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Erickson, Harold Paul

Overview:

Cytoskeleton: It is now clear that the actin and microtubule cytoskeleton originated in bacteria. Our major research is on FtsZ, the bacterial tubulin homolog, which assembles into a contractile ring that divides the bacterium. We have studied FtsZ assembly in vitro, and found that it assembles into thin protofilaments. Dozens of these protofilaments are further clustered to form the contractile Z-ring in vivo. Recent discoveries include:

• The Z ring is very dynamic, exchanging subunits with a half time of 8 s.
• Reconstitution of Z rings in vitro. We provided FtsZ with a membrane tether, and found that when incorporated inside liposomes, membrane-targeted FtsZ can assemble Z rings without any other proteins.
• These reconstituted Z rings can also generate a constriction force on the membranes, again without any other proteins (no motor molecules).
• The constriction force is generated by a curved conformation of the protofilaments generating a bending force on the membrane.
• Negative stain EM of artificial Z rings shows ribbons of protofilaments, contradicting the prevailing view from cryoEM tomography of scattered protofilaments

Our long term goals are two-fold. First, to understand the mechanism of bacterial cell division. Second, to learn basic principles of assembly and mechanics that will apply to both FtsZ and tubulin.

Extracellular Matrix: A second interest of our lab is extracellular matrix and cell adhesion, focusing now on fibronectin. We have discovered that the FN matrix is very elastic, with fibrils stretching up to four-fold over their relaxed length. We have two possible mechanisms to explain the elasticity of FN, and are currently developing experimental tests to resolve the mechanism. We are also studying the molecular structure of FN matrix fibrils and the mechanism of assembly. Assembly of "super FN" is providing important new insights.

Positions:

James B. Duke Professor of Medicine

Cell Biology
School of Medicine

Professor of Cell Biology

Cell Biology
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. 1968

Ph.D. — Johns Hopkins University

News:

Grants:

Differentiation Induced Changes in Centrosomes and Microtubule Organization

Administered By
Dermatology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
August 01, 2014
End Date
July 31, 2018

Structure and Assembly Dynamics of FtsZ

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2002
End Date
May 31, 2018

University Training Program in Biomolecular and Tissue Engineering

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1994
End Date
June 30, 2017

Extracellular Matrix and Cell Attachment Proteins

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2012
End Date
May 31, 2017

Fluoride and human health: Assessing novel biomarkers in detecting bone disorder

Administered By
Earth and Ocean Sciences
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
April 20, 2015
End Date
March 31, 2017

Assembly, Dynamics and Regulation of Chloroplast FtsZ

Administered By
Cell Biology
AwardedBy
Michigan State University
Role
Principal Investigator
Start Date
August 01, 2011
End Date
April 30, 2016

Structure and Assembly Dynamics of FtsZ

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2002
End Date
June 30, 2014

A Genomic and Structural Study of FTsZ Constriction in Cell Divison

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2010
End Date
May 30, 2013

Extracellular Matrix and Cell Attachment Proteins

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1979
End Date
March 31, 2012

A Fluorescence-Based Biosensor for Measurement of Cell Derived Forces

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2009
End Date
June 30, 2011

Live cell widefield fluorescence microscope with activation and bleaching lasers

Administered By
Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
April 22, 2010
End Date
April 21, 2011

Training in Biomolecular and Tissue Engineering

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 20, 2003
End Date
June 30, 2009

Graduate training in Biologically Inspired Materials

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
December 15, 2002
End Date
November 30, 2007

Regulation of Germline Stem Cell Division in Drosophila

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
February 01, 1996
End Date
June 07, 2006

Zeiss LSM510 META confocal-fluorescence spectroscopy

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

Development and Construction of Single Molecule Spectrometers for Research and Student Training

Administered By
Mechanical Engineering and Materials Science
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
August 15, 2001
End Date
July 31, 2003

Structure and Assembly of Cytoskeletal Filaments

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1981
End Date
April 30, 2002

Center for FEL Research in the Medical, Biological, and Materials Sciences

Administered By
Physics
AwardedBy
Air Force Office of Scientific Research
Role
Co-Principal Investigator
Start Date
September 15, 2000
End Date
October 31, 2001

Multivalent Ligands To Enhance Cell Receptor Binding

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1998
End Date
January 31, 2000

Structure & Assembly Of Cytoskeletal Filaments

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1981
End Date
March 31, 1999

Tenascin Regulation Of Cell-Matrix Interactions In Skin

Administered By
Dermatology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
January 01, 1996
End Date
December 31, 1997

Tenascin Regulation Of Cell Matrix Interactions In Skin

Administered By
Dermatology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
January 01, 1995
End Date
December 31, 1997

Extracellular Matrix And Cell Attachment Proteins

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 01, 1996
End Date
November 30, 1997

Structure And Assembly Of Cytoskeletal Filamients

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1986
End Date
June 01, 1989

Electron Microscopy Of Plasma And Cell Surface Proteins

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1986
End Date
April 01, 1988

Electron Microscopy Of Plasma And Cell Surface Protein

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1986
End Date
April 01, 1987

434uisition Of Philips Em 420 Microscope With Goniometer

Administered By
Cell Biology
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
February 01, 1984
End Date
July 01, 1985

Phailips Em420 Microscope With Goniometer & Cryo Stage

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1984
End Date
December 01, 1984
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Publications:

FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings.

FtsZ protofilaments (pfs) form the bacterial cytokinetic Z ring. Previous work suggested that a conformational change from straight to curved pfs generated the constriction force. In the simplest model, the C-terminal membrane tether is on the outside of the curved pf, facing the membrane. Tubulin, a homologue of FtsZ, also forms pfs with a curved conformation. However, it is well-established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In this study, we explored the FtsZ curvature direction by fusing large protein tags to the FtsZ termini. Thin section electron microscopy showed that the C-terminal tag was on the outside, consistent with the bending pf model. This has interesting implications for the evolution of tubulin. Tubulin likely began with the curvature of FtsZ, but evolution managed to reverse direction to produce outward-curving rings, which are useful for pulling chromosomes.

Authors
Housman, M; Milam, SL; Moore, DA; Osawa, M; Erickson, HP
MLA Citation
Housman, M, Milam, SL, Moore, DA, Osawa, M, and Erickson, HP. "FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings." Biochemistry 55.29 (July 2016): 4085-4091.
PMID
27368355
Source
epmc
Published In
Biochemistry
Volume
55
Issue
29
Publish Date
2016
Start Page
4085
End Page
4091
DOI
10.1021/acs.biochem.6b00479

How the kinetochore couples microtubule force and centromere stretch to move chromosomes.

The Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule- and MAP-binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule-binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule-binding interactions with the Ndc80 and Dam1 complexes.

Authors
Suzuki, A; Badger, BL; Haase, J; Ohashi, T; Erickson, HP; Salmon, ED; Bloom, K
MLA Citation
Suzuki, A, Badger, BL, Haase, J, Ohashi, T, Erickson, HP, Salmon, ED, and Bloom, K. "How the kinetochore couples microtubule force and centromere stretch to move chromosomes." Nature cell biology 18.4 (April 2016): 382-392.
PMID
26974660
Source
epmc
Published In
Nature Cell Biology
Volume
18
Issue
4
Publish Date
2016
Start Page
382
End Page
392
DOI
10.1038/ncb3323

The Presence and Anti-HIV-1 Function of Tenascin C in Breast Milk and Genital Fluids.

Tenascin-C (TNC) is a newly identified innate HIV-1-neutralizing protein present in breast milk, yet its presence and potential HIV-inhibitory function in other mucosal fluids is unknown. In this study, we identified TNC as a component of semen and cervical fluid of HIV-1-infected and uninfected individuals, although it is present at a significantly lower concentration and frequency compared to that of colostrum and mature breast milk, potentially due to genital fluid protease degradation. However, TNC was able to neutralize HIV-1 after exposure to low pH, suggesting that TNC could be active at low pH in the vaginal compartment. As mucosal fluids are complex and contain a number of proteins known to interact with the HIV-1 envelope, we further studied the relationship between the concentration of TNC and neutralizing activity in breast milk. The amount of TNC correlated only weakly with the overall innate HIV-1-neutralizing activity of breast milk of uninfected women and negatively correlated with neutralizing activity in milk of HIV-1 infected women, indicating that the amount of TNC in mucosal fluids is not adequate to impede HIV-1 transmission. Moreover, the presence of polyclonal IgG from milk of HIV-1 infected women, but not other HIV-1 envelope-binding milk proteins or monoclonal antibodies, blocked the neutralizing activity of TNC. Finally, as exogenous administration of TNC would be necessary for it to mediate measurable HIV-1 neutralizing activity in mucosal compartments, we established that recombinantly produced TNC has neutralizing activity against transmitted/founder HIV-1 strains that mimic that of purified TNC. Thus, we conclude that endogenous TNC concentration in mucosal fluids is likely inadequate to block HIV-1 transmission to uninfected individuals.

Authors
Mansour, RG; Stamper, L; Jaeger, F; McGuire, E; Fouda, G; Amos, J; Barbas, K; Ohashi, T; Alam, SM; Erickson, H; Permar, SR
MLA Citation
Mansour, RG, Stamper, L, Jaeger, F, McGuire, E, Fouda, G, Amos, J, Barbas, K, Ohashi, T, Alam, SM, Erickson, H, and Permar, SR. "The Presence and Anti-HIV-1 Function of Tenascin C in Breast Milk and Genital Fluids." PloS one 11.5 (January 2016): e0155261-.
PMID
27182834
Source
epmc
Published In
PloS one
Volume
11
Issue
5
Publish Date
2016
Start Page
e0155261
DOI
10.1371/journal.pone.0155261

FtsZ filament capping by MciZ, a developmental regulator of bacterial division.

Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.

Authors
Bisson-Filho, AW; Discola, KF; Castellen, P; Blasios, V; Martins, A; Sforça, ML; Garcia, W; Zeri, ACM; Erickson, HP; Dessen, A; Gueiros-Filho, FJ
MLA Citation
Bisson-Filho, AW, Discola, KF, Castellen, P, Blasios, V, Martins, A, Sforça, ML, Garcia, W, Zeri, ACM, Erickson, HP, Dessen, A, and Gueiros-Filho, FJ. "FtsZ filament capping by MciZ, a developmental regulator of bacterial division." Proceedings of the National Academy of Sciences of the United States of America 112.17 (April 6, 2015): E2130-E2138.
PMID
25848052
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
17
Publish Date
2015
Start Page
E2130
End Page
E2138
DOI
10.1073/pnas.1414242112

Irisin - a myth rather than an exercise-inducible myokine.

The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Authors
Albrecht, E; Norheim, F; Thiede, B; Holen, T; Ohashi, T; Schering, L; Lee, S; Brenmoehl, J; Thomas, S; Drevon, CA; Erickson, HP; Maak, S
MLA Citation
Albrecht, E, Norheim, F, Thiede, B, Holen, T, Ohashi, T, Schering, L, Lee, S, Brenmoehl, J, Thomas, S, Drevon, CA, Erickson, HP, and Maak, S. "Irisin - a myth rather than an exercise-inducible myokine." Scientific reports 5 (March 9, 2015): 8889-.
Website
http://hdl.handle.net/10161/9501
PMID
25749243
Source
epmc
Published In
Scientific Reports
Volume
5
Publish Date
2015
Start Page
8889
DOI
10.1038/srep08889

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin.

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction.

Authors
Spahich, NA; Kenjale, R; McCann, J; Meng, G; Ohashi, T; Erickson, HP; St Geme, JW
MLA Citation
Spahich, NA, Kenjale, R, McCann, J, Meng, G, Ohashi, T, Erickson, HP, and St Geme, JW. "Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin." Microbiology (Reading, England) 160.Pt 6 (June 2014): 1182-1190.
PMID
24687948
Source
epmc
Published In
Microbiology (Reading, England)
Volume
160
Issue
Pt 6
Publish Date
2014
Start Page
1182
End Page
1190
DOI
10.1099/mic.0.077784-0

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature- sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.

Authors
Kiro, R; Molshanski-Mor, S; Yosef, I; Milam, SL; Erickson, HP; Qimron, U
MLA Citation
Kiro, R, Molshanski-Mor, S, Yosef, I, Milam, SL, Erickson, HP, and Qimron, U. "Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division." Proceedings of the National Academy of Sciences of the United States of America 110.48 (November 26, 2013): 19549-19554.
PMID
24218612
Source
scopus
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
48
Publish Date
2013
Start Page
19549
End Page
19554
DOI
10.1073/pnas.1314096110

The structure of irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: implications for receptor activation.

