page
7766

Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes.

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.

Authors
Agre, P; Gardner, K; Bennett, V
MLA Citation
Agre, P, Gardner, K, and Bennett, V. "Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes." J Biol Chem 258.10 (May 25, 1983): 6258-6265.
PMID
6133862
Source
pubmed
Published In
The Journal of biological chemistry
Volume
258
Issue
10
Publish Date
1983
Start Page
6258
End Page
6265

Detection of pulmonary venous flow by pulsed Doppler echocardiography in children.

Parasternal pulsed Doppler echocardiographic examinations of the left atrium were prospectively performed in 14 pediatric patients, aged 2 weeks to 8 years, in order to characterize the left atrial flow pattern in children. None of the patients had clinical or cardiac catheterization evidence (9 of 14 patients) of either mitral regurgitation, right-to-left shunts at the atrial level, or anomalies of pulmonary venous drainage. In all 14 patients, a systolic-diastolic positive velocity pattern could be recorded within the left atrium. Furthermore, this pattern was more readily detected in those children with left-to-right shunts. We conclude that continuous positive velocity patterns are commonly recorded in the left atria of children and probably represent pulmonary venous return. Demonstration of this signal is contingent on proper orientation of the transducer with respect to pulmonary venous flow.

Authors
Pickoff, AS; Bennett, V; Soler, P; Ferrer, PL
MLA Citation
Pickoff, AS, Bennett, V, Soler, P, and Ferrer, PL. "Detection of pulmonary venous flow by pulsed Doppler echocardiography in children." Am Heart J 105.5 (May 1983): 826-829.
PMID
6846126
Source
pubmed
Published In
American Heart Journal
Volume
105
Issue
5
Publish Date
1983
Start Page
826
End Page
829

Spectrin and ankyrin in brain

Further similarity between mammalian erythrocyte spectrin and pig brain spectrin has been demonstrated by (a) formation of hybrid molecules with brain α‐chains and erythrocyte β‐chains and by (b) identification of an ankyrin protein in brain membranes. Hybrid spectrin molecules prepared from brain α‐chains and erythrocyte β‐chains were visualized by low‐angle rotary shadowing as double‐stranded rods (dimers) 100 nM in length. 125 I‐labeled brain α‐chain that was hybridized with erythrocyte β‐subunit acquired ability to bind to ankyrin sites on erythrocyte membranes. 125 I‐labeled brain α‐chain bound only to β‐subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate (SDS) gels. Thus brain spectrin and mammalian erythrocyte spectrin have shared functional sites involved in association of their subunits. Additional evidence for similarity of brain and erythrocyte membranes is the finding of a 210,000 M r membrane protein in brain that cross‐reacts with erythrocyte ankyrin and has a water‐soluble domain of 72,000 M r that is produced by protease digestion. The 72,000 M r domain of brain ankyrin has been isolated by affinity chromatography on erythrocyte spectrin‐Sepharose, and was demonstrated to bind directly to erythrocyte and brain spectrin. The brain 72,000 M r fragment has distinct peptide maps from the erythrocyte 72,000 M r ankyrin fragment and thus is not a result of erythrocyte contamination. Copyright © 1983 Wiley‐Liss, Inc.

Authors
Benett, V; Davis, J
MLA Citation
Benett, V, and Davis, J. "Spectrin and ankyrin in brain." Cell Motility 3.5 (January 1, 1983): 623-633.
Source
scopus
Published In
Cell Motility
Volume
3
Issue
5
Publish Date
1983
Start Page
623
End Page
633
DOI
10.1002/cm.970030527

Spectrin and ankyrin in brain.

Further similarity between mammalian erythrocyte spectrin and pig brain spectrin has been demonstrated by (a) formation of hybrid molecules with brain alpha-chains and erythrocyte beta-chains and by (b) identification of an ankyrin protein in brain membranes. Hybrid spectrin molecules prepared from brain alpha-chains and erythrocyte beta-chains were visualized by low-angle rotary shadowing as double-stranded rods (dimers) 100 nM in length. 125I-labeled brain alpha-chain that was hybridized with erythrocyte beta-subunit acquired ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain alpha-chain bound only to beta-subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate (SDS) gels. Thus brain spectrin and mammalian erythrocyte spectrin have shared functional sites involved in association of their subunits. Additional evidence for similarity of brain and erythrocyte membranes is the finding of a 210,000 Mr membrane protein in brain that cross-reacts with erythrocyte ankyrin and has a water-soluble domain of 72,000 Mr that is produced by protease digestion. The 72,000 Mr domain of brain ankyrin has been isolated by affinity chromatography on erythrocyte spectrin-Sepharose, and was demonstrated to bind directly to erythrocyte and brain spectrin. The brain 72,000 Mr fragment has distinct peptide maps from the erythrocyte 72,000 Mr ankyrin fragment and thus is not a result of erythrocyte contamination.

Authors
Bennett, V; Davis, J
MLA Citation
Bennett, V, and Davis, J. "Spectrin and ankyrin in brain." Cell Motil 3.5-6 (1983): 623-633.
PMID
6229335
Source
pubmed
Published In
Cell Motility
Volume
3
Issue
5-6
Publish Date
1983
Start Page
623
End Page
633

Proteins involved in membrane--cytoskeleton association in human erythrocytes: spectrin, ankyrin, and band 3.

Authors
Bennett, V
MLA Citation
Bennett, V. "Proteins involved in membrane--cytoskeleton association in human erythrocytes: spectrin, ankyrin, and band 3." Methods Enzymol 96 (1983): 313-324.
PMID
6228705
Source
pubmed
Published In
Methods in Enzymology
Volume
96
Publish Date
1983
Start Page
313
End Page
324

Immunoreactive forms of erythrocyte spectrin and ankyrin in brain.

Authors
Bennett, V; Davis, J; Fowler, WE
MLA Citation
Bennett, V, Davis, J, and Fowler, WE. "Immunoreactive forms of erythrocyte spectrin and ankyrin in brain." Philos Trans R Soc Lond B Biol Sci 299.1095 (November 4, 1982): 301-312.
PMID
6129664
Source
pubmed
Published In
Philosophical Transactions B
Volume
299
Issue
1095
Publish Date
1982
Start Page
301
End Page
312

Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin.

An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes. This protein co-sediments with and cross-links actin filaments, associates with spectrin-binding sites on erythrocyte membranes, and has been visualized by rotary shadowing as an extended, flexible rod. The brain spectrin comprises 3% of the total membrane protein, and may have a major role in mediating linkage of actin to membranes.

Authors
Bennett, V; Davis, J; Fowler, WE
MLA Citation
Bennett, V, Davis, J, and Fowler, WE. "Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin." Nature 299.5879 (September 9, 1982): 126-131.
PMID
7110333
Source
pubmed
Published In
Nature
Volume
299
Issue
5879
Publish Date
1982
Start Page
126
End Page
131

Isolation of an ankyrin-band 3 oligomer from human erythrocyte membranes.

Authors
Bennett, V
MLA Citation
Bennett, V. "Isolation of an ankyrin-band 3 oligomer from human erythrocyte membranes." Biochim Biophys Acta 689.3 (August 12, 1982): 475-484.
PMID
6215064
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
689
Issue
3
Publish Date
1982
Start Page
475
End Page
484

Microtubule-associated protein 2, a microtubule-associated protein from brain, is immunologically related to the alpha subunit of erythrocyte spectrin.

Authors
Davis, J; Bennett, V
MLA Citation
Davis, J, and Bennett, V. "Microtubule-associated protein 2, a microtubule-associated protein from brain, is immunologically related to the alpha subunit of erythrocyte spectrin." J Biol Chem 257.10 (May 25, 1982): 5816-5820.
PMID
6175631
Source
pubmed
Published In
The Journal of biological chemistry
Volume
257
Issue
10
Publish Date
1982
Start Page
5816
End Page
5820

Deficient red-cell spectrin in severe, recessively inherited spherocytosis.