Irisin was recently identified as a putative myokine that is induced by exercise. Studies suggest that it is produced by cleavage of the FNDC5 (fibronectin domain-containing protein 5) receptor; irisin corresponds to the extracellular receptor ectodomain. Data suggesting that irisin stimulates white-to-brown fat conversion have led to the hypothesis that it does so by binding an unknown receptor, thus functioning as a myokine. As brown fat promotes energy dissipation, myokines that elicit the transformation of white to brown fat have potentially profound benefits in the treatment of obesity and metabolic disorders. Understanding the molecular basis for such exercise-induced phenomena is thus of considerable interest. Moreover, FNDC5-like receptors are highly conserved and have been shown to be critical for neuronal development. However, the structural and molecular mechanisms utilized by these proteins are currently unknown. Here, we describe the crystal structure and biochemical characterization of the FNDC5 ectodomain, corresponding to the irisin myokine. The 2.28 Å structure shows that irisin consists of an N-terminal fibronectin III (FNIII)-like domain attached to a flexible C-terminal tail. Strikingly, the FNIII-like domain forms a continuous intersubunit β-sheet dimer, previously unobserved for any FNIII protein. Biochemical data confirm that irisin is a dimer and that dimerization is unaffected by glycosylation. This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors.

Authors
Schumacher, MA; Chinnam, N; Ohashi, T; Shah, RS; Erickson, HP
MLA Citation
Schumacher, MA, Chinnam, N, Ohashi, T, Shah, RS, and Erickson, HP. "The structure of irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: implications for receptor activation." J Biol Chem 288.47 (November 22, 2013): 33738-33744.
PMID
24114836
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
47
Publish Date
2013
Start Page
33738
End Page
33744
DOI
10.1074/jbc.M113.516641

Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk.

Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.

Authors
Fouda, GG; Jaeger, FH; Amos, JD; Ho, C; Kunz, EL; Anasti, K; Stamper, LW; Liebl, BE; Barbas, KH; Ohashi, T; Moseley, MA; Liao, H-X; Erickson, HP; Alam, SM; Permar, SR
MLA Citation
Fouda, GG, Jaeger, FH, Amos, JD, Ho, C, Kunz, EL, Anasti, K, Stamper, LW, Liebl, BE, Barbas, KH, Ohashi, T, Moseley, MA, Liao, H-X, Erickson, HP, Alam, SM, and Permar, SR. "Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk." Proc Natl Acad Sci U S A 110.45 (November 5, 2013): 18220-18225.
PMID
24145401
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
45
Publish Date
2013
Start Page
18220
End Page
18225
DOI
10.1073/pnas.1307336110

Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?

FNDC5 (fibronectin domain-containing [protein] 5) was initially discovered and characterized by two groups in 2002. In 2011 FNDC5 burst into prominence as the parent of irisin, a small protein containing the fibronectin type III domain. Irisin was proposed to be secreted by skeletal muscle cells in response to exercise, and to circulate to fat tissue where it induced a transition to brown fat. Since brown fat results in dissipation of energy, this pathway is of considerable interest for metabolism and obesity. Here I review the original discoveries of FNDC5 and the more recent discovery of irisin. I note in particular three problems in the characterization of irisin: the antibodies used to detect irisin in plasma lack validity; the recombinant protein used to demonstrate activity in cell culture was severely truncated; and the degree of shedding of soluble irisin from the cell surface has not been quantitated. The original discovery proposing that FNDC5 may be a transmembrane receptor may deserve a new look.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?." Adipocyte 2.4 (October 1, 2013): 289-293.
Website
http://hdl.handle.net/10161/9280
PMID
24052909
Source
pubmed
Published In
Adipocyte
Volume
2
Issue
4
Publish Date
2013
Start Page
289
End Page
293
DOI
10.4161/adip.26082

Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2.

FtsZ from most bacteria assembles rapidly in vitro, reaching a steady-state plateau in 5-10 s after addition of GTP. A recent study used a novel dynamic light-scattering technique to assay the assembly of FtsZ from Caulobacter crescentus (CcFtsZ) and reported that assembly required 10 min, ∼100 times slower than for related bacteria. Previous studies had indicated normal, rapid assembly of CcFtsZ. We have reinvestigated the assembly kinetics using a mutant L72W, where assembly of subunits into protofilaments results in a significant increase in tryptophan fluorescence. We found that assembly reached a plateau in 5-10 s and showed no change in the following 10 min. This was confirmed by 90° light scattering and negative-stain electron microscopy. The very slow kinetics in the dynamic light-scattering study may be related to a refractory state induced when the FtsZ protein is stored without nucleotide, a phenomenon that we had observed in a previous study of EcFtsZ. We conclude that CcFtsZ is not an outlier, but shows rapid assembly kinetics similar to FtsZ from related bacteria.

Authors
Milam, SL; Erickson, HP
MLA Citation
Milam, SL, and Erickson, HP. "Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2." J Biol Chem 288.33 (August 16, 2013): 23675-23679.
PMID
23824192
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
33
Publish Date
2013
Start Page
23675
End Page
23679
DOI
10.1074/jbc.M113.491845

Liposome division by a simple bacterial division machinery.

We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en face at the top of the liposome, many of the patches were seen to be small Z rings, which still maintained the concave depressions. We also succeeded in reconstituting the more natural, two-protein system, with FtsA and FtsZ-YFP (having the FtsA-binding peptide instead of the mts). Unilamellar liposomes incorporating FtsA and FtsZ-YFP showed a variety of distributions, including foci and linear arrays. A small fraction of liposomes had obvious Z rings. These Z rings could constrict the liposomes and in some cases appeared to complete the division, leaving a clear septum between the two daughter liposomes. Because complete liposome divisions were not seen with FtsZ-mts, FtsA may be critical for the final membrane scission event. We demonstrate that reconstituted cell division machinery apparently divides the liposome in vitro.

Authors
Osawa, M; Erickson, HP
MLA Citation
Osawa, M, and Erickson, HP. "Liposome division by a simple bacterial division machinery." Proc Natl Acad Sci U S A 110.27 (July 2, 2013): 11000-11004.
PMID
23776220
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
27
Publish Date
2013
Start Page
11000
End Page
11004
DOI
10.1073/pnas.1222254110

The C-terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide.

The tubulin homologue FtsZ provides the cytoskeletal framework and constriction force for bacterial cell division. FtsZ has an 50-amino-acid (aa) linker between the protofilament-forming globular domain and the C-terminal (Ct) peptide that binds FtsA and ZipA, tethering FtsZ to the membrane. This Ct-linker is widely divergent across bacterial species and thought to be an intrinsically disordered peptide (IDP). We confirmed that the Ct-linkers from three bacterial species behaved as IDPs in vitro by circular dichroism and trypsin proteolysis. We made chimeras, swapping the Escherichia coli linker for Ct-linkers from other bacteria, and even for an unrelated IDP from human α-adducin. Most substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter. With few exceptions, almost any sequence appears to work. Length, however, was important: IDPs shorter than 43 or longer than 95 aa had compromised or no function. We conclude that the Ct-linker functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA/ZipA at the membrane. Modelling the Ct-linker as a worm-like chain, we predict that it functions as a stiff entropic spring linking the bending protofilaments to the membrane.

Authors
Gardner, KAJA; Moore, DA; Erickson, HP
MLA Citation
Gardner, KAJA, Moore, DA, and Erickson, HP. "The C-terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide." Mol Microbiol 89.2 (July 2013): 264-275.
PMID
23714328
Source
pubmed
Published In
Molecular Microbiology
Volume
89
Issue
2
Publish Date
2013
Start Page
264
End Page
275
DOI
10.1111/mmi.12279

The C-terminal linker of Escherichia coliFtsZ functions as an intrinsically disordered peptide

The tubulin homologue FtsZ provides the cytoskeletal framework and constriction force for bacterial cell division. FtsZ has an ~50-amino-acid (aa) linker between the protofilament-forming globular domain and the C-terminal (Ct) peptide that binds FtsA and ZipA, tethering FtsZ to the membrane. This Ct-linker is widely divergent across bacterial species and thought to be an intrinsically disordered peptide (IDP). We confirmed that the Ct-linkers from three bacterial species behaved as IDPsin vitro by circular dichroism and trypsin proteolysis. We made chimeras, swapping the Escherichia coli linker for Ct-linkers from other bacteria, and even for an unrelated IDP from human α-adducin. Most substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter. With few exceptions, almost any sequence appears to work. Length, however, was important: IDPs shorter than 43 or longer than 95aa had compromised or no function. We conclude that the Ct-linker functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA/ZipA at the membrane. Modelling the Ct-linker as a worm-like chain, we predict that it functions as a stiff entropic spring linking the bending protofilaments to the membrane. © 2013 John Wiley & Sons Ltd.

Authors
Gardner, KAJA; Moore, DA; Erickson, HP
MLA Citation
Gardner, KAJA, Moore, DA, and Erickson, HP. "The C-terminal linker of Escherichia coliFtsZ functions as an intrinsically disordered peptide." Molecular Microbiology 89.2 (2013): 264-275.
Source
scival
Published In
Molecular Microbiology
Volume
89
Issue
2
Publish Date
2013
Start Page
264
End Page
275
DOI
10.1111/mmi.12279

Bacterial actin homolog ParM: arguments for an apolar, antiparallel double helix.

The bacterial actin homolog ParM has always been modeled as a polar filament, comprising two parallel helical strands, like actin itself. I present arguments here that ParM may be an apolar filament, in which the two helical strands are antiparallel.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Bacterial actin homolog ParM: arguments for an apolar, antiparallel double helix." J Mol Biol 422.4 (September 28, 2012): 461-463.
PMID
22651984
Source
pubmed
Published In
Journal of Molecular Biology
Volume
422
Issue
4
Publish Date
2012
Start Page
461
End Page
463
DOI
10.1016/j.jmb.2012.05.019

Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.

FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.

Authors
Milam, SL; Osawa, M; Erickson, HP
MLA Citation
Milam, SL, Osawa, M, and Erickson, HP. "Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments." Biophys J 103.1 (July 3, 2012): 59-68.
PMID
22828332
Source
pubmed
Published In
Biophysical Journal
Volume
103
Issue
1
Publish Date
2012
Start Page
59
End Page
68
DOI
10.1016/j.bpj.2012.05.035

SulA inhibits assembly of FtsZ by a simple sequestration mechanism.

We have investigated the inhibition by SulA of the assembly of Escherichia coli FtsZ. Using quantitative GTPase and fluorescence assays, we found that SulA inhibition resulted in an increase in the apparent critical concentration for FtsZ assembly. The increase in apparent critical concentration was always less than the total amount of SulA added, suggesting that the association of SulA and FtsZ was of modest affinity. Isothermal titration calorimetry gave a value of 0.78 μM for the dissociation constant of the FtsZ-SulA complex, similar in magnitude to the 0.72 μM critical concentration of FtsZ protofilament assembly at steady state. We modeled the reaction as an equilibrium competition between (a) FtsZ subunits assembling onto protofilaments or (b) binding SulA. When FtsZ was assembled in GMPCPP or in EDTA, the inhibition by SulA was reduced. The reduced inhibition could be explained by a 3- and 10-fold weaker binding of SulA to FtsZ. The mutant D212G, which has no GTPase activity and therefore minimal subunit cycling, was shown here to assemble one-stranded protofilaments, and the assembly was blocked by SulA. We also assayed the SulA and FtsZ proteins from Pseudomonas. The SulA inhibition was stronger than with the E. coli proteins, and the model indicated a 5-fold higher affinity of Pseudomonas SulA for FtsZ.

Authors
Chen, Y; Milam, SL; Erickson, HP
MLA Citation
Chen, Y, Milam, SL, and Erickson, HP. "SulA inhibits assembly of FtsZ by a simple sequestration mechanism." Biochemistry 51.14 (April 10, 2012): 3100-3109.
PMID
22432817
Source
pubmed
Published In
Biochemistry
Volume
51
Issue
14
Publish Date
2012
Start Page
3100
End Page
3109
DOI
10.1021/bi201669d

Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain.

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.

Authors
Ohashi, T; Erickson, HP
MLA Citation
Ohashi, T, and Erickson, HP. "Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain." J Biol Chem 286.45 (November 11, 2011): 39188-39199.
PMID
21949131
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
45
Publish Date
2011
Start Page
39188
End Page
39199
DOI
10.1074/jbc.M111.262337

Probing the folded state of fibronectin type III domains in stretched fibrils by measuring buried cysteine accessibility.

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.

Authors
Lemmon, CA; Ohashi, T; Erickson, HP
MLA Citation
Lemmon, CA, Ohashi, T, and Erickson, HP. "Probing the folded state of fibronectin type III domains in stretched fibrils by measuring buried cysteine accessibility." J Biol Chem 286.30 (July 29, 2011): 26375-26382.
PMID
21652701
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
30
Publish Date
2011
Start Page
26375
End Page
26382
DOI
10.1074/jbc.M111.240028

Inside-out Z rings--constriction with and without GTP hydrolysis.

The bacterial tubulin homologue FtsZ forms a ring-like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ-YFP-mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts-FtsZ-YFP, where the membrane tether is switched to the opposite side of the protofilament. This assembled 'inside-out' Z rings that wrapped around the outside surface of tubular liposomes. The inside-out Z rings were highly dynamic, and generated a constriction force that squeezed the tubular liposomes from outside. This is consistent with models where the constriction force is generated by curved protofilaments bending the membrane. We used this system to test how GTP hydrolysis by FtsZ is involved in Z-ring constriction. Without GTP hydrolysis, Z rings could still assemble and generate an initial constriction. However, the constriction quickly stopped, suggesting that Z rings became rigidly stabilized in the absence of GTP hydrolysis. We propose that remodelling of the Z ring, mediated by GTP hydrolysis and exchange of subunits, is necessary for the continuous constriction.

Authors
Osawa, M; Erickson, HP
MLA Citation
Osawa, M, and Erickson, HP. "Inside-out Z rings--constriction with and without GTP hydrolysis." Mol Microbiol 81.2 (July 2011): 571-579.
PMID
21631604
Source
pubmed
Published In
Molecular Microbiology
Volume
81
Issue
2
Publish Date
2011
Start Page
571
End Page
579
DOI
10.1111/j.1365-2958.2011.07716.x

Conformational changes of FtsZ reported by tryptophan mutants.

E. coli FtsZ has no native tryptophan. We showed previously that the mutant FtsZ L68W gave a 2.5-fold increase in trp fluorescence when assembly was induced by GTP. L68 is probably buried in the protofilament interface upon assembly, causing the fluorescence increase. In the present study we introduced trp residues at several other locations and examined them for assembly-induced fluorescence changes. L189W, located on helix H7 and buried between the N- and C-terminal subdomains, showed a large fluorescence increase, comparable to L68W. This may reflect a shift or rotation of the two subdomains relative to each other. L160W showed a smaller increase in fluorescence, and Y222W a decrease in fluorescence, upon assembly. These two are located on the surface of the N and C subdomains, near the domain boundary. The changes in fluorescence may reflect movements of the domains or of nearby side chains. We prepared a double mutant Y222W/S151C and coupled ATTO-655 to the cys. The Cα of trp in the C-terminal subdomain was 10 Å away from that of the cys in the N-terminal subdomain, permitting the ATTO to make van der Waals contact with the trp. The ATTO fluorescence showed strong tryptophan-induced quenching. The quenching was reduced following assembly, consistent with a movement apart of the two subdomains. Movements of one to several angstroms are probably sufficient to account for the changes in trp fluorescence and trp-induced quenching of ATTO. Assembly in GDP plus DEAE dextran produces tubular polymers that are related to the highly curved, mini-ring conformation. No change in trp fluorescence was observed upon assembly of these tubes, suggesting that the mini-ring conformation is the same as that of a relaxed, monomeric FtsZ.

Authors
Chen, Y; Erickson, HP
MLA Citation
Chen, Y, and Erickson, HP. "Conformational changes of FtsZ reported by tryptophan mutants." Biochemistry 50.21 (May 31, 2011): 4675-4684.
PMID
21510681
Source
pubmed
Published In
Biochemistry
Volume
50
Issue
21
Publish Date
2011
Start Page
4675
End Page
4684
DOI
10.1021/bi200106d

Race disparity in grants: Check the citations

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Race disparity in grants: Check the citations." Science 334.6058 (2011): 899--.
PMID
22096167
Source
scival
Published In
Science
Volume
334
Issue
6058
Publish Date
2011
Start Page
899-
DOI
10.1126/science.334.6058.899-a

FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one.

FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator. Here we review light microscope observations of how Z rings assemble in bacteria. Assembly begins with long-pitch helices that condense into the Z ring. Once formed, the Z ring can transition to short-pitch helices that are suggestive of its structure. FtsZ assembles in vitro into short protofilaments that are ∼30 subunits long. We present models for how these protofilaments might be further assembled into the Z ring. We discuss recent experiments on assembly dynamics of FtsZ in vitro, with particular attention to how two regulatory proteins, SulA and MinC, inhibit assembly. Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation.

Authors
Erickson, HP; Anderson, DE; Osawa, M
MLA Citation
Erickson, HP, Anderson, DE, and Osawa, M. "FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one." Microbiol Mol Biol Rev 74.4 (December 2010): 504-528. (Review)
PMID
21119015
Source
pubmed
Published In
Microbiology and molecular biology reviews : MMBR
Volume
74
Issue
4
Publish Date
2010
Start Page
504
End Page
528
DOI
10.1128/MMBR.00021-10

Cell division without FtsZ--a variety of redundant mechanisms.

Authors
Erickson, HP; Osawa, M
MLA Citation
Erickson, HP, and Osawa, M. "Cell division without FtsZ--a variety of redundant mechanisms." Mol Microbiol 78.2 (October 2010): 267-270.
PMID
20979330
Source
pubmed
Published In
Molecular Microbiology
Volume
78
Issue
2
Publish Date
2010
Start Page
267
End Page
270

Suprastructures and dynamic properties of Mycobacterium tuberculosis FtsZ.

Tuberculosis causes the most death in humans by any bacterium. Drug targeting of bacterial cytoskeletal proteins requires detailed knowledge of the various filamentous suprastructures and dynamic properties. Here, we have investigated by high resolution electron microscopy the assembly of cell division protein and microtubule homolog FtsZ from Mycobacterium tuberculosis (MtbFtsZ) in vitro in the presence of various monovalent salts, crowding agents and polycations. Supramolecular structures, including two-dimensional rings, three-dimensional toroids, and multistranded helices formed in the presence of molecular crowding, were similar to those observed by fluorescence microscopy in bacteria in vivo. Dynamic properties of MtbFtsZ filaments were visualized by light scattering and real time total internal reflection fluorescence microscopy. Interestingly, MtbFtsZ revealed a form of dynamic instability at steady state. Cation-induced condensation phenomena of bacterial cytomotive polymers have not been investigated in any detail, although it is known that many bacteria can contain high amounts of polycations, which may modulate the prokaryotic cytoskeleton. We find that above a threshold concentration of polycations which varied with the valence of the cation, ionic strength, and pH, MtbFtsZ mainly formed sheets. The general features of these cation-induced condensation phenomena could be explained in the framework of the Manning condensation theory. Chirality and packing defects limited the dimensions of sheets and toroids at steady state as predicted by theoretical models. In first approximation simple physical principles seem to govern the formation of MtbFtsZ suprastructures.

Authors
Popp, D; Iwasa, M; Erickson, HP; Narita, A; Maéda, Y; Robinson, RC
MLA Citation
Popp, D, Iwasa, M, Erickson, HP, Narita, A, Maéda, Y, and Robinson, RC. "Suprastructures and dynamic properties of Mycobacterium tuberculosis FtsZ." J Biol Chem 285.15 (April 9, 2010): 11281-11289.
PMID
20139085
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
15
Publish Date
2010
Start Page
11281
End Page
11289
DOI
10.1074/jbc.M109.084079

Curved FtsZ protofilaments generate bending forces on liposome membranes.

We have created FtsZ-YFP-mts where an amphipathic helix on the C-terminus tethers FtsZ to the membrane. When incorporated inside multi-lamellar tubular liposomes, FtsZ-YFP-mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ-YFP-mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposomes. Prominent membrane tubules were then extruded at the intersections of concave depressions. We tested the effect of moving the membrane-targeting sequence (mts) from the C-terminus to the N-terminus, which is approximately 180 degrees from the C-terminal tether. When mts-FtsZ-YFP was applied to the outside of liposomes, it generated convex bulges, bending the membrane in the direction opposite to the concave depressions. We conclude that FtsZ protofilaments have a fixed direction of curvature, and the direction of membrane bending depends on which side of the bent protofilament the mts is attached to. This supports models in which the FtsZ constriction force is generated by protofilament bending.

Authors
Osawa, M; Anderson, DE; Erickson, HP
MLA Citation
Osawa, M, Anderson, DE, and Erickson, HP. "Curved FtsZ protofilaments generate bending forces on liposome membranes." EMBO J 28.22 (November 18, 2009): 3476-3484.
PMID
19779463
Source
pubmed
Published In
EMBO Journal
Volume
28
Issue
22
Publish Date
2009
Start Page
3476
End Page
3484
DOI
10.1038/emboj.2009.277

Structural determinants of autoproteolysis of the Haemophilus influenzae Hap autotransporter.

Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapbeta C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.

Authors
Kenjale, R; Meng, G; Fink, DL; Juehne, T; Ohashi, T; Erickson, HP; Waksman, G; St Geme, JW
MLA Citation
Kenjale, R, Meng, G, Fink, DL, Juehne, T, Ohashi, T, Erickson, HP, Waksman, G, and St Geme, JW. "Structural determinants of autoproteolysis of the Haemophilus influenzae Hap autotransporter." Infect Immun 77.11 (November 2009): 4704-4713.
PMID
19687208
Source
pubmed
Published In
Infection and immunity
Volume
77
Issue
11
Publish Date
2009
Start Page
4704
End Page
4713
DOI
10.1128/IAI.00598-09

BtubA-BtubB heterodimer is an essential intermediate in protofilament assembly.

BACKGROUND: BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament. METHODOLOGY/PRINCIPAL FINDINGS: We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1:1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity. CONCLUSIONS: An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.

Authors
Sontag, CA; Sage, H; Erickson, HP
MLA Citation
Sontag, CA, Sage, H, and Erickson, HP. "BtubA-BtubB heterodimer is an essential intermediate in protofilament assembly. (Published online)" PLoS One 4.9 (September 29, 2009): e7253-.
PMID
19787042
Source
pubmed
Published In
PloS one
Volume
4
Issue
9
Publish Date
2009
Start Page
e7253
DOI
10.1371/journal.pone.0007253

FtsZ filament dynamics at steady state: subunit exchange with and without nucleotide hydrolysis.

We have measured three aspects of FtsZ filament dynamics at steady state: rates of GTP hydrolysis, subunit exchange between protofilaments, and disassembly induced by dilution or excess GDP. All three reactions were slowed with an increase in the potassium concentration from 100 to 500 mM, via replacement of potassium with rubidium, or with an increase in the magnesium concentration from 5 to 20 mM. Electron microscopy showed that the polymers assembled under the conditions of fastest assembly were predominantly short, one-stranded protofilaments, whereas under conditions of slower dynamics, the protofilaments tended to associate into long, thin bundles. We suggest that exchange of subunits between protofilaments at steady state involves two separate mechanisms: (1) fragmentation or dissociation of subunits from protofilament ends following GTP hydrolysis and (2) reversible association and dissociation of subunits from protofilament ends independent of hydrolysis. Exchange of nucleotides on these recycling subunits could give the appearance of exchange directly into the polymer. Several of our observations suggest that exchange of nucleotide can take place on these recycling subunits, but not directly into the FtsZ polymer. Annealing of protofilaments was demonstrated for the L68W mutant in EDTA buffer but not in Mg buffer, where rapid cycling of subunits may obscure the effect of annealing. We also reinvestigated the nucleotide composition of FtsZ polymers at steady state. We found that the GDP:GTP ratio was 50:50 for concentrations of GTP >100 microM, significantly higher than the 20:80 ratio previously reported at 20 microM GTP.

Authors
Chen, Y; Erickson, HP
MLA Citation
Chen, Y, and Erickson, HP. "FtsZ filament dynamics at steady state: subunit exchange with and without nucleotide hydrolysis." Biochemistry 48.28 (July 21, 2009): 6664-6673.
PMID
19527070
Source
pubmed
Published In
Biochemistry
Volume
48
Issue
28
Publish Date
2009
Start Page
6664
End Page
6673
DOI
10.1021/bi8022653

Modeling the physics of FtsZ assembly and force generation.