Authors
Agre, P; Orringer, EP; Bennett, V
MLA Citation
Agre, P, Orringer, EP, and Bennett, V. "Deficient red-cell spectrin in severe, recessively inherited spherocytosis." N Engl J Med 306.19 (May 13, 1982): 1155-1161.
PMID
7070419
Source
pubmed
Published In
The New England journal of medicine
Volume
306
Issue
19
Publish Date
1982
Start Page
1155
End Page
1161
DOI
10.1056/NEJM198205133061906

The molecular basis for membrane - cytoskeleton association in human erythrocytes.

Spectrin, the major cytoskeletal protein in erythrocytes, is localized on the inner membrane surface in association with membrane-spanning glycoproteins and with intramembrane particles. The presence of a specific, high-affinity protein binding site for spectrin on the cytoplasmic surface of the membrane has been established by measurement of reassociation of spectrin with spectrin-depleted inside-out vesicles. A 72,000 Mr proteolytic fragment of this attachment protein has been purified, which bound to spectrin in solution and competed for reassociation of spectrin with vesicles. A 215,000 Mr polypeptide has been identified as the precursor of the spectrin-binding fragment. The membrane attachment protein for spectrin was named ankyrin, and has been purified and characterized. Ankyrin has been demonstrated to be tightly associated in detergent extracts of vesicles with band 3, a major membrane-spanning polypeptide, and to bind directly to a proteolytic fragment derived from the cytoplasmic domain of band 3. Ankyrin is thus an example of a protein that directly links a cytoplasmic structural protein to an integral membrane protein. The organization of the erythrocyte membrane has implications for more complex cell types since immunoreactive forms of ankyrin distinct from myosin or filamin have been detected by radioimmunoassay in a variety of cells and tissues. Indirect immunofluorescent staining of cultured cells reveals immunoreactive forms of ankyrin in a cytoplasmic meshwork and in a punctate distribution over nuclei. The staining changes dramatically during mitosis, with concentration of stain at the spindle poles in metaphase and intense staining of the cleavage furrow during cytokinesis.

Authors
Bennett, V
MLA Citation
Bennett, V. "The molecular basis for membrane - cytoskeleton association in human erythrocytes." J Cell Biochem 18.1 (1982): 49-65. (Review)
PMID
6461664
Source
pubmed
Published In
Journal of Cellular Biochemistry
Volume
18
Issue
1
Publish Date
1982
Start Page
49
End Page
65
DOI
10.1002/jcb.1982.240180106

Immunoreactive forms of human erythrocyte ankyrin are localized in mitotic structures in cultured cells and are associated with microtubules in brain.

Authors
Bennett, V; Davis, J
MLA Citation
Bennett, V, and Davis, J. "Immunoreactive forms of human erythrocyte ankyrin are localized in mitotic structures in cultured cells and are associated with microtubules in brain." Cold Spring Harb Symp Quant Biol 46 Pt 2 (1982): 647-657.
PMID
6213353
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
46 Pt 2
Publish Date
1982
Start Page
647
End Page
657

Isolation of an ankyrin-band 3 oligomer from human erythrocyte membranes

A cytoskeleton-associated population of band 3 has been isolated in milligram quantities from human erythrocyte membranes as a stable complex with ankyrin. The major population of band 3 (free band 3) was solubilized from ghosts with 0.1 M KCl/Triton X-100. A detergent-insoluble assembly of proteins (cytoskeletons) contained 10-15% of the band 3, as well as ankyrin, spectrin, band 4.1, actin and other minor polypeptides. The remaining band 3 and ankyrin were extracted in a 1:1 molar ratio from the cytoskeletons with 1 M KCl/Triton X-100, and were copurified with the same 1:1 stoichiometry during DEAE-chromatography, and gel filtration. Free band 3 was isolated by the same procedures, and was clearly resolved from ankyrin-associated band 3 on DEAE-chromatography and gel filtration. Direct evidence that ankyrin and band 3 were associated in a complex was provided by immunoprecipitation with anti-ankyrin IgG of band 3 from the native complex, but not of free band 3 or after denaturation of the complex. Ankyrin-associated band 3 contained a reactive site for H2DIDS (the dihydroanalog of 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid) and thus has an anion transport site. Comparison of 125I-labeled α-chymotryptic peptide fragments of free band 3 and ankyrin-associated band 3 revealed extensive homology with 28 out of 30 identical fragments. The ankyrin-band 3 oligomer is arranged as an αβ dimer with one polypeptide chain of each component, based on the molecular weight calculated from hydrodynamic parameters in dilute solution. Free band 3 behaved under the same conditions as a homodimer. Ankyrin-associated band 3 was capable of band 3 dimerization at concentrations of 1-3 μM, since chemical cross-linking of the oligomer with Cu2+/ophenanthroline produced a 190 000 Mr band 3 dimer on SDS gels. © 1982.

Authors
Bennett, V
MLA Citation
Bennett, V. "Isolation of an ankyrin-band 3 oligomer from human erythrocyte membranes." BBA - Biomembranes 689.3 (1982): 475-484.
Source
scival
Published In
BBA - Biomembranes
Volume
689
Issue
3
Publish Date
1982
Start Page
475
End Page
484

A molecular defect in two families with hemolytic poikilocytic anemia: reduction of high affinity membrane binding sites for ankyrin.

Patients from two families with chronic hemolytic anemia have been studied. The erythrocytes are very fragile and appear microcytic with a great variety of shapes. Clinical evaluation failed to identify traditionally recognized causes of hemolysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed no significant abnormality of the major polypeptide bands. Erythrocytes spectrin-ankyrin and ankyrin-membrane interactions were analyzed with 125I-labeled spectrin, 125I-labeled ankyrin, and inside-out vesicles. Patients' vesicles bound 125I-spectrin normally. Likewise, patients' spectrin and ankyrin competed normally for the binding sites on control membranes. None of the individual components appeared to have abnormal thermal sensitivity. Ankyrin-stripped, inside-out vesicles prepared from the patients bound less 125I-ankyrin than did vesicles prepared from normals (P less than 0.05 for all corresponding points in the high-affinity region). Scatchard analysis showed the most significant abnormality to be a 50% reduction in the high affinity ankyrin binding sites. Similar experiments were performed with blood from patients with spherocytosis and splenectomized controls, but no abnormalities were detected. The water soluble 43,000-dalton fragments of band 3 (the high-affinity ankyrin binding sites) were prepared from one of the patients and competed normally for 125I-ankyrin binding in solution. This suggests that the primary structural defect is a reduction in the number of high affinity membrane binding sites for ankyrin, and is consistent with an abnormal organization of band 3 in the membrane.

Authors
Agre, P; Orringer, EP; Chui, DH; Bennett, V
MLA Citation
Agre, P, Orringer, EP, Chui, DH, and Bennett, V. "A molecular defect in two families with hemolytic poikilocytic anemia: reduction of high affinity membrane binding sites for ankyrin." J Clin Invest 68.6 (December 1981): 1566-1576.
PMID
6459341
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
68
Issue
6
Publish Date
1981
Start Page
1566
End Page
1576

Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain.