The tubulin homolog FtsZ is the major cytoskeletal protein in bacterial cytokinesis. It can generate a constriction force on the bacterial membrane or inside tubular liposomes. Several models have recently been proposed for how this force might be generated. These fall into 2 categories. The first is based on a conformational change from a straight to a curved protofilament. The simplest "hydrolyze and bend" model proposes a 22 degrees bend at every interface containing a GDP. New evidence suggests another curved conformation with a 2.5 degrees bend at every interface and that the relation of curvature to GTP hydrolysis is more complicated than previously thought. However, FtsZ protofilaments do appear to be mechanically rigid enough to bend membranes. A second category of models is based on lateral bonding between protofilaments, postulating that a contraction could be generated when protofilaments slide to increase the number of lateral bonds. Unfortunately these lateral bond models have ignored the contribution of subunit entropy when adding bond energies; if included, the mechanism is seen to be invalid. Finally, I address recent models that try to explain how protofilaments 1-subunit-thick show a cooperative assembly.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Modeling the physics of FtsZ assembly and force generation." Proc Natl Acad Sci U S A 106.23 (June 9, 2009): 9238-9243.
PMID
19478069
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
23
Publish Date
2009
Start Page
9238
End Page
9243
DOI
10.1073/pnas.0902258106

Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.

We previously reported that the fibronectin (FN) type III domains of FN may unfold to interact with anastellin and form FN aggregates. In the present study, we have focused on the interaction between anastellin and the third FN type III domain (III3), which is a key anastellin binding site on FN. Anastellin binding to III3 was monitored by 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. ANS binding to anastellin dramatically increased its emission intensity, but this was reduced to half by the addition of III3, suggesting that ANS and III3 share a common hydrophobic binding site on anastellin. An engineered mutant of III3 that was stabilized by an intrachain disulfide bond did not interact with anastellin, as seen by its failure to interfere with ANS binding to anastellin. We also mutated hydrophobic core residues to destabilize III3 and found that these mutants were still capable of interacting with anastellin. Anastellin binding to III3 was also monitored using an intramolecular green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) construct, in which III3 was flanked by two GFP variants (III3-FRET). Anastellin bound to III3-FRET and caused an increase in the FRET signal. The dissociation constant was estimated to be approximately 210 nM. The binding kinetics of anastellin to III3-FRET fit a first-order reaction with a half-time of approximately 30 s; the kinetics with destabilized III3 mutants were even faster. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry suggested that the middle part of III3 became destabilized and protease sensitive upon anastellin binding. Thus, the stability of III3 seems to be a key factor in anastellin binding.

Authors
Ohashi, T; Augustus, AM; Erickson, HP
MLA Citation
Ohashi, T, Augustus, AM, and Erickson, HP. "Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin." Biochemistry 48.19 (May 19, 2009): 4189-4197.
PMID
19320499
Source
pubmed
Published In
Biochemistry
Volume
48
Issue
19
Publish Date
2009
Start Page
4189
End Page
4197
DOI
10.1021/bi900001g

Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy.

An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elongated. It is recalled that a gel filtration column fractionates proteins on the basis of their Stokes radius, not molecular weight. The molecular weight can be determined by combining gradient sedimentation and gel filtration, techniques available in most biochemistry laboratories, as originally proposed by Siegel and Monte. Finally, rotary shadowing and negative stain electron microscopy are powerful techniques for resolving the size and shape of single protein molecules and complexes at the nanometer level. A combination of hydrodynamics and electron microscopy is especially powerful.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. (Published online)" Biol Proced Online 11 (May 15, 2009): 32-51.
PMID
19495910
Source
pubmed
Published In
Biol Proced Online
Volume
11
Publish Date
2009
Start Page
32
End Page
51
DOI
10.1007/s12575-009-9008-x

FtsZ condensates: an in vitro electron microscopy study.

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto-filaments. Although FtsZ assembles into single proto-filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z-ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy.

Authors
Popp, D; Iwasa, M; Narita, A; Erickson, HP; Maéda, Y
MLA Citation
Popp, D, Iwasa, M, Narita, A, Erickson, HP, and Maéda, Y. "FtsZ condensates: an in vitro electron microscopy study." Biopolymers 91.5 (May 2009): 340-350.
PMID
19137575
Source
pubmed
Published In
Biopolymers
Volume
91
Issue
5
Publish Date
2009
Start Page
340
End Page
350
DOI
10.1002/bip.21136

Revisiting the mystery of fibronectin multimers: the fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds.

Fibronectin (FN) matrix fibrils have long been thought to be formed by disulfide-bonded FN multimers, although there is no direct evidence that they are covalently linked with each other. To understand the biochemical properties of these fibrils, we extracted a crude FN matrix from FN-YPet transfected 3T3 cell culture using 0.2% deoxycholate and DNase. The insoluble extracted matrix preserved fibrillar structures and a major portion of the extracted proteins migrated as FN monomers on an SDS gel under reducing conditions. Under non-reducing conditions, some FN molecules appeared to be trapped at the top of the stacking gel. We tested this by mixing fluorescently labeled FN dimers with the extracted matrix just before loading on an SDS gel, and found that most of them were trapped with the extracted proteins at the top of the stacking gel. These results suggested that some components of the extracted matrix plugged the stacking gel and FN dimers were trapped with them. Rotary shadowing electron microscopy showed that the extracted matrix had some fibers that resembled fibrillin microfibrils. Peptide mass fingerprinting confirmed the presence of fibrillin in the extracted matrix. Fibrillin is known to form disulfide-bonded multimers and it is likely to be one of the components that plug the stacking gel and trap FN molecules in this system. The phenomenon by which FN molecules appear to migrate as multimers on SDS gels is thus an artifact rising from the presence of other large components in the extract. We conclude that FN matrix fibrils are made of FN dimers that are further cross-linked by non-covalent protein-protein bonds.

Authors
Ohashi, T; Erickson, HP
MLA Citation
Ohashi, T, and Erickson, HP. "Revisiting the mystery of fibronectin multimers: the fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds." Matrix Biol 28.3 (April 2009): 170-175.
PMID
19285555
Source
pubmed
Published In
Matrix Biology
Volume
28
Issue
3
Publish Date
2009
Start Page
170
End Page
175
DOI
10.1016/j.matbio.2009.03.002

Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

Authors
Xu, J; Bae, E; Zhang, Q; Annis, DS; Erickson, HP; Mosher, DF
MLA Citation
Xu, J, Bae, E, Zhang, Q, Annis, DS, Erickson, HP, and Mosher, DF. "Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin." PLoS One 4.1 (2009): e4113-.
Website
http://hdl.handle.net/10161/4509
PMID
19119318
Source
pubmed
Published In
PloS one
Volume
4
Issue
1
Publish Date
2009
Start Page
e4113
DOI
10.1371/journal.pone.0004113

Chapter 1 - Tubular liposomes with variable permeability for reconstitution of FtsZ rings.

We have developed a system for producing tubular multilamellar liposomes that incorporate the protein FtsZ on the inside. We start with a mixture of spherical multilamellar liposomes with FtsZ initially on the outside. Shearing forces generated by applying a coverslip most likely distort some of the spherical liposomes into a tubular shape, and causes some to leak and incorporate FtsZ inside. We describe protocols for liposome preparation, and for preparing membrane-targeted FtsZ that can assemble contractile Z rings inside the tubular liposomes. We also describe the characterization of the multilamellar liposomes in terms of the permeability or leakiness for a small fluorescent dye and larger protein molecules. These liposomes may be useful for reconstitution of other biological systems.

Authors
Osawa, M; Erickson, HP
MLA Citation
Osawa, M, and Erickson, HP. "Chapter 1 - Tubular liposomes with variable permeability for reconstitution of FtsZ rings." Methods Enzymol 464 (2009): 3-17.
PMID
19903547
Source
pubmed
Published In
Methods in Enzymology
Volume
464
Publish Date
2009
Start Page
3
End Page
17
DOI
10.1016/S0076-6879(09)64001-5

The coiled coils of cohesin are conserved in animals, but not in yeast.

BACKGROUND: The SMC proteins are involved in DNA repair, chromosome condensation, and sister chromatid cohesion throughout Eukaryota. Long, anti-parallel coiled coils are a prominent feature of SMC proteins, and are thought to serve as spacer rods to provide an elongated structure and to separate domains. We reported recently that the coiled coils of mammalian condensin (SMC2/4) showed moderate sequence divergence (approximately 10-15%) consistent with their functioning as spacer rods. The coiled coils of mammalian cohesins (SMC1/3), however, were very highly constrained, with amino acid sequence divergence typically <0.5%. These coiled coils are among the most highly conserved mammalian proteins, suggesting that they make extensive contacts over their entire surface. METHODOLOGY/PRINCIPAL FINDINGS: Here, we broaden our initial analysis of condensin and cohesin to include additional vertebrate and invertebrate organisms and multiple species of yeast. We found that the coiled coils of SMC1/3 are highly constrained in Drosophila and other insects, and more generally across all animal species. However, in yeast they are no more constrained than the coils of SMC2/4 and Ndc80/Nuf2p, suggesting that they are serving primarily as spacer rods. CONCLUSIONS/SIGNIFICANCE: SMC1/3 functions for sister chromatid cohesion in all species. Since its coiled coils apparently serve only as spacer rods in yeast, it is likely that this is sufficient for sister chromatid cohesion in all species. This suggests an additional function in animals that constrains the sequence of the coiled coils. Several recent studies have demonstrated that cohesin has a role in gene expression in post-mitotic neurons of Drosophila, and other animal cells. Some variants of human Cornelia de Lange Syndrome involve mutations in human SMC1/3. We suggest that the role of cohesin in gene expression may involve intimate contact of the coiled coils of SMC1/3, and impose the constraint on sequence divergence.

Authors
White, GE; Erickson, HP
MLA Citation
White, GE, and Erickson, HP. "The coiled coils of cohesin are conserved in animals, but not in yeast." PLoS One 4.3 (2009): e4674-.
PMID
19262687
Source
pubmed
Published In
PloS one
Volume
4
Issue
3
Publish Date
2009
Start Page
e4674
DOI
10.1371/journal.pone.0004674

Reconstitution of contractile FtsZ rings in liposomes.

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation.

Authors
Osawa, M; Anderson, DE; Erickson, HP
MLA Citation
Osawa, M, Anderson, DE, and Erickson, HP. "Reconstitution of contractile FtsZ rings in liposomes." Science 320.5877 (May 9, 2008): 792-794.
PMID
18420899
Source
pubmed
Published In
Science
Volume
320
Issue
5877
Publish Date
2008
Start Page
792
End Page
794
DOI
10.1126/science.1154520

In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.

Proteins with a weak sequence similarity to tubulin and FtsZ are expressed from large plasmids of Bacillus anthracis and Bacillus thuringiensis and are probably involved in plasmid segregation. Previously designated RepX and TubZ, we designate them here as TubZ-Ba and TubZ-Bt. We have expressed and purified the proteins for in vitro studies. TubZ-Ba and TubZ-Bt share only 21% amino acid identity, but they have remarkably similar biochemical properties. They both assemble into two-stranded filaments and larger bundles above a critical concentration, and they hydrolyze GTP at a very high rate, approximately 20 GTP min(-1) TubZ(-1). Assembly is also supported by GTPgammaS. A tiny amount of GTPgammaS stabilizes polymers assembled in GTP and inhibits the GTPase by a mechanism involving cooperativity. The nucleotide in the polymers is almost 100% GDP, which is similar to microtubules but very different from the 20-30% GDP in FtsZ polymers. This suggests that the TubZ polymers have a capping mechanism that may be related to the GTP cap that produces dynamic instability of microtubules.

Authors
Chen, Y; Erickson, HP
MLA Citation
Chen, Y, and Erickson, HP. "In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism." J Biol Chem 283.13 (March 28, 2008): 8102-8109.
PMID
18198178
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
13
Publish Date
2008
Start Page
8102
End Page
8109
DOI
10.1074/jbc.M709163200

Synergistic actions of hematopoietic and mesenchymal stem/progenitor cells in vascularizing bioengineered tissues.

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34(+) hematopoietic cells. Co-transplantation of CD34(+) HSCs and CD34(-) MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromised mice yielded vascularized tissue. The average vascular number of co-transplanted CD34(+) and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34(+) cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34(+) cells. Based on additional in vitro results of endothelial differentiation of CD34(+) cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34(+) and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34(+) cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.

Authors
Moioli, EK; Clark, PA; Chen, M; Dennis, JE; Erickson, HP; Gerson, SL; Mao, JJ
MLA Citation
Moioli, EK, Clark, PA, Chen, M, Dennis, JE, Erickson, HP, Gerson, SL, and Mao, JJ. "Synergistic actions of hematopoietic and mesenchymal stem/progenitor cells in vascularizing bioengineered tissues." PLoS One 3.12 (2008): e3922-.
PMID
19081793
Source
pubmed
Published In
PloS one
Volume
3
Issue
12
Publish Date
2008
Start Page
e3922
DOI
10.1371/journal.pone.0003922

Reconstitution of contractile FtsZ rings in liposomes

FtsZ, a tubulin homolog, is the major protein involved in cell division in bacteria and archaea. FtsZ assembles along with a number of other proteins into the Z ring, which constricts to divide the cell. This investigation examined the nature of the FtsZ filaments and a Z ring that it forms in bacterial cells, FtsZ attachment to the membrane, and the generation of the constriction force essential for cell division to occur. It had previously been shown that attachment of FtsZ to the membrane is through another protein, FtsA, which has an amphipatic helix that anchors FtsA to the cell membrane. The authors thus constructed a membrane-targeted FtsZ, called FtsZ-mts, in which the segment of FtsZ that binds FtsA was replaced with yellow fluorescent protein (YFP) followed by an amphipathic helix. The authors showed that in Escherichia coli and in a liposome system in vitro, FtsZ attachment is dependent only on the amphipatic anchor to the membrane (i.e., intact FtsA is not required for Z ring formation). Addition of FtsZ-mts and GTP in tubular liposome preparations resulted in the incorporation of FtsZ and the GTP-dependent formation of multiple dim Z rings that moved laterally, coalescing into brighter Z rings. Formation of bright Z rings was succeeded by the appearance of constrictions in the liposome walls (Fig. 1). The force to create constrictions in the liposome was thus generated by FtsZ, either through the alteration of its conformation upon GTP hydrolysis or by creating force in the membrane through the insertion of the anchor of amphipatic helices. The force distorts the membrane, causing the protofilaments to form closed rings that oriented perpendicular to the axis of the tubular liposome. The authors thus concluded that Z ring formation and constriction of the membrane required only FtsZ and attachment to the membrane, which may be mediated by any amphipatic helix. © 2008 Data Trace Publishing Company.

Authors
Osawa, M; Anderson, DA; Erickson, HP
MLA Citation
Osawa, M, Anderson, DA, and Erickson, HP. "Reconstitution of contractile FtsZ rings in liposomes." Chemtracts 21.6 (2008): 222-223.
Source
scival
Published In
Chemtracts
Volume
21
Issue
6
Publish Date
2008
Start Page
222
End Page
223

Assembly dynamics of Mycobacterium tuberculosis FtsZ.

We have investigated the assembly of FtsZ from Mycobacterium tuberculosis (MtbFtsZ). Electron microscopy confirmed the previous observation that MtbFtsZ assembled into long, two-stranded filaments at pH 6.5. However, we found that assembly at pH 7.2 or 7.7 produced predominantly short, one-stranded protofilaments, similar to those of Escherichia coli FtsZ (EcFtsZ). Near pH 7, which is close to the pH of M. tuberculosis cytoplasm, MtbFtsZ formed a mixture of single- and two-stranded filaments. We developed a fluorescence resonance energy transfer assay to measure the kinetics of initial assembly and the dynamic properties at steady state. Assembly of MtbFtsZ reached a plateau after 60-100 s, about 10 times slower than EcFtsZ. The initial assembly kinetics were similar at pH 6.5 and 7.7, despite the striking difference in the polymer structures. Both were fit with a cooperative assembly mechanism involving a weak dimer nucleus, similar to EcFtsZ but with slower kinetics. Subunit turnover and GTPase at steady state were also about 10 times slower for MtbFtsZ than for EcFtsZ. Specifically, the half-time for subunit turnover in vitro at pH 7.7 was 42 s for MtbFtsZ compared with 5.5 s for EcFtsZ. Photobleaching studies in vivo showed a range of turnover half-times with an average of 25 s for MtbFtsZ as compared with 9 s for EcFtsZ.

Authors
Chen, Y; Anderson, DE; Rajagopalan, M; Erickson, HP
MLA Citation
Chen, Y, Anderson, DE, Rajagopalan, M, and Erickson, HP. "Assembly dynamics of Mycobacterium tuberculosis FtsZ." J Biol Chem 282.38 (September 21, 2007): 27736-27743.
PMID
17644520
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
38
Publish Date
2007
Start Page
27736
End Page
27743
DOI
10.1074/jbc.M703788200

The RGD motif in fibronectin is essential for development but dispensable for fibril assembly.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

Authors
Takahashi, S; Leiss, M; Moser, M; Ohashi, T; Kitao, T; Heckmann, D; Pfeifer, A; Kessler, H; Takagi, J; Erickson, HP; Fässler, R
MLA Citation
Takahashi, S, Leiss, M, Moser, M, Ohashi, T, Kitao, T, Heckmann, D, Pfeifer, A, Kessler, H, Takagi, J, Erickson, HP, and Fässler, R. "The RGD motif in fibronectin is essential for development but dispensable for fibril assembly." J Cell Biol 178.1 (July 2, 2007): 167-178.
PMID
17591922
Source
pubmed
Published In
The Journal of Cell Biology
Volume
178
Issue
1
Publish Date
2007
Start Page
167
End Page
178
DOI
10.1083/jcb.200703021

Evolution of the cytoskeleton.

The eukaryotic cytoskeleton appears to have evolved from ancestral precursors related to prokaryotic FtsZ and MreB. FtsZ and MreB show 40-50% sequence identity across different bacterial and archaeal species. Here I suggest that this represents the limit of divergence that is consistent with maintaining their functions for cytokinesis and cell shape. Previous analyses have noted that tubulin and actin are highly conserved across eukaryotic species, but so divergent from their prokaryotic relatives as to be hardly recognizable from sequence comparisons. One suggestion for this extreme divergence of tubulin and actin is that it occurred as they evolved very different functions from FtsZ and MreB. I will present new arguments favoring this suggestion, and speculate on pathways. Moreover, the extreme conservation of tubulin and actin across eukaryotic species is not due to an intrinsic lack of variability, but is attributed to their acquisition of elaborate mechanisms for assembly dynamics and their interactions with multiple motor and binding proteins. A new structure-based sequence alignment identifies amino acids that are conserved from FtsZ to tubulins. The highly conserved amino acids are not those forming the subunit core or protofilament interface, but those involved in binding and hydrolysis of GTP.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Evolution of the cytoskeleton." Bioessays 29.7 (July 2007): 668-677. (Review)
PMID
17563102
Source
pubmed
Published In
Bioessays
Volume
29
Issue
7
Publish Date
2007
Start Page
668
End Page
677
DOI
10.1002/bies.20601

An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins.

We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.

Authors
Ohashi, T; Galiacy, SD; Briscoe, G; Erickson, HP
MLA Citation
Ohashi, T, Galiacy, SD, Briscoe, G, and Erickson, HP. "An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins." Protein Sci 16.7 (July 2007): 1429-1438.
PMID
17586775
Source
pubmed
Published In
Protein Science
Volume
16
Issue
7
Publish Date
2007
Start Page
1429
End Page
1438
DOI
10.1110/ps.072845607

Designing an extracellular matrix protein with enhanced mechanical stability.

The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by approximately 20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions.

Authors
Ng, SP; Billings, KS; Ohashi, T; Allen, MD; Best, RB; Randles, LG; Erickson, HP; Clarke, J
MLA Citation
Ng, SP, Billings, KS, Ohashi, T, Allen, MD, Best, RB, Randles, LG, Erickson, HP, and Clarke, J. "Designing an extracellular matrix protein with enhanced mechanical stability." Proc Natl Acad Sci U S A 104.23 (June 5, 2007): 9633-9637.
PMID
17535921
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
23
Publish Date
2007
Start Page
9633
End Page
9637
DOI
10.1073/pnas.0609901104

FtsZ from divergent foreign bacteria can function for cell division in Escherichia coli.

FtsZs from Mycoplasma pulmonis (MpuFtsZ) and Bacillus subtilis (BsFtsZ) are only 46% and 53% identical in amino acid sequence to FtsZ from Escherichia coli (EcFtsZ). In the present study we show that MpuFtsZ and BsFtsZ can function for cell division in E. coli provided we make two modifications. First, we replaced their C-terminal tails with that from E. coli, giving the foreign FtsZ the binding site for E. coli FtsA and ZipA. Second, we selected for mutations in the E. coli genome that facilitated division by the foreign FtsZs. These suppressor strains arose at a relatively high frequency of 10(-3) to 10(-5), suggesting that they involve loss-of-function mutations in multigene pathways. These pathways may be negative regulators of FtsZ or structural pathways that facilitate division by slightly defective FtsZ. Related suppressor strains were obtained for EcFtsZ containing certain point mutations or insertions of yellow fluorescent protein. The ability of highly divergent FtsZs to function for division in E. coli is consistent with a two-part mechanism. FtsZ assembles the Z ring, and perhaps generates the constriction force, through self interactions; the downstream division proteins remodel the peptidoglycan wall by interacting with each other and the wall. The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling.

Authors
Osawa, M; Erickson, HP
MLA Citation
Osawa, M, and Erickson, HP. "FtsZ from divergent foreign bacteria can function for cell division in Escherichia coli." J Bacteriol 188.20 (October 2006): 7132-7140.
PMID
17015652
Source
pubmed
Published In
Journal of bacteriology
Volume
188
Issue
20
Publish Date
2006
Start Page
7132
End Page
7140
DOI
10.1128/JB.00647-06

Sequence divergence of coiled coils--structural rods, myosin filament packing, and the extraordinary conservation of cohesins.

The amino acid sequences of the long, anti-parallel coiled coils of the cohesin subunits SMC1 and SMC3 are almost totally conserved in mammals. To understand this exceptional conservation more broadly, we analyzed amino acid sequence variation for several groups of coiled-coil proteins. Some long coiled coils, including giantin, NuMA, and Ndc80p/Nuf2p diverge approximately 20% from humans to rodents, suggesting they function as spacer rods, whose sequence divergence is constrained only by the need to maintain the coiled-coil structure. Other coiled coils such as skeletal muscle myosin, intermediate filaments, and the lamins diverge only 1-3%. We suggest that this sequence divergence is constrained by the extensive packing contacts over the entire surface of the coiled-coil. The coiled coils of SMC5/6 and SMC2/4 (condensin) are slightly more constrained than the presumed spacer rods, diverging 10-15%. Conversely, the coiled coils of SMC1/3 (cohesin) diverge only 0.0-1.0%. This extreme constraint suggests that the entire surface of the coiled coil is intimately involved in the mechanism of sister chromatid cohesion. Direct binding of the coiled coils to chromatin, or perhaps the need to avoid such binding, are two possible mechanisms. Finally, analysis of the heptad repeat shows that the a and d positions are more constrained in spacer rods, and the bcefg positions more constrained in skeletal muscle myosin.

Authors
White, GE; Erickson, HP
MLA Citation
White, GE, and Erickson, HP. "Sequence divergence of coiled coils--structural rods, myosin filament packing, and the extraordinary conservation of cohesins." J Struct Biol 154.2 (May 2006): 111-121.
PMID
16495084
Source
pubmed
Published In
Journal of Structural Biology
Volume
154
Issue
2
Publish Date
2006
Start Page
111
End Page
121
DOI
10.1016/j.jsb.2006.01.001

Understanding the elasticity of fibronectin fibrils: unfolding strengths of FN-III and GFP domains measured by single molecule force spectroscopy.

While it is well established that fibronectin (FN) matrix fibrils are elastic, the mechanism of fibril elasticity during extension is still debated. To investigate the molecular origin of FN fibril elasticity, we used single molecule force spectroscopy (SMFS) to determine the unfolding behavior of a recombinant FN-III protein construct that contained eight FN-III domains ((1-8)FN-III) and two green fluorescent protein (GFP) domains. FN-III domains were distinguished from GFP domains by their shorter unfolding lengths. The unfolding strengths of both domains were determined for a wide range of pulling rates (50 to 1,745 nm/s). We found that the mechanical stabilities of FN-III and GFP domains were very similar to each other over the entire range of pulling speeds. FN fibrils containing GFP remain brightly fluorescent, even when stretched, meaning that GFP domains remain largely folded. Since GFP and FN-III have equal unfolding strengths, this suggests that FN-III domains are not extensively unraveled in stretched FN fibrils. Our results thus favor an alternative model, which invokes a conformational change from a compact to an extended conformation, as the basis for FN fibril elasticity.