Human erythrocyte ankyrin, the membrane attachment protein for spectrin, has been detected by radioimmunoassay in a variety of cells and tissues. This report identifies polypeptides crossreacting with ankyrin in brain and HeLa cells and demonstrates that one function of these ankyrin analogues involves association with microtubules. Ankyrin immunoreactivity was localized by indirect immunofluorescence in a colchicine- and detergent-sensitive cytoplasmic meshwork in interphase cells. There also was specific nuclear staining, localized in a bright spots, which was displaced entirely by ankyrin or by high molecular weight microtubule-associated proteins (MAPs) from brain. In dividing cells, the punctate nuclear staining and the meshwork disappeared. Fluorescence was localized at the spindle pole during metaphase and was redistributed to the cleavage furrow in later stages of mitosis. An immunoreactive Mr 370,000 polypeptide comigrating with MAP1 was identified in brain extracts and copolymerized with microtubules through repeated cycles of polymerization and depolymerization. Finally, erythrocyte ankyrin associated with microtubules prepared from pure tubulin, and this binding was displaced by brain MAPs.

Authors
Bennett, V; Davis, J
MLA Citation
Bennett, V, and Davis, J. "Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain." Proc Natl Acad Sci U S A 78.12 (December 1981): 7550-7554.
PMID
6461004
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
78
Issue
12
Publish Date
1981
Start Page
7550
End Page
7554

Proteolytic domains of the epidermal growth factor receptor of human placenta.

Microsomal membranes form human placenta, which bind 5-20 pmol of 125I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with 125I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.

Authors
O'Keefe, EJ; Battin, T; Bennett, V
MLA Citation
O'Keefe, EJ, Battin, T, and Bennett, V. "Proteolytic domains of the epidermal growth factor receptor of human placenta." J Supramol Struct Cell Biochem 15.1 (1981): 15-27.
PMID
6265704
Source
pubmed
Published In
Journal of Supramolecular and Cellular Biochemistry
Volume
15
Issue
1
Publish Date
1981
Start Page
15
End Page
27
DOI
10.1002/jsscb.1981.380150103

Erythrocyte ankyrin: Immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain

Authors
Bennett, V; Davis, J
MLA Citation
Bennett, V, and Davis, J. "Erythrocyte ankyrin: Immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain." Proceedings of the National Academy of Sciences of the United States of America 78.12 II (1981): 7550-7554.
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
78
Issue
12 II
Publish Date
1981
Start Page
7550
End Page
7554

Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane.

Authors
Bennett, V; Stenbuck, PJ
MLA Citation
Bennett, V, and Stenbuck, PJ. "Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane." J Biol Chem 255.13 (July 10, 1980): 6424-6432.
PMID
6446557
Source
pubmed
Published In
The Journal of biological chemistry
Volume
255
Issue
13
Publish Date
1980
Start Page
6424
End Page
6432

Human erythrocyte ankyrin. Purification and properties.

Authors
Bennett, V; Stenbuck, PJ
MLA Citation
Bennett, V, and Stenbuck, PJ. "Human erythrocyte ankyrin. Purification and properties." J Biol Chem 255.6 (March 25, 1980): 2540-2548.
PMID
6444633
Source
pubmed
Published In
The Journal of biological chemistry
Volume
255
Issue
6
Publish Date
1980
Start Page
2540
End Page
2548

Pulsed Doppler echocardiographic findings in total anomalous pulmonary venous drainage to the coronary sinus.

The pulsed doppler echocardiographic (PDE) findings in a premature cyanotic infant with total anomalous pulmonary venous return to the coronary sinus are reported. Features that suggested the diagnosis of total anomalous pulmonary venous return were 1) an area of systolic-diastolic turbulent flow posterior to the apparent left atrial border, which was interpreted as representing the convergence of pulmonary venous return to a common pulmonary vein, and 2) systolic and diastolic turbulent flow within the right atrium suggesting pulmonary venous return through the coronary sinus. We suggest that PDE should be included as part of the evaluation of cyanotic infants in whom the diagnosis of total anomalous pulmonary venous return is considered.

Authors
Pickoff, AS; Sequeira, R; Ferrer, PL; Tamer, D; Bennett, V; Fojaco, R; Gelband, H
MLA Citation
Pickoff, AS, Sequeira, R, Ferrer, PL, Tamer, D, Bennett, V, Fojaco, R, and Gelband, H. "Pulsed Doppler echocardiographic findings in total anomalous pulmonary venous drainage to the coronary sinus." Cathet Cardiovasc Diagn 6.3 (1980): 247-254.
PMID
7448856
Source
pubmed
Published In
Catheterization and Cardiovascular Diagnosis
Volume
6
Issue
3
Publish Date
1980
Start Page
247
End Page
254

Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.

Authors
Bennett, V
MLA Citation
Bennett, V. "Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues." Nature 281.5732 (October 18, 1979): 597-599.
PMID
492324
Source
pubmed
Published In
Nature
Volume
281
Issue
5732
Publish Date
1979
Start Page
597
End Page
599

The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes.

Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10--15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.

Authors
Bennett, V; Stenbuck, PJ
MLA Citation
Bennett, V, and Stenbuck, PJ. "The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes." Nature 280.5722 (August 9, 1979): 468-473.
PMID
379653
Source
pubmed
Published In
Nature
Volume
280
Issue
5722
Publish Date
1979
Start Page
468
End Page
473

Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin.

Authors
Bennett, V; Stenbuck, PJ
MLA Citation
Bennett, V, and Stenbuck, PJ. "Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin." J Biol Chem 254.7 (April 10, 1979): 2533-2541.
PMID
372182
Source
pubmed
Published In
The Journal of biological chemistry
Volume
254
Issue
7
Publish Date
1979
Start Page
2533
End Page
2541

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of 'leaky' erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (>70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V. Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of 32P-spectrin with inside-out vesicles with a K(i) of 10-7M, and causes rapid dissociation of 32P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.

Authors
Fowler, V; Bennett, V
MLA Citation
Fowler, V, and Bennett, V. "Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins." Progress in Clinical and Biological Research No 30 (1979): 25-31.
Source
scival
Published In
Progress in Clinical and Biological Research
Volume
No 30
Publish Date
1979
Start Page
25
End Page
31

Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin.

Authors
Bennett, V
MLA Citation
Bennett, V. "Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin." J Biol Chem 253.7 (April 10, 1978): 2292-2299.
PMID
632270
Source
pubmed
Published In
The Journal of biological chemistry
Volume
253
Issue
7
Publish Date
1978
Start Page
2292
End Page
2299

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Authors
Fowler, V; Bennett, V
MLA Citation
Fowler, V, and Bennett, V. "Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins." Journal of Supramolecular and Cellular Biochemistry 8.2 (1978): 215-221.
Source
scival
Published In
Journal of Supramolecular and Cellular Biochemistry
Volume
8
Issue
2
Publish Date
1978
Start Page
215
End Page
221

Human erythrocyte spectrin: phosphorylation in intact cells and purification of the 32P-labeled protein in a non-aggregated state.

Authors
Bennett, V
MLA Citation
Bennett, V. "Human erythrocyte spectrin: phosphorylation in intact cells and purification of the 32P-labeled protein in a non-aggregated state." Life Sci 21.3 (August 1, 1977): 433-440.
PMID
895375
Source
pubmed
Published In
Life Sciences
Volume
21
Issue
3
Publish Date
1977
Start Page
433
End Page
440

Topographic separation of adenylate cyclase and hormone receptors in the plasma membrane of toad erythrocyte ghosts.