Authors
Abu-Lail, NI; Ohashi, T; Clark, RL; Erickson, HP; Zauscher, S
MLA Citation
Abu-Lail, NI, Ohashi, T, Clark, RL, Erickson, HP, and Zauscher, S. "Understanding the elasticity of fibronectin fibrils: unfolding strengths of FN-III and GFP domains measured by single molecule force spectroscopy." Matrix Biol 25.3 (April 2006): 175-184.
PMID
16343877
Source
pubmed
Published In
Matrix Biology
Volume
25
Issue
3
Publish Date
2006
Start Page
175
End Page
184
DOI
10.1016/j.matbio.2005.10.007

Probing the domain structure of FtsZ by random truncation and insertion of GFP.

Random transposon-mediated mutagenesis has been used to create truncations and insertions of green fluorescent protein (GFP), and Venus-yellow fluorescent protein (YFP), in Escherichia coli FtsZ. Sixteen unique insertions were obtained, and one of them, in the poorly conserved C-terminal spacer, was functional for cell division with the Venus-YFP insert. The insertion of enhanced GFP (eGFP) at this same site was not functional; Venus-YFP was found to be superior to eGFP in other respects too. Testing the constructs for dominant negative effects led to the following general conclusion. The N-terminal domain, aa 1-195, is an independently folding domain that can poison Z-ring function when expressed without a functional C-terminal domain. The effects were weak, requiring expression of the mutant at 3-5 times the level of wild-type FtsZ. The C-terminal domain, aa 195-383, was also independently folding, but had no activity in vivo. The differential activity of the N- and C-terminal domains suggests that FtsZ protofilament assembly is directional, with subunits adding primarily at the bottom of the protofilament. Directional assembly could occur by either a treadmilling or a dynamic instability mechanism.

Authors
Osawa, M; Erickson, HP
MLA Citation
Osawa, M, and Erickson, HP. "Probing the domain structure of FtsZ by random truncation and insertion of GFP." Microbiology 151.Pt 12 (December 2005): 4033-4043.
PMID
16339948
Source
pubmed
Published In
Microbiology (Reading, England)
Volume
151
Issue
Pt 12
Publish Date
2005
Start Page
4033
End Page
4043
DOI
10.1099/mic.0.28219-0

Domain unfolding plays a role in superfibronectin formation.

Superfibronectin (sFN) is a fibronectin (FN) aggregate that is formed by mixing FN with anastellin, a fragment of the first type III domain of FN. However, the mechanism of this aggregation has not been clear. In this study, we found that anastellin co-precipitated with FN in a ratio of approximately 4:1, anastellin:FN monomer. The primary binding site for anastellin was in the segment (III)1-3, which bound three molecules of anastellin and was able to form a precipitate without the rest of the FN molecule. Anastellin binding to (III)3 caused a conformational change in that domain that exposed a cryptic thermolysin-sensitive site. An additional anastellin binds to (III)11, where it enhances thermolysin digestion of (III)11. An engineered disulfide bond in (III)3 inhibited both aggregation and protease digestion, suggesting that the stability of (III)3 is a key factor in sFN formation. We propose a three-step model for sFN formation: 1) FN-III domains spontaneously unfold and refold; 2) anastellin binds to an unfolded domain, preventing its refolding and leaving it with exposed hydrophobic surfaces and beta-sheet edges; and 3) these exposed elements bind to similar exposed elements on other molecules, leading to aggregation. The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix.

Authors
Ohashi, T; Erickson, HP
MLA Citation
Ohashi, T, and Erickson, HP. "Domain unfolding plays a role in superfibronectin formation." J Biol Chem 280.47 (November 25, 2005): 39143-39151.
PMID
16195231
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
47
Publish Date
2005
Start Page
39143
End Page
39151
DOI
10.1074/jbc.M509082200

Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer.

We have developed an assay for the assembly of FtsZ based on fluorescence resonance energy transfer (FRET). We mutated an innocuous surface residue to cysteine and labeled separate pools with fluorescein (donor) and tetramethylrhodamine (acceptor). When the pools were mixed and GTP was added, assembly produced a FRET signal that was linearly proportional to FtsZ concentration from 0.7 microm (the critical concentration (C(c))) to 3 microm. At concentrations greater than 3 microm, an enhanced FRET signal was observed with both GTP and GDP, indicating additional assembly above this second C(c). This second C(c) varied with Mg(2+) concentration, whereas the 0.7 microm C(c) did not. We used the FRET assay to measure the kinetics of initial assembly by stopped flow. The data were fit by the simple kinetic model used previously: monomer activation, a weak dimer nucleus, and elongation, although with some differences in kinetic parameters from the L68W mutant. We then studied the rate of turnover at steady state by pre-assembling separate pools of donor and acceptor protofilaments. When the pools were mixed, a FRET signal developed with a half-time of 7 s, demonstrating a rapid and continuous disassembly and reassembly of protofilaments at steady state. This is comparable with the 9-s half-time for FtsZ turnover in vivo and the 8-s turnover time of GTP hydrolysis in vitro. Finally, we found that an excess of GDP caused disassembly of protofilaments with a half-time of 5 s. Our new data suggest that GDP does not exchange into intact protofilaments. Rather, our interpretation is that subunits are released following GTP hydrolysis, and then they exchange GDP for GTP and reassemble into new protofilaments, all on a time scale of 7 s. The mechanism may be related to the dynamic instability of microtubules.

Authors
Chen, Y; Erickson, HP
MLA Citation
Chen, Y, and Erickson, HP. "Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer." J Biol Chem 280.23 (June 10, 2005): 22549-22554.
PMID
15826938
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
23
Publish Date
2005
Start Page
22549
End Page
22554
DOI
10.1074/jbc.M500895200

In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB.

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35-36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4-7 protofilaments wide (20-30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4-1.0 microM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

Authors
Sontag, CA; Staley, JT; Erickson, HP
MLA Citation
Sontag, CA, Staley, JT, and Erickson, HP. "In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB." J Cell Biol 169.2 (April 25, 2005): 233-238.
PMID
15851515
Source
pubmed
Published In
The Journal of Cell Biology
Volume
169
Issue
2
Publish Date
2005
Start Page
233
End Page
238
DOI
10.1083/jcb.200410027

Mutants of FtsZ targeting the protofilament interface: effects on cell division and GTPase activity.

The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 microM, in contrast to the high GTPase activity of wild-type FtsZ above 1 muM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 microM K(d) for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.

Authors
Redick, SD; Stricker, J; Briscoe, G; Erickson, HP
MLA Citation
Redick, SD, Stricker, J, Briscoe, G, and Erickson, HP. "Mutants of FtsZ targeting the protofilament interface: effects on cell division and GTPase activity." J Bacteriol 187.8 (April 2005): 2727-2736.
PMID
15805519
Source
pubmed
Published In
Journal of bacteriology
Volume
187
Issue
8
Publish Date
2005
Start Page
2727
End Page
2736
DOI
10.1128/JB.187.8.2727-2736.2005

A rapid fluorescence assay for FtsZ assembly indicates cooperative assembly with a dimer nucleus.

FtsZ is the major cytoskeletal protein operating in bacterial cell division. FtsZ assembles into protofilaments in vitro, and there has been some controversy over whether the assembly is isodesmic or cooperative. Assembly has been assayed previously by sedimentation and light scattering. However, these techniques will under-report small polymers. We have now produced a mutant of Escherichia coli FtsZ, L68W, which gives a 250% increase in tryptophan fluorescence upon polymerization. This provides a real-time assay of polymer that is directly proportional to the concentration of subunit interfaces. FtsZ-L68W is functional for cell division, and should therefore be a valid model for studying the thermodynamics and kinetics of FtsZ assembly. We assayed assembly at pH 7.7 and pH 6.5, in 2.5 mM EDTA. EDTA blocks GTP hydrolysis and should give an assembly reaction that is not complicated by the irreversible hydrolysis step. Assembly kinetics was determined with a stopped-flow device for a range of FtsZ concentrations. When assembly was initiated by adding 0.2 mM GTP, fluorescence increase showed a lag, followed by nucleation, elongation, and a plateau. The assembly curves were fit to a cooperative mechanism that included a monomer activation step, a weak dimer nucleus, and elongation. Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?

Authors
Chen, Y; Bjornson, K; Redick, SD; Erickson, HP
MLA Citation
Chen, Y, Bjornson, K, Redick, SD, and Erickson, HP. "A rapid fluorescence assay for FtsZ assembly indicates cooperative assembly with a dimer nucleus." Biophys J 88.1 (January 2005): 505-514.
PMID
15475583
Source
pubmed
Published In
Biophysical Journal
Volume
88
Issue
1
Publish Date
2005
Start Page
505
End Page
514
DOI
10.1529/biophysj.104.044149

Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins.

FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (approximately 9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an approximately 3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, approximately 8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

Authors
Anderson, DE; Gueiros-Filho, FJ; Erickson, HP
MLA Citation
Anderson, DE, Gueiros-Filho, FJ, and Erickson, HP. "Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins." J Bacteriol 186.17 (September 2004): 5775-5781.
PMID
15317782
Source
pubmed
Published In
Journal of bacteriology
Volume
186
Issue
17
Publish Date
2004
Start Page
5775
End Page
5781
DOI
10.1128/JB.186.17.5775-5781.2004

Localization of a cryptic binding site for tenascin on fibronectin.

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.

Authors
Ingham, KC; Brew, SA; Erickson, HP
MLA Citation
Ingham, KC, Brew, SA, and Erickson, HP. "Localization of a cryptic binding site for tenascin on fibronectin." J Biol Chem 279.27 (July 2, 2004): 28132-28135.
PMID
15123658
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
27
Publish Date
2004
Start Page
28132
End Page
28135
DOI
10.1074/jbc.M312785200

The disulfide bonding pattern in ficolin multimers.

Ficolin is a plasma lectin, consisting of a short N-terminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin alpha in which the N-terminal cysteines were substituted by serines (Cys4, Cys24, and Cys4/Cys24). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfide-linked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers. The Cys4 mutant also formed 12-mers, but Cys24 and Cys4/Cys24 mutants formed only trimers. This means that protein interfaces containing Cys4 are stable as noncovalent protein-protein interactions and do not require disulfides, whereas those containing Cys24-Cys24 require the disulfides for stability. Proteins were also analyzed by nonreducing SDS-PAGE to show the covalent structure under denaturing conditions. Wild-type ficolin was covalently linked into 12-mers, whereas elimination of either Cys4 or Cys24 gave dimers and monomers. We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers.

Authors
Ohashi, T; Erickson, HP
MLA Citation
Ohashi, T, and Erickson, HP. "The disulfide bonding pattern in ficolin multimers." J Biol Chem 279.8 (February 20, 2004): 6534-6539.
PMID
14660572
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
8
Publish Date
2004
Start Page
6534
End Page
6539
DOI
10.1074/jbc.M310555200

In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function.

We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.

Authors
Stricker, J; Erickson, HP
MLA Citation
Stricker, J, and Erickson, HP. "In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function." J Bacteriol 185.16 (August 2003): 4796-4805.
PMID
12896999
Source
pubmed
Published In
Journal of bacteriology
Volume
185
Issue
16
Publish Date
2003
Start Page
4796
End Page
4805

Apparent cooperative assembly of the bacterial cell division protein FtsZ demonstrated by isothermal titration calorimetry.

The assembly dynamics of FtsZ, a prokaryotic homolog of tubulin, are important for their role in bacterial cytokinesis. Here we used isothermal titration calorimetry (ITC) to measure the heat of FtsZ self-association under various conditions. The measurements were designed to test whether FtsZ protofilaments are assembled by an isodesmic (linear aggregates in which each bond has an identical equilibrium constant) or a cooperative (aggregates only become stable after forming a oligomeric nucleus) assembly process. The isodesmic model can fit the assembly in GDP closely but cannot fit the assembly in GTP. FtsZ-GTP without Mg(2+) exhibits an apparent critical concentration, which is indicative of cooperative assembly, near 2.9 microm. With 2.5 mm Mg(2+) (which allows FtsZ to hydrolyze GTP) the critical concentration is reduced 10-fold to approximately 0.31 microm. Both with and without Mg(2+) there is no evidence for assembly below the critical concentration, but there is an abrupt transition to full assembly above. The ITC data are highly suggestive of a cooperative assembly, although this is difficult to reconcile with the 1-subunit-thick protofilaments observed by electron microscopy.