Brief sonication of whole erythrocyte plasma membranes (ghosts) from toads at 4 degrees does not inactivate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] or destroy the receptor binding properties of hydroxybenzylpindolol or insulin. The hormonal (but not the fluoride-induced) stimulation of this enzyme is, however, lost. Fractionation of the small, resealed membrane fragments (vesicles) on discontinuous sucrose gradients results in the separation of vesicle populations differing grossly in size and protein composition. In addition, the distribution of the beta-adrenergic receptor, an insulin binding site, and adenylate cyclase among these vesicles fractions differs. The pattern of distribution of these functional structures can be altered differentially by manipulations of the ghosts before sonication. For example, brief preincubation with isoproterenol leads to a change in the relative distribution of beta-receptor (but not adenylate cyclase) among the various vesicle fractions; this effect is not obtained with beta-receptor antagonists, which block the isoproterenol effect. Exposure of the ghosts to different temperatures, changes in the divalent cation composition of the medium, or the addition of ATP also leads to changes in the distribution of surface markers of the subsequently formed vesicles. The results indicate gross asymmetries in the distribution of protein components within the plane of the membrane and raise important questions regarding the manner whereby functionally related and coupled components, such as hormone receptors and adenylate cyclase, interact.

Authors
Sahyoun, N; Hollenberg, MD; Bennett, V; Cuatrecasas, P
MLA Citation
Sahyoun, N, Hollenberg, MD, Bennett, V, and Cuatrecasas, P. "Topographic separation of adenylate cyclase and hormone receptors in the plasma membrane of toad erythrocyte ghosts." Proc Natl Acad Sci U S A 74.7 (July 1977): 2860-2864.
PMID
197522
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
74
Issue
7
Publish Date
1977
Start Page
2860
End Page
2864

Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.

A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.

Authors
Bennett, V; Branton, D
MLA Citation
Bennett, V, and Branton, D. "Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin." J Biol Chem 252.8 (April 25, 1977): 2753-2763.
PMID
15998
Source
pubmed
Published In
The Journal of biological chemistry
Volume
252
Issue
8
Publish Date
1977
Start Page
2753
End Page
2763

Kinetics of irreversible activation of adenylate cyclase of fat cell membranes by phosphonium and phosphoramidate analogs of gtp1.

The ability of guanylylimidodiphosphate (GMP=P(NH)P) and guanylylmethylenediphosphonate (GMP-P(CH2)P) to activate adenylate cyclase activity has been studied by incubating these analogs with fat cell membranes followed by thorough washing of the membranes before assay of enzyme activity. GMP-P(NH)P is hydrolyzed by membrane preparations from several tissues. A pyruvate kinase regenerating system maintains the concentration of GMP-P(NH)P and thereby augments the ability of suboptimal concentrations of GMP-P(NH)P to activate adenylate cyclase. GTP inhibits activation of fat cell membrane adenylate cyclase by GMP-P(NH)P but this inhibition is overcome by time. This is consistent with the virtually irreversible nature of the GMP-P(NH)P activation, and with the inability of GTP to reverse the stimulated state of the enzyme. Although the initial rate of enzyme activation is highly dependent on the concentration of GMP-P(NH)P, with increasing times of incubation nearly the same maximal extent of activation is seen over a wide range of concentrations. Thus, it is not possible to estimate true affinity constants (at equilibrium) for GMP-P(NH)P, as anticipated from the virtually irreversible character of the activation process.

Authors
Jacobs, S; Bennett, V; Cuatrecasas, P
MLA Citation
Jacobs, S, Bennett, V, and Cuatrecasas, P. "Kinetics of irreversible activation of adenylate cyclase of fat cell membranes by phosphonium and phosphoramidate analogs of gtp1." J Cyclic Nucleotide Res 2.4 (July 1976): 205-223.
PMID
184121
Source
pubmed
Published In
Journal of Cyclic Nucleotide Research
Volume
2
Issue
4
Publish Date
1976
Start Page
205
End Page
223

Irreversible activation of adenylate cyclase of toad erythrocyte plasma membrane by 5'-guanylylimidodiphosphate.

The irreversible activation of adenylate-cyclase by 5'guanylylimidodiphosphate, a phosphoramidate analog of 5'GTP, has been examined in toad (Bufus marinus) plasma membranes using the technique of preincubating the membranes with the nucleotide under various controlled conditions followed by washing and subsequent assay of enzyme activity. Activation of adenylate cyclase by Gpp(NH)p, but not GTP, is essentially permanent and persists following extensive washing, prolonged incubation at 30 degrees C in the absence of the nucleotide, and after dissolution of the membranes with Lubrol PX. (-)-Isoproterenol increases the activation observed with maximal concentrations of Gpp(NH)p from eight- to 10-fold (in the absence of hormone) to 50- to 100-fold; final activities as high as 10-15 nmoles of cyclic AMP per min per mg protein are achieved. The activated state obtained with isoproterenol and Gpp(NH)p is also permanent and is not inhibited by propranolol. The synergism between Gpp(NH)p and hormone requires the simultaneous presence of these compounds, and the time-dependent enhancement of activation with (-)-isoproterenol may be interrupted by addition of propranolol. The stimulation is slow, and may proceed for as long as 45 min at 30 degrees C in the presence of maximal concentrations of Gpp(NH)p and (-)-isoproterenol. Very little activation occurs at 0 degrees C. The time course of activation at 30 degrees C exhibits an accelerating phase lasting from 5 to 30 min when Gpp(NH)p is added directly during assay of cyclase activity or when the membranes are preincubated for various times and washed prior to assay for a fixed time. The lag period occurs in the presence and absence of (-)-isoproterenol, although the rate of increase in velocity is greater with hormone. The length of the accelerating phase decreases with increasing concentrations of Gpp(NH)p, although it is still evident with maximal levels of Gpp(NH)p and hormone. However, prewarming the membranes at 30 degrees C for 10 min in the absence of Gpp(NH)p or (-)-isoproterenol results in an immediate onset of linear activation at a rate which is achieved in untreated membranes only after about 10 min. The events occurring during prewarming at 30 degrees C are readily reversible since chilling the warmed membranes to 0 degrees C results in a time course of activation identical to that of membranes maintained at 0 degrees C until addition of Gpp(NH)p. Activation is proportional to the concentration of Gpp(NH)p within the range of 10(-8) to 10(-4) mM. The apparent affinity for Gpp(NH)p increases with increasing time of incubation. The primary effect of increasing the concentration of Gpp(NH)p is to decrease the time required to obtain a maximal rate of activation. The possible relevance of these findings to the mechanism of action of Gpp(NH)p, adenylate cyclase and hormones is discussed within the context of current views of biological membranes which recognize the lateral mobility of membrane molecules.

Authors
Bennett, V; Cuatrecasas, P
MLA Citation
Bennett, V, and Cuatrecasas, P. "Irreversible activation of adenylate cyclase of toad erythrocyte plasma membrane by 5'-guanylylimidodiphosphate." J Membr Biol 27.3 (1976): 207-232.
PMID
820859
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
27
Issue
3
Publish Date
1976
Start Page
207
End Page
232

Structure and function of cholera toxin and hormone receptors.

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active 125I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside GM1. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyne to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the "active" subunit of the toxin can specifically absorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergent-solubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct perturbation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.

Authors
Bennett, V; Craig, S; Hollenberg, MD; O'Keefe, E; Sahyoun, N; Cuatrecasas, P
MLA Citation
Bennett, V, Craig, S, Hollenberg, MD, O'Keefe, E, Sahyoun, N, and Cuatrecasas, P. "Structure and function of cholera toxin and hormone receptors." J Supramol Struct 4.1 (1976): 99-120.
PMID
176537
Source
pubmed
Published In
Journal of supramolecular structure
Volume
4
Issue
1
Publish Date
1976
Start Page
99
End Page
120
DOI
10.1002/jss.400040110

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones.

The influence of Vibrio cholerae enterotoxin (choleragen) on the response of adenylate cyclase to hormones and GTP, and on the binding of 125I-labeled glucagon to membranes, has been examined primarily in rat adipocytes, but also in guinea pig ileal mucosa and rat liver. Incubation of fat cells with choleragen converts adenylate cyclase to a GTP-responsive state; (-)-isoproterenol has a similar effect when added directly to membranes. Choleragen also increases by two- to fivefold the apparent affinity of (-)-isoproterenol, ACTH, glucagon, and vasoactive intestinal polypeptide for the activation of adenylate cyclase. This effect on vasoactive intestinal polypeptide action is also seen with the enzyme of guinea pig ileal mucosa; the toxin-induced sensitivity to VIP may be relevant in the pathogenesis of cholera diarrhea. The apparent affinity of binding of 125I-labeled glucagon is increased about 1.5- to twofold in choleragen-treated liver and fat cell membranes. The effects of choleragen on the response of adenylate cyclase to hormones are independent of protein synthesis, and they are not simply a consequence to protracted stimulation of the enzyme in vivo or during preparation of the membranes. Activation of cyclase in rat erythrocytes by choleragen is not impaired by agents which disrupt microtubules or microfilaments, and it is still observed in cultured fibroblasts after completely suppressing protein synthesis with diphtheria toxin. Choleragen does not interact directly with hormone receptor sites. Simple occupation of the choleragen binding sites with the analog, choleragenoid, does not lead to any of the biological effects of the toxin.

Authors
Bennett, V; Mong, L; Cuatrecasas, P
MLA Citation
Bennett, V, Mong, L, and Cuatrecasas, P. "Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones." J Membr Biol 24.2 (November 7, 1975): 107-129.
PMID
172636
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
24
Issue
2
Publish Date
1975
Start Page
107
End Page
129

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin.

The kinetics and properties of the activation of adenylate cyclase by cholera enterotoxin have been examined primarily in toad erythrocytes, but also in avian erythrocytes, rat fat cells and cultured melanoma cells. When cholera toxin is incubated with intact cells it stimulates adenylate cyclase activity, as measured in the subsequently isolated plasma membranes, according to a triphasic time course. This consists of a true lag period of about 30 min, followed by a stage of exponentially increasing adenylate cyclase activity which continues for 110 to 130 min, and finally a period of slow activation which may extend as long as 30 hr in cultured melanoma cells. The progressive activation of adenylate cyclase activity by cholera toxin is interrupted by cell lysis; continued incubation of the isolated membranes under nearly identical conditions does not lead to further activation of the enzyme. The delay in the action of the toxin is not grossly dependent of the number of toxin-receptor (GM1 ganglioside) complexes, and is still seen upon adding a second dose of toxin to partially stimulated cells. Direct measurements indicate negligible intracellular levels of biologically active radioiodinated toxin in either a soluble or a nuclear-bound form. The effects are not prevented by Actinomycin D (20 mug/ml), uromycin (30 mug/ml), cycloheximide (30 mug/ml), sodium fluoride (10 mM) or sodium azide (1 mM); KCN, however, almost completely prevents the action of cholera toxin. The action of the toxin is temperature dependent, occurring at very slow or negligible rates below certain critical temperatures, the values of which depend on the specific animal species. Thetransition for toad erythrocytes occurs at 15 to 17 degrees C, while rat adipocytes and turkey erythrocytes demonstrate a discontinuity at 26 to 30 degrees C. The temperature effects are evident during the lag period as well as during the exponential phase of activation. The rate of decay of the stimulated adenylate cyclase activity of cultured melanoma cells indicates a half-time of about 36 hr. The rate of exponentially increasing activity and extent of enzyme activation are related to the number of bound toxin molecules according to a Langmuir adsorption isotherm and are half-maximal when about 2000 molecules of toxin are bound per cell. It is proposed that initially cholera toxin binds ineffectively, but that it is converted to an active form during the lag phase. This process may involve lateral motion of toxin-GM1 ganglioside complex within the plane of the membrane. The kinetics of adenylate cyclase activation are consistent with the possibility that during the exponential phase a bimolecular association is proceeding between the active form of the cholera toxin and some other membrane component. The possibility is considered that the cholera toxin molecule may bind directly to adenylate cyclase. These considerations may prove useful in understanding the possible interactions of active hormone-receptor complexes with adenylate cyclase in cell membranes.

Authors
Bennett, V; Cuatrecasas, P
MLA Citation
Bennett, V, and Cuatrecasas, P. "Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin." J Membr Biol 22.1 (June 3, 1975): 29-52.
PMID
805248
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
22
Issue
1
Publish Date
1975
Start Page
29
End Page
52

Mechanism of action of Vibrio cholerae enterotoxin. Effects on adenylate cyclase of toad and rat erythrocyte plasma membranes.

The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30 degrees C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximate Km for ATP, Mg++ increases from about 1.8 to 3.4 mMin the toxin-stimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg++ concentrations, from 20 percent at low levels (0.2 mM) to 500 percent at high concentrations (greater than 3 mM). Addition of GTP, Mg++ (0.2 mM) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation of Vmax. GTP enhances the toxin-treated enzyme activity two- to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6 mM ATP and 5 mM MgC12 the apparent K alpha for GTP, Mg++ is 5 to 10 muM. The control(unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide, 5'-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg++, decreasing the apparent K alpha from about 25 to 5 mM. (minus)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparent K alpha for free metal. Sodium fluoride, which causes a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparent K alpha for Mg++; moreover,it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three to fivefold stimulation by toxin becomes a 30 to 60 percent inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (minus)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.

Authors
Bennett, V; Cuatrecasas, P
MLA Citation
Bennett, V, and Cuatrecasas, P. "Mechanism of action of Vibrio cholerae enterotoxin. Effects on adenylate cyclase of toad and rat erythrocyte plasma membranes." J Membr Biol 22.1 (June 3, 1975): 1-28.
PMID
805247
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
22
Issue
1
Publish Date
1975
Start Page
1
End Page
28

Activation of adenylate cyclase by phosphoramidate and phosphonate analogs of GTP: possible role of covalent enzyme-substrate intermediates in the mechanism of hormonal activation.

Incubation of rat fat pad membranes with 5-guanylyliminodiphosphonate [Gpp-(NH)p] and 5-guanylylmethylenediphosphonate [Gpp(CH2)p], but not GTP (with or without hormones), at 24 degrees or 30 degrees (but not at 4 degrees) greatly stimulates adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] measured after thoroughly washing the membranes. The rate of activation is relatively slow, even with very high (and saturating) concentrations of the analogs. Binding alone appears to be insufficient for activation. Hormones (catecholamines, glucagon) increase the rate but not the extent of activation, even when saturating analog concentrations are used. The dependence on analog concentration (apparent Km) varies with the time of incubation. GTP and very high concentrations of ATP inhibit the activation by Gpp(NH)p, but this effect is dependent on the length of incubation and can be overcome with time. The activated state is not reversed upon incubation of the washed membranes at 30 degrees, even in the presence of GTP, or by solubilization with nonionic detergents. Also, Gpp(NH)p can directly stimulate the control, solubilized enzyme. The activated state of the solubilized enzyme persists upon specific adsorption to and subsequent elution from an organomercurial-agarose column. It is suggested that after forming reversible Michaelis complexes of relatively low affinity, these analogs may react irreversibly with the GTP regulatory site of the enzyme, perhaps forming p(NH)p- and p(CH2)p-covalent enzyme intermediates which capture the activated state of the enzyme. GTP, after binding, may normally activate the enzyme by forming a "labile" pyrophosphoryl enzyme intermediate, and hormone receptors may function to increase the rate of formation (and thus concentration) of this active state of the enzyme.