Authors
Caplan, MR; Erickson, HP
MLA Citation
Caplan, MR, and Erickson, HP. "Apparent cooperative assembly of the bacterial cell division protein FtsZ demonstrated by isothermal titration calorimetry." J Biol Chem 278.16 (April 18, 2003): 13784-13788.
PMID
12566454
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
16
Publish Date
2003
Start Page
13784
End Page
13788
DOI
10.1074/jbc.M300860200

Force measurements of the alpha(5)beta(1) integrin-fibronectin interaction

Authors
Li, FY; Redick, SD; Erickson, HP; Moy, VT
MLA Citation
Li, FY, Redick, SD, Erickson, HP, and Moy, VT. "Force measurements of the alpha(5)beta(1) integrin-fibronectin interaction." BIOPHYSICAL JOURNAL 84.2 (February 2003): 1252-1262.
Source
wos-lite
Published In
Biophysical Journal
Volume
84
Issue
2
Publish Date
2003
Start Page
1252
End Page
1262

Force measurements of the alpha5beta1 integrin-fibronectin interaction.

The interaction of the alpha(5)beta(1) integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the alpha(5)beta(1)/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between alpha(5)beta(1) and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an alpha(5)beta(1) expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the alpha(5)beta(1)/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual alpha(5)beta(1)/FN7-10 interactions. The dynamic rupture force of the alpha(5)beta(1)/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the alpha(5)beta(1)/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.

Authors
Li, F; Redick, SD; Erickson, HP; Moy, VT
MLA Citation
Li, F, Redick, SD, Erickson, HP, and Moy, VT. "Force measurements of the alpha5beta1 integrin-fibronectin interaction." Biophys J 84.2 Pt 1 (February 2003): 1252-1262.
PMID
12547805
Source
pubmed
Published In
Biophysical Journal
Volume
84
Issue
2 Pt 1
Publish Date
2003
Start Page
1252
End Page
1262
DOI
10.1016/S0006-3495(03)74940-6

Macromolecular assemblages - Many structures to function

Authors
Cate, JHD; Erickson, HP
MLA Citation
Cate, JHD, and Erickson, HP. "Macromolecular assemblages - Many structures to function." Current Opinion in Structural Biology 13.2 (2003): 203-205.
Source
scival
Published In
Current Opinion in Structural Biology
Volume
13
Issue
2
Publish Date
2003
Start Page
203
End Page
205
DOI
10.1016/S0959-440X(03)00043-5

Force measurements of the α5β1 integrin-fibronectin interaction

The interaction of the α5β1 integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α5β1/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α5β1 and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α5β1 expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α5β1/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α5β1/FN7-10 interactions. The dynamic rupture force of the α5β1/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α5β1/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.

Authors
Li, F; Redick, SD; Erickson, HP; Moy, VT
MLA Citation
Li, F, Redick, SD, Erickson, HP, and Moy, VT. "Force measurements of the α5β1 integrin-fibronectin interaction." Biophysical Journal 84.2 I (2003): 1252-1262.
Source
scival
Published In
Biophysical Journal
Volume
84
Issue
2 I
Publish Date
2003
Start Page
1252
End Page
1262
DOI
10.1016/S0006-3495(03)74940-6

Structural evidence that the P/Q domain of ZipA is an unstructured, flexible tether between the membrane and the C-terminal FtsZ-binding domain.

The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.

Authors
Ohashi, T; Hale, CA; de Boer, PAJ; Erickson, HP
MLA Citation
Ohashi, T, Hale, CA, de Boer, PAJ, and Erickson, HP. "Structural evidence that the P/Q domain of ZipA is an unstructured, flexible tether between the membrane and the C-terminal FtsZ-binding domain." J Bacteriol 184.15 (August 2002): 4313-4315.
PMID
12107152
Source
pubmed
Published In
Journal of bacteriology
Volume
184
Issue
15
Publish Date
2002
Start Page
4313
End Page
4315

Tenascin-C expression and distribution in cultured human chondrocytes and chondrosarcoma cells.

Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix with several distinct isoforms variably expressed during embryogenesis, tumorogenesis, angiogenesis and wound healing. In the normal human adult, TNC is found in large concentrations in articular cartilage, suggesting tissue-specific function. The purpose of this study was to determine the specific in vitro TNC splicing patterns of articular chondrocytes and a human chondrosarcoma cell line. Cells were cultured in a three-dimensional bead system and TNC splice variant expression and distribution were examined with the use of Western blotting techniques, semi-quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. At both the transcriptional and post-translational levels, the chondrocytes were found to express significantly higher levels of the smaller 220 kDa isoform (P < 0.01), which was predominantly incorporated into the matrix. The splicing pattern of the malignant cells was characterized by a higher proportion of the larger 320 kDa isoform which was extruded into the media. In vivo studies are necessary to verify the expression of the large TNC isoform in chondrosarcoma and the production and integration of the smaller isoform in normal chondroid matrix. In addition, elucidation of the biologic functions of the two major TNC isoforms may lead to the development of novel diagnostic and therapeutic approaches to chondrosarcoma.

Authors
Ghert, MA; Qi, W-N; Erickson, HP; Block, JA; Scully, SP
MLA Citation
Ghert, MA, Qi, W-N, Erickson, HP, Block, JA, and Scully, SP. "Tenascin-C expression and distribution in cultured human chondrocytes and chondrosarcoma cells." J Orthop Res 20.4 (July 2002): 834-841.
PMID
12168675
Source
pubmed
Published In
Journal of Orthopaedic Research
Volume
20
Issue
4
Publish Date
2002
Start Page
834
End Page
841
DOI
10.1016/S0736-0266(01)00172-3

Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation.

Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10, on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton.

Authors
Coussen, F; Choquet, D; Sheetz, MP; Erickson, HP
MLA Citation
Coussen, F, Choquet, D, Sheetz, MP, and Erickson, HP. "Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation." J Cell Sci 115.Pt 12 (June 15, 2002): 2581-2590.
PMID
12045228
Source
pubmed
Published In
Journal of cell science
Volume
115
Issue
Pt 12
Publish Date
2002
Start Page
2581
End Page
2590

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding.

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.

Authors
Takagi, J; DeBottis, DP; Erickson, HP; Springer, TA
MLA Citation
Takagi, J, DeBottis, DP, Erickson, HP, and Springer, TA. "The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding." Biochemistry 41.13 (April 2, 2002): 4339-4347.
PMID
11914080
Source
pubmed
Published In
Biochemistry
Volume
41
Issue
13
Publish Date
2002
Start Page
4339
End Page
4347

Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants.

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire length. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

Authors
Ohashi, T; Kiehart, DP; Erickson, HP
MLA Citation
Ohashi, T, Kiehart, DP, and Erickson, HP. "Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants." J Cell Sci 115.Pt 6 (March 15, 2002): 1221-1229.
PMID
11884521
Source
pubmed
Published In
Journal of cell science
Volume
115
Issue
Pt 6
Publish Date
2002
Start Page
1221
End Page
1229

Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching.

FtsZ, the major cytoskeletal component of the bacterial cell-division machine, assembles into a ring (the Z-ring) that contracts at septation. FtsZ is a bacterial homolog of tubulin, with similar tertiary structure, GTP hydrolysis, and in vitro assembly. We used green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching to show that the E. coli Z-ring is extremely dynamic, continually remodeling itself with a half-time of 30 s. ZipA, a membrane protein involved in cell division that colocalizes with FtsZ, was equally dynamic. The Z-ring of the mutant ftsZ84, which has 1/10 the guanosine triphosphatase activity of wild-type FtsZ in vitro, showed a 9-fold slower turnover in vivo. This finding implies that assembly dynamics are determined primarily by GTP hydrolysis. Despite the greatly reduced assembly dynamics, the ftsZ84 cells divide with a normal cell-cycle time.

Authors
Stricker, J; Maddox, P; Salmon, ED; Erickson, HP
MLA Citation
Stricker, J, Maddox, P, Salmon, ED, and Erickson, HP. "Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching." Proc Natl Acad Sci U S A 99.5 (March 5, 2002): 3171-3175.
PMID
11854462
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
5
Publish Date
2002
Start Page
3171
End Page
3175
DOI
10.1073/pnas.052595099

Condensin and cohesin display different arm conformations with characteristic hinge angles.

Structural maintenance of chromosomes (SMC) proteins play central roles in higher-order chromosome dynamics from bacteria to humans. In eukaryotes, two different SMC protein complexes, condensin and cohesin, regulate chromosome condensation and sister chromatid cohesion, respectively. Each of the complexes consists of a heterodimeric pair of SMC subunits and two or three non-SMC subunits. Previous studies have shown that a bacterial SMC homodimer has a symmetrical structure in which two long coiled-coil arms are connected by a flexible hinge. A catalytic domain with DNA- and ATP-binding activities is located at the distal end of each arm. We report here the visualization of vertebrate condensin and cohesin by electron microscopy. Both complexes display the two-armed structure characteristic of SMC proteins, but their conformations are remarkably different. The hinge of condensin is closed and the coiled-coil arms are placed close together. In contrast, the hinge of cohesin is wide open and the coiled-coils are spread apart from each other. The non-SMC subunits of both condensin and cohesin form a globular complex bound to the catalytic domains of the SMC heterodimers. We propose that the "closed" conformation of condensin and the "open" conformation of cohesin are important structural properties that contribute to their specialized biochemical and physiological functions.

Authors
Anderson, DE; Losada, A; Erickson, HP; Hirano, T
MLA Citation
Anderson, DE, Losada, A, Erickson, HP, and Hirano, T. "Condensin and cohesin display different arm conformations with characteristic hinge angles." J Cell Biol 156.3 (February 4, 2002): 419-424.
PMID
11815634
Source
pubmed
Published In
The Journal of Cell Biology
Volume
156
Issue
3
Publish Date
2002
Start Page
419
End Page
424
DOI
10.1083/jcb.200111002

Stretching fibronectin.

Fibronectin (FN) matrix fibrils assembled in cell culture have been observed to stretch in response to cell movements, and when broken relax to 1/3 to 1/4 of their rest length. Two molecular mechanisms have been proposed, for the elasticity. One proposes that FN molecules in relaxed fibers are bent and looped into a compact conformation, and stretching pulls the molecules into the extended conformation but domains remain folded. The second proposes that molecules in fibrils are already extended, and stretching is produced by force-induced unfolding of FN type III domains. Experimental observations that may help distinguish these two possibilities are discussed.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Stretching fibronectin." J Muscle Res Cell Motil 23.5-6 (2002): 575-580. (Review)
PMID
12785106
Source
pubmed
Published In
Journal of Muscle Research and Cell Motility
Volume
23
Issue
5-6
Publish Date
2002
Start Page
575
End Page
580

Trimers of the fivronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation

Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10, on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton.

Authors
Coussen, F; Choquet, D; Sheetz, MP; Erickson, HP
MLA Citation
Coussen, F, Choquet, D, Sheetz, MP, and Erickson, HP. "Trimers of the fivronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation." Journal of Cell Science 115.12 (2002): 2581-2590.
Source
scival
Published In
Journal of Cell Science
Volume
115
Issue
12
Publish Date
2002
Start Page
2581
End Page
2590

Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire length. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

Authors
Ohashi, T; Kiehart, DP; Erickson, HP
MLA Citation
Ohashi, T, Kiehart, DP, and Erickson, HP. "Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants." Journal of Cell Science 115.6 (2002): 1221-1229.
Source
scival
Published In
Journal of Cell Science
Volume
115
Issue
6
Publish Date
2002
Start Page
1221
End Page
1229

Disulfide-mediated dimerization of L1 Ig domains.

The neural cell adhesion molecule L1 contains immunoglobulin-like (Ig) domains in its extracellular region that mediate homophilic binding, neurite outgrowth and other activities relevant to CNS development. To correlate conformations of these domains to biological function, several L1-Fc fusion proteins whose bioactivities were previously characterized were analyzed by rotary shadowing electron microscopy. We found that bioactive L1-Fcs containing Ig domains 1-4 or 1-6 exhibited extended, branched structures. In contrast, inactive L1-Fcs containing only the first two or three Ig domains assumed compact shapes that suggested interactions between the L1 arms of these proteins. Analysis of an untagged L1 fragment composed of Ig domains 1-3 demonstrated a mixture of monomeric and dimeric forms. Surprisingly, these dimers were stabilized by intermolecular disulfide bonds. Finally, cell surface L1-GFP fusion proteins containing only the first two or three Ig domains in the extracellular region also engaged in disulfide-mediated dimerization. These results suggest a novel mechanism by which mutations in L1 could interfere with its biological functioning.