Authors
Cuatrecasas, P; Jacobs, S; Bennett, V
MLA Citation
Cuatrecasas, P, Jacobs, S, and Bennett, V. "Activation of adenylate cyclase by phosphoramidate and phosphonate analogs of GTP: possible role of covalent enzyme-substrate intermediates in the mechanism of hormonal activation." Proc Natl Acad Sci U S A 72.5 (May 1975): 1739-1743.
PMID
1057166
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
72
Issue
5
Publish Date
1975
Start Page
1739
End Page
1743

Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin.

Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 10--9 M to 10--10 M) of a highly radioactive (125-I, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 125-I-Labeled insulin, cholera toxin and the plant lictins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Plasma membranes are labeled before homogenization by incubating intact cells briefly at 24 degrees or 4 degrees, or by very brief in situ perfusion of the organ, with the 125-I-Labeled marker. After removing the free 125-I-labeled ligand from the medium by washing (at 4 degrees), the membrane-marker complex remains intact over prolonged (days) periods of time at 4 degrees. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. When desired, the complex can be dissociated deliberately by manipulating experimental conditions such as temperature or by adding specific simple sugars. The most generally suitable marker appears to be WGA. At least in certain tissues (e. g. fat cells) labeling of the plasma membrane with 125-I-WGA and 125-I-isnulin can be performed equally well and selectively in homogenates as in the intact cell. 125-I-Cholera toxin cannot be used in homogenates because of significant binding to nuclei. The use of 125-I-labeled WGA as a specific plasma membrane marker is illustrated in following the course of fractionations, and in quantitating the yield and purity, of plasma membranes from fat cells, lymphocytes, and liver. The results are compared with simultaneous measurements of the plasma membrane enzyme "markers," ATPase, 5-nucleotidase, and basal as well as hormone-stimulated adenylate cyclase activities. The fractionation of liver plasma membranes by aqueous dextran-polyethylene glycol two-phase polymer systems and by conventional differential centrifugation procedures arealso quantitated with the marker, 125I-WGA. Substantial quantities of plasma membrane material are no recovered in the interphase of the two-phase polymer system. Conventional liver fractionation procedures which retain, for further purification, only the readily sedimented pellet (2000 times g, 15 min) discard a very large (at least 70%) questenal hy

Authors
Chang, KJ; Bennett, V; Cuatrecasas, P
MLA Citation
Chang, KJ, Bennett, V, and Cuatrecasas, P. "Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin." J Biol Chem 250.2 (January 25, 1975): 488-500.
PMID
163229
Source
pubmed
Published In
The Journal of biological chemistry
Volume
250
Issue
2
Publish Date
1975
Start Page
488
End Page
500

Mechanism of action of cholera toxin and the mobile receptor theory of hormone receptor-adenylate cyclase interactions.

Rat liver membrane adenylate cyclase (EC 4.6.1.1) that has been stimulated more than 10-fold by cholera toxin (choleragen) has a 3-fold greater sensitivity to stimulation by glucagon. Choleragen similarly increases the sensitivity of cyclase to other peptide (ACTH, vasoactive intestinal polypeptide) and nonpeptide (catecholamines) hormones in this and other tissues. The rate of 125I-labeled glucagon-membrane dissociation is decreased about 2-fold in toxin-treated liver membranes. Toxin-activated cyclase activity of fat cell membranes is retained upon solubilization with Lubrol PX. Provided 125I-labeled choleragen is first incubated with cells under conditions resulting in enzyme activation, the solubilized cyclase activity migrates with a component of 125I-labeled choleragen on gel filtration chromatography. Agarose derivatives containing the "active" subunit (molecular weight 36,000) of the toxin can specifically adsorb solubilized adenylate cyclase. Toxin-stimulated cyclase can be immunoprecipitated with antitoxin or anti-"active" subunit antibodies. There is a large excess of membrane receptors (ganglioside GM1) which, with the use of choleragenoid, can be shown to be functionally equivalent with respect to cyclase activation. Choleragenoid, an inactive competitive antagonist of toxin binding, can occupy and block a large proportion of toxin receptors without affecting toxin activity. A scheme of toxin action is proposed that involves lateral membrane diffusion of the initially inactive toxin-receptor complex with subsequent direct interaction with and modulation of adenylate cyclase. The basic features of this scheme may be pertinent to the mechanisms by which hormone receptors normally modulate adenylate cyclase.

Authors
Bennett, V; O'Keefe, E; Cuatrecasaş, P
MLA Citation
Bennett, V, O'Keefe, E, and Cuatrecasaş, P. "Mechanism of action of cholera toxin and the mobile receptor theory of hormone receptor-adenylate cyclase interactions." Proc Natl Acad Sci U S A 72.1 (January 1975): 33-37.
PMID
164020
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
72
Issue
1
Publish Date
1975
Start Page
33
End Page
37

Hormone receptor complexes and their modulation of membrane function.

Authors
Cuatrecasas, P; Hollenberg, MD; Chang, KJ; Bennett, V
MLA Citation
Cuatrecasas, P, Hollenberg, MD, Chang, KJ, and Bennett, V. "Hormone receptor complexes and their modulation of membrane function." Recent Prog Horm Res 31 (1975): 37-94. (Review)
PMID
172994
Source
pubmed
Published In
Recent progress in hormone research
Volume
31
Publish Date
1975
Start Page
37
End Page
94

Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP.

The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.

Authors
Cuatrecasas, P; Bennett, V; Jacobs, S
MLA Citation
Cuatrecasas, P, Bennett, V, and Jacobs, S. "Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP." J Membr Biol 23.3-4 (1975): 249-278.
PMID
172635
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
23
Issue
3-4
Publish Date
1975
Start Page
249
End Page
278

Cholera toxin and cell growth: role of membrane gangliosides.

The binding of cholera toxin to three transformed mouse cell lines derived from the same parent strain, and the effects of the toxin on DNA synthesis and adenylate cyclase activity, vary in parallel with the ganglioside composition of the cells. TAL/N cells of early passage, which contain large quantities of gangliosides G(M3), G(M2), G(M1), and G(Dla), as well as the glycosyltransferases necessary for the synthesis of these gangliosides, bind the most cholera toxin and are the most sensitive to its action. TAL/N cells of later passage, which lack chemically detectable G(M1) and G(Dla) and which have no UDP-Gal:G(M2) galactosyltransferase activity, are intermediate in binding and response to the toxin. SVS AL/N cells, which lack G(M2) in addition to G(M1) and G(Dla) and which have little detectable UDP-GalNAc:G(M3)N-acetylgalactosaminyltransferase activity, bind the least amount of toxin. The SVS AL/N cells are the least responsive to inhibition of DNA synthesis and stimulation of adenylate cyclase activity by cholera toxin. Gangliosides (especially G(M1)), which appear to be the natural membrane receptors for cholera toxin, may normally have important roles in the regulation of cell growth and cAMP-mediated responses.

Authors
Hollenberg, MD; Fishman, PH; Bennett, V; Cuatrecasas, P
MLA Citation
Hollenberg, MD, Fishman, PH, Bennett, V, and Cuatrecasas, P. "Cholera toxin and cell growth: role of membrane gangliosides." Proc Natl Acad Sci U S A 71.10 (October 1974): 4224-4228.
PMID
4530298
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
71
Issue
10
Publish Date
1974
Start Page
4224
End Page
4228

Properties of the cholera toxin stimulated adenylate cyclase of toad and rat erythrocyte plasma membranes and the mechanism of action of cholera toxin

Authors
Bennett, V; Cuatrecasas, P
MLA Citation
Bennett, V, and Cuatrecasas, P. "Properties of the cholera toxin stimulated adenylate cyclase of toad and rat erythrocyte plasma membranes and the mechanism of action of cholera toxin." Federation Proceedings 33.5 II (1974): no.-756.
Source
scival
Published In
Federation Proceedings
Volume
33
Issue
5 II
Publish Date
1974
Start Page
no.
End Page
756

Preparation of inverted plasma membrane vesicles from isolated adipocytes.

Authors
Bennett, V; Cuatrecasas, P
MLA Citation
Bennett, V, and Cuatrecasas, P. "Preparation of inverted plasma membrane vesicles from isolated adipocytes." Biochim Biophys Acta 311.3 (July 6, 1973): 362-380.
PMID
4354129
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
311
Issue
3
Publish Date
1973
Start Page
362
End Page
380

Insulin receptor of fat cells in insulin-resistant metabolic states

A diminished response to insulin is exhibited by isolated fat cells obtained from rats that have been either starved, or treated with prednisone, or made diabetic by administration of streptozotocin. This decrease in response is not accompanied by changes in the quantity of insulin receptor of these cells or in the affinity of these receptors for insulin. Similarly, the decreased responsiveness to insulin of fat cells obtained from certain species (hamster, rabbit, mouse, guinea pig) is not explainable in terms of alterations of the insulin receptor.

Authors
Bennett, GV; Cuatrecasas, P
MLA Citation
Bennett, GV, and Cuatrecasas, P. "Insulin receptor of fat cells in insulin-resistant metabolic states." Science 176.4036 (1972): 805-806.
PMID
4260623
Source
scival
Published In
Science
Volume
176
Issue
4036
Publish Date
1972
Start Page
805
End Page
806

Phytochrome control of maize leaf inorganic pyrophosphatase and adenylate kinase.

Brief exposure of etiolated maize seedlings to light induces large increases in adenylate kinase and inorganic pyrophosphatase activity of the leaf in the following 48 hr in the dark. Red light is more effective than white or far red light, and far red reverses the effect of red light, indicating phytochrome control. Out of several tested, only these 2 enzymes appear to be coordinately induced, which is consistant with their close functional relationship. For inorganic pyrophosphatase, light treatment induces biosynthesis of a distinctive form of the enzyme characteristic of chloroplasts, readily separable from the enzyme characteristic of etiolated tissue.

Authors
Butler, LG; Bennett, V
MLA Citation
Butler, LG, and Bennett, V. "Phytochrome control of maize leaf inorganic pyrophosphatase and adenylate kinase." Plant Physiol 44.9 (September 1969): 1285-1290.
PMID
5379106
Source
pubmed
Published In
Plant physiology
Volume
44
Issue
9
Publish Date
1969
Start Page
1285
End Page
1290
Show More

Research Areas:

  • 3T3 Cells
  • Acetylglucosamine
  • Actin Cytoskeleton
  • Actinin
  • Actins
  • Action Potentials
  • Adaptor Proteins, Vesicular Transport
  • Adenylate Cyclase
  • Adherens Junctions
  • Adipocytes
  • Adipose Tissue
  • Adrenergic beta-2 Receptor Antagonists
  • Adult
  • Aging
  • Alcohols
  • Alkaloids
  • Alternative Splicing
  • Amino Acid Motifs
  • Amino Acid Substitution
  • Anemia
  • Anemia, Hemolytic
  • Anemia, Hemolytic, Congenital
  • Animals
  • Animals, Genetically Modified
  • Animals, Newborn
  • Anion Exchange Protein 1, Erythrocyte
  • Anion Transport Proteins
  • Anisotropy
  • Ankyrin Repeat
  • Ankyrins
  • Antibodies
  • Antigen-Antibody Complex
  • Arrhythmias, Cardiac
  • Ataxia
  • Attachment Sites, Microbiological
  • Avian Proteins
  • Avidin
  • Axonal Transport
  • Axons
  • Bacteriophage lambda
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • Biocompatible Materials
  • Biological Evolution
  • Biomechanics
  • Blood Platelets
  • Blood Proteins
  • Blotting, Southern
  • Bone Marrow Cells
  • Brain
  • Brain Ischemia
  • Ca(2+) Mg(2+)-ATPase
  • Caenorhabditis elegans
  • Caenorhabditis elegans Proteins
  • Calcium
  • Calcium Channel Blockers
  • Calcium Channels
  • Calcium Signaling
  • Calcium-Binding Proteins
  • Calcium-Transporting ATPases
  • Calmodulin
  • Calmodulin-Binding Proteins
  • Carbachol
  • Cardiovascular Diseases
  • Carrier Proteins
  • Catecholamines
  • Cattle
  • Cell Adhesion
  • Cell Adhesion Molecules
  • Cell Adhesion Molecules, Neuronal
  • Cell Aggregation
  • Cell Communication
  • Cell Compartmentation
  • Cell Cycle
  • Cell Death
  • Cell Differentiation
  • Cell Fractionation
  • Cell Line
  • Cell Membrane
  • Cell Movement
  • Cell Polarity
  • Cell Survival
  • Cells, Cultured
  • Central Nervous System
  • Cerebellum
  • Cerebral Cortex
  • Chemical Phenomena
  • Chemistry, Physical
  • Child
  • Chloride-Bicarbonate Antiporters
  • Chloroplasts
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Chromosome Mapping
  • Chromosomes, Human, Pair 1
  • Chromosomes, Human, Pair 2
  • Chromosomes, Human, Pair 7
  • Chymotrypsin
  • Cilia
  • Circular Dichroism
  • Clathrin
  • Cleavage Stage, Ovum
  • Cloning, Molecular
  • Coated Pits, Cell-Membrane
  • Community Health Nursing
  • Coronary Circulation
  • Cross-Linking Reagents
  • Cyanides
  • Cyclic Nucleotide-Gated Cation Channels
  • Cytoplasmic Granules
  • Cytoskeletal Proteins
  • Cytoskeleton
  • DNA Primers
  • DNA, Neoplasm
  • Death, Sudden, Cardiac
  • Dendrites
  • Desmoplakins
  • Desmosomes
  • Detergents
  • Diabetes Mellitus, Type 1
  • Diabetes Mellitus, Type 2
  • Dimerization
  • Dithiothreitol
  • Dogs
  • Dose-Response Relationship, Drug
  • Drosophila Proteins
  • Ductus Arteriosus, Patent
  • Edetic Acid
  • Education, Nursing
  • Egtazic Acid
  • Electrocardiography
  • Electrophoresis, Polyacrylamide Gel
  • Embryo Transfer
  • Emergencies
  • Endocytosis
  • Endopeptidases
  • Endosomes
  • Endotoxins
  • Enterotoxins
  • Epidermal Growth Factor
  • Epididymis
  • Epithelial Cells
  • Epithelium
  • Epitopes
  • Erythrocyte Membrane
  • Erythrocytes
  • Erythrocytes, Abnormal
  • Erythroid Precursor Cells
  • Escherichia coli
  • Ethylmaleimide
  • Evolution, Molecular
  • Exons
  • Female
  • Fertilization in Vitro
  • Fetus
  • Fibroblasts
  • Fibronectins
  • Fluoresceins
  • Fluorescent Dyes
  • Fluorides
  • Forkhead Transcription Factors
  • Fungal Proteins
  • GTPase-Activating Proteins
  • Galactose
  • Ganglia, Spinal
  • Gelsolin
  • Gene Library
  • Genes
  • Genetic Linkage
  • Genetic Variation
  • Giant Cells
  • Glass
  • Glucose
  • Glycosphingolipids
  • Gonads
  • Green Fluorescent Proteins
  • Guanosine Triphosphate
  • HSP70 Heat-Shock Proteins
  • Heart
  • Heart Defects, Congenital
  • Heart Diseases
  • Heart Rate
  • Heat-Shock Proteins
  • Helminth Proteins
  • Hemolysis
  • Hepatocyte Growth Factor
  • Hexosyltransferases
  • Hippocampus
  • History, 20th Century
  • Homeostasis
  • Housing
  • Humans
  • Hydrogen-Ion Concentration
  • Hydroquinones
  • Hydroxylamine
  • Hydroxylamines
  • Hypoglycemia
  • Immune Sera
  • Immunoblotting
  • Immunoglobulin G
  • Immunohistochemistry
  • Immunosorbent Techniques
  • In Situ Hybridization, Fluorescence
  • Infant
  • Infertility
  • Inositol 1,4,5-Trisphosphate
  • Inositol 1,4,5-Trisphosphate Receptors
  • Insulin
  • Insulin-Secreting Cells
  • Intercellular Junctions
  • Intermediate Filaments
  • Intestine, Small
  • Intracellular Membranes
  • Invertebrates
  • Iodine Radioisotopes
  • Ion Channels
  • Isoelectric Focusing
  • Isoproterenol
  • Kidney
  • Kidney Tubules, Collecting
  • Kinetics
  • Lectins
  • Lens, Crystalline
  • Leukocyte L1 Antigen Complex
  • Leupeptins
  • Libido
  • Light
  • Lipid Mobilization
  • Liposomes
  • Long QT Syndrome
  • Lymphocytes
  • Macromolecular Substances
  • Malaria
  • Mathematics
  • Mechanical Processes
  • Mechanoreceptors
  • Melanoma
  • Membrane Fluidity
  • Membrane Glycoproteins
  • Membrane Lipids
  • Membrane Microdomains
  • Membrane Proteins
  • Membranes
  • Mesencephalon
  • Methanol
  • Mice
  • Mice, Congenic
  • Mice, Inbred C57BL
  • Mice, Inbred ICR
  • Mice, Jimpy
  • Mice, Knockout
  • Mice, Mutant Strains
  • Mice, Neurologic Mutants
  • Mice, Transgenic
  • Microfilament Proteins
  • Microscopy, Atomic Force
  • Microscopy, Confocal
  • Microscopy, Electron
  • Microscopy, Electron, Scanning
  • Microscopy, Fluorescence
  • Microscopy, Immunoelectron
  • Microsomes
  • Microtubule-Associated Proteins
  • Microtubules
  • Models, Biological
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Molecular Weight
  • Moths
  • Multigene Family
  • Muscle, Skeletal
  • Muscles
  • Muscular Dystrophy, Animal
  • Mutagenesis
  • Mutagenesis, Site-Directed
  • Mutagenicity Tests
  • Mutation
  • Mutation, Missense
  • Myelin Sheath
  • Myelin-Associated Glycoprotein
  • Myocardial Contraction
  • Myocardial Ischemia
  • Myocardium
  • Myocytes, Cardiac
  • Myosins
  • Nanostructures
  • Nanotechnology
  • Nerve Degeneration
  • Nerve Fibers, Myelinated
  • Nerve Growth Factors
  • Nerve Tissue Proteins
  • Nervous System
  • Nervous System Diseases
  • Nervous System Malformations
  • Neural Cell Adhesion Molecules
  • Neural Stem Cells
  • Neurogenesis
  • Neuroglia
  • Neuromuscular Junction
  • Neurons
  • Neuropeptides
  • Neutrophils
  • Nevus, Pigmented
  • Nucleic Acid Hybridization
  • Nurse Midwives
  • Oligodendroglia
  • Oligopeptides
  • Oligospermia
  • Oocytes
  • Optic Nerve
  • Organ Specificity
  • Organophosphonates
  • Osmotic Fragility
  • Palmitic Acid
  • Parasympathetic Nervous System
  • Patient Care Team
  • Peptide Fragments
  • Peptide Hydrolases
  • Peptide Mapping
  • Peripheral Nerves
  • Phenanthrolines
  • Phenotype
  • Phosphates
  • Phosphatidylinositol 4,5-Diphosphate
  • Phospholipases
  • Phospholipids
  • Phosphopeptides
  • Phosphoproteins
  • Phosphorus Radioisotopes
  • Phosphoserine
  • Phosphotransferases
  • Photoreceptor Cells
  • Placenta
  • Plant Proteins
  • Plasmids
  • Poly A
  • Polymorphism, Single Nucleotide
  • Potassium Chloride
  • Pregnancy
  • Prospective Studies
  • Protein Binding
  • Protein Conformation
  • Protein Denaturation
  • Protein Engineering
  • Protein Folding
  • Protein Isoforms
  • Protein Kinase C
  • Protein Multimerization
  • Protein Refolding
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Unfolding
  • Protein-Serine-Threonine Kinases
  • Protein-Tyrosine Kinases
  • Proteins
  • Proto-Oncogenes
  • Pulmonary Veins
  • Purkinje Cells
  • Putamen
  • Pyrrolidines
  • RNA
  • RNA Precursors
  • RNA Splicing
  • RNA, Antisense
  • RNA, Messenger
  • RNA, Small Interfering
  • Radioimmunoassay
  • Ranvier's Nodes
  • Rats
  • Rats, Inbred Strains
  • Rats, Sprague-Dawley
  • Receptor, Epidermal Growth Factor
  • Receptors, Adrenergic
  • Receptors, Adrenergic, beta
  • Receptors, Cell Surface
  • Receptors, Cytoplasmic and Nuclear
  • Receptors, Drug
  • Receptors, Fibroblast Growth Factor
  • Receptors, Transferrin
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Regulatory Sequences, Nucleic Acid
  • Repetitive Sequences, Amino Acid
  • Repetitive Sequences, Nucleic Acid
  • Reproductive Techniques
  • Restriction Mapping
  • Reticulocytes
  • Retinal Ganglion Cells
  • Reverse Transcriptase Polymerase Chain Reaction
  • Rhodopsin
  • Risk
  • Risk Factors
  • Risk-Taking
  • Rod Cell Outer Segment
  • Ryanodine Receptor Calcium Release Channel
  • SOS Response (Genetics)
  • Sarcolemma
  • Sarcoplasmic Reticulum
  • Schwann Cells
  • Sciatic Nerve
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Serine
  • Sexual Behavior
  • Signal Transduction
  • Social Work
  • Sodium Channels
  • Sodium-Calcium Exchanger
  • Sodium-Potassium-Exchanging ATPase
  • Solubility
  • Spectrin
  • Spermatozoa
  • Spherocytosis, Hereditary
  • Spinal Cord
  • Splenectomy
  • Stem Cell Niche
  • Streptavidin
  • Structure-Activity Relationship
  • Subcellular Fractions
  • Substrate Specificity
  • Succinimides
  • Surface Properties
  • Surface-Active Agents
  • Symporters
  • Synapsins
  • Synaptic Membranes
  • Synaptic Vesicles
  • Synaptosomes
  • Syndrome
  • Talc
  • Testis
  • Thalamus
  • Thermodynamics
  • Thiocyanates
  • Thrombin
  • Time Factors
  • Tissue Distribution
  • Tongue
  • Toxins, Biological
  • Transcription, Genetic
  • Transfection
  • Transient Receptor Potential Channels
  • Treatment Failure
  • Trifluoperazine
  • Tritium
  • Tropomodulin
  • Tropomyosin
  • Trypsin
  • Tubulin
  • Turkeys
  • Tyrosine
  • Ultrasonography
  • Up-Regulation
  • Urban Health
  • Vasodilator Agents
  • Vibrio cholerae
  • Vinculin
  • Virus Activation
  • Voluntary Workers
  • Wheat Germ Agglutinins
  • X Chromosome
  • Yeasts
  • Zona Pellucida
  • beta-Adrenergic Receptor Kinases
  • rho-Associated Kinases
  • von Willebrand Factor