Authors
Haspel, J; Schürmann, G; Jacob, J; Erickson, HP; Grumet, M
MLA Citation
Haspel, J, Schürmann, G, Jacob, J, Erickson, HP, and Grumet, M. "Disulfide-mediated dimerization of L1 Ig domains." J Neurosci Res 66.3 (November 1, 2001): 347-355.
PMID
11746352
Source
pubmed
Published In
Journal of Neuroscience Research
Volume
66
Issue
3
Publish Date
2001
Start Page
347
End Page
355
DOI
10.1002/jnr.1227

Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy.

The RAD50 gene of Saccharomyces cerevisiae is one of several genes required for recombinational repair of double-strand DNA breaks during vegetative growth and for initiation of meiotic recombination. Rad50 forms a complex with two other proteins, Mre11 and Xrs2, and this complex is involved in double-strand break formation and processing. Rad50 has limited sequence homology to the structural maintenance of chromosomes (SMC) family of proteins and shares the same domain structure as SMCs: N- and C-terminal globular domains separated by two long coiled-coils. However, a notable difference is the much smaller non-coil hinge region between the two coiled-coils. We report here a structural analysis of full-length S. cerevisiae Rad50, alone and in a complex with yeast Mre11 by electron microscopy. Our results confirm that yeast Rad50 does have the same antiparallel coiled-coil structure as SMC proteins, but with no detectable globular hinge domain. However, the molecule is still able to bend sharply in the middle to bring the two catalytic domains together, indicating that the small hinge domain is flexible. We also demonstrate that Mre11 binds as a dimer between the catalytic domains of Rad50, bringing the nuclease activities of Mre11 in close proximity to the ATPase and DNA binding activities of Rad50.

Authors
Anderson, DE; Trujillo, KM; Sung, P; Erickson, HP
MLA Citation
Anderson, DE, Trujillo, KM, Sung, P, and Erickson, HP. "Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy." J Biol Chem 276.40 (October 5, 2001): 37027-37033.
PMID
11470800
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
40
Publish Date
2001
Start Page
37027
End Page
37033
DOI
10.1074/jbc.M106179200

Multiple conformations of PEVK proteins detected by single-molecule techniques.

An important component of muscle elasticity is the PEVK region of titin, so named because of the preponderance of these amino acids. However, the PEVK region, similar to other elastomeric proteins, is thought to form a random coil and therefore its structure cannot be determined by standard techniques. Here we combine single-molecule electron microscopy and atomic force microscopy to examine the conformations of the human cardiac titin PEVK region. In contrast to a simple random coil, we have found that cardiac PEVK shows a wide range of elastic conformations with end-to-end distances ranging from 9 to 24 nm and persistence lengths from 0.4 to 2.5 nm. Individual PEVK molecules retained their distinctive elastic conformations through many stretch-relaxation cycles, consistent with the view that these PEVK conformers cannot be interconverted by force. The multiple elastic conformations of cardiac PEVK may result from varying degrees of proline isomerization. The single-molecule techniques demonstrated here may help elucidate the conformation of other proteins that lack a well-defined structure.

Authors
Li, H; Oberhauser, AF; Redick, SD; Carrion-Vazquez, M; Erickson, HP; Fernandez, JM
MLA Citation
Li, H, Oberhauser, AF, Redick, SD, Carrion-Vazquez, M, Erickson, HP, and Fernandez, JM. "Multiple conformations of PEVK proteins detected by single-molecule techniques." Proc Natl Acad Sci U S A 98.19 (September 11, 2001): 10682-10686.
PMID
11526214
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
19
Publish Date
2001
Start Page
10682
End Page
10686
DOI
10.1073/pnas.191189098

Cytoskeleton. Evolution in bacteria.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Cytoskeleton. Evolution in bacteria." Nature 413.6851 (September 6, 2001): 30-.
PMID
11544510
Source
pubmed
Published In
Nature
Volume
413
Issue
6851
Publish Date
2001
Start Page
30
DOI
10.1038/35092655

Cytoskeleton. Evolution in bacteria.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Cytoskeleton. Evolution in bacteria." Nature 413.6851 (September 2001): 30-. (Academic Article)
Source
manual
Published In
Nature
Volume
413
Issue
6851
Publish Date
2001
Start Page
30

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1).

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).

Authors
Jun, CD; Carman, CV; Redick, SD; Shimaoka, M; Erickson, HP; Springer, TA
MLA Citation
Jun, CD, Carman, CV, Redick, SD, Shimaoka, M, Erickson, HP, and Springer, TA. "Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1)." J Biol Chem 276.31 (August 3, 2001): 29019-29027.
PMID
11390397
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
31
Publish Date
2001
Start Page
29019
End Page
29027
DOI
10.1074/jbc.M103394200

XMAP215 is a long thin molecule that does not increase microtubule stiffness.

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60+/-18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4 degrees C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8+/-1.8 nm compared with 5.6+/-1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally, sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

Authors
Cassimeris, L; Gard, D; Tran, PT; Erickson, HP
MLA Citation
Cassimeris, L, Gard, D, Tran, PT, and Erickson, HP. "XMAP215 is a long thin molecule that does not increase microtubule stiffness." J Cell Sci 114.Pt 16 (August 2001): 3025-3033.
PMID
11686305
Source
pubmed
Published In
Journal of cell science
Volume
114
Issue
Pt 16
Publish Date
2001
Start Page
3025
End Page
3033

Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans. It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end. It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC. By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms. This unique structure brings about bimodal regulation of the SMC ATPase cycle. Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode). When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode). Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.

Authors
Hirano, M; Anderson, DE; Erickson, HP; Hirano, T
MLA Citation
Hirano, M, Anderson, DE, Erickson, HP, and Hirano, T. "Bimodal activation of SMC ATPase by intra- and inter-molecular interactions." EMBO J 20.12 (June 15, 2001): 3238-3250.
PMID
11406600
Source
pubmed
Published In
EMBO Journal
Volume
20
Issue
12
Publish Date
2001
Start Page
3238
End Page
3250
DOI
10.1093/emboj/20.12.3238

Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin.

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that has been found to have both adhesive and anti-adhesive properties for cells. Recent elucidation of the two major TNC splice variants (320 kDa and 220 kDa) has shed light on the possibility of varying functions of the molecule based on its splicing pattern. Tenascin-C is prominently expressed in embryogenesis and in pathologic conditions such as tumorogenesis and wound healing. Fibronectin is a prominent adhesive molecule of the extracellular matrix that is often co-localized with tenascin-C in these processes. We studied the chondrosarcoma cell line JJ012 with enzyme-linked immunoabsorbance assays, cell attachment assays and antibody-blocking assays to determine the adhesive/anti-adhesive properties of the two major tenascin-C splice variants with respect to fibronectin and their effect on chondrosarcoma cell attachment. We found that the small tenascin-C splice variant (220 kDa) binds to fibronectin, whereas the large tenascin-C splice variant (320 kDa) does not. In addition, the small tenascin-C splice variant was found to decrease adhesion for cells when bound to fibronectin, but contributed to adhesion when bound to plastic in fibronectin-coated wells. Antibody blocking experiments confirmed that both the small tenascin-C splice variant and fibronectin contribute to cell adhesion when bound to plastic. The large tenascin-C splice variant did not promote specific cell attachment. We hypothesize that the biologic activity of tenascin-C is dependent on the tissue-specific splicing pattern. The smaller tenascin-C isoform likely plays a structural and adhesive role, whereas the larger isoform, preferentially expressed in malignant tissue, likely plays a role in cell egress and metastasis.

Authors
Ghert, MA; Qi, WN; Erickson, HP; Block, JA; Scully, SP
MLA Citation
Ghert, MA, Qi, WN, Erickson, HP, Block, JA, and Scully, SP. "Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin." Cell Struct Funct 26.3 (June 2001): 179-187.
PMID
11565810
Source
pubmed
Published In
Cell structure and function
Volume
26
Issue
3
Publish Date
2001
Start Page
179
End Page
187

Cell adhesion molecule L1 in folded (horseshoe) and extended conformations.

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

Authors
Schürmann, G; Haspel, J; Grumet, M; Erickson, HP
MLA Citation
Schürmann, G, Haspel, J, Grumet, M, and Erickson, HP. "Cell adhesion molecule L1 in folded (horseshoe) and extended conformations." Mol Biol Cell 12.6 (June 2001): 1765-1773.
PMID
11408583
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
12
Issue
6
Publish Date
2001
Start Page
1765
End Page
1773

C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1.

Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by 'inside-out signaling', in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long, approximately 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, alpha5beta1 integrin heterodimer extracellular fragments in which interactions between alpha- and beta-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an approximately 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.

Authors
Takagi, J; Erickson, HP; Springer, TA
MLA Citation
Takagi, J, Erickson, HP, and Springer, TA. "C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1." Nat Struct Biol 8.5 (May 2001): 412-416.
PMID
11323715
Source
pubmed
Published In
Nature Structural Biology
Volume
8
Issue
5
Publish Date
2001
Start Page
412
End Page
416
DOI
10.1038/87569

Polymerization of Ftsz, a bacterial homolog of tubulin. is assembly cooperative?

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis. In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or "isodesmic") assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolar K(D).

Authors
Romberg, L; Simon, M; Erickson, HP
MLA Citation
Romberg, L, Simon, M, and Erickson, HP. "Polymerization of Ftsz, a bacterial homolog of tubulin. is assembly cooperative?." J Biol Chem 276.15 (April 13, 2001): 11743-11753.
PMID
11152458
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
15
Publish Date
2001
Start Page
11743
End Page
11753
DOI
10.1074/jbc.M009033200

Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis.

Fibronectin performs essential roles in embryonic development and is prominently expressed during tissue repair. Two forms of fibronectin have been identified: plasma fibronectin (pFn), which is expressed by hepatocytes and secreted in soluble form into plasma; and cellular fibronectin (cFn), an insoluble form expressed locally by fibroblasts and other cell types and deposited and assembled into the extracellular matrix. To investigate the role of pFn in vivo, we generated pFn-deficient adult mice using Cre-loxP conditional gene-knockout technology. Here we show that pFn-deficient mice show increased neuronal apoptosis and larger infarction areas following transient focal cerebral ischemia. However, pFn is dispensable for skin-wound healing and hemostasis.

Authors
Sakai, T; Johnson, KJ; Murozono, M; Sakai, K; Magnuson, MA; Wieloch, T; Cronberg, T; Isshiki, A; Erickson, HP; Fässler, R
MLA Citation
Sakai, T, Johnson, KJ, Murozono, M, Sakai, K, Magnuson, MA, Wieloch, T, Cronberg, T, Isshiki, A, Erickson, HP, and Fässler, R. "Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis." Nat Med 7.3 (March 2001): 324-330.
PMID
11231631
Source
pubmed
Published In
Nature Medicine
Volume
7
Issue
3
Publish Date
2001
Start Page
324
End Page
330
DOI
10.1038/85471

The FtsZ protofilament and attachment of ZipA--structural constraints on the FtsZ power stroke.

Bacterial cell division protein FtsZ forms protofilaments in vitro that can shift from a straight to a curved conformation. The inside of the curved protofilaments, which corresponds to the carboxyl terminus, should face the center of the cell as curvature increases during constriction of the Z-ring. ZipA, a membrane-tethered division protein, binds to a highly conserved short peptide on the carboxyl terminus of FtsZ. A model is proposed here for how membrane-bound ZipA can reach around the FtsZ protofilament to bind the carboxy-terminal peptide, which faces away from the membrane.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "The FtsZ protofilament and attachment of ZipA--structural constraints on the FtsZ power stroke." Curr Opin Cell Biol 13.1 (February 2001): 55-60. (Review)
PMID
11163134
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
13
Issue
1
Publish Date
2001
Start Page
55
End Page
60

Site-specific mutations of FtsZ--effects on GTPase and in vitro assembly.

BACKGROUND: FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli. RESULTS: The mutants were grouped into three classes. Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro. GTP contact mutations had less than 10% of wild type GTPase activity. They could all assemble in vitro, and several of these mutants could complement ftsZ84. A third, and newly discovered, class of mutations mapped to the sides of the protofilaments. These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84. The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects. CONCLUSIONS: Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function. All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division. These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.

Authors
Lu, C; Stricker, J; Erickson, HP
MLA Citation
Lu, C, Stricker, J, and Erickson, HP. "Site-specific mutations of FtsZ--effects on GTPase and in vitro assembly." BMC Microbiol 1 (2001): 7-.
PMID
11394965
Source
pubmed
Published In
BMC Microbiology
Volume
1
Publish Date
2001
Start Page
7

The clinical significance of tenascin-C splice variant expression in chondrosarcoma.

OBJECTIVES: Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix that is prominently expressed in malignant tumors. The purpose of this study was: (1) to determine the in vitro TNC splicing pattern in cultured human chondrocytes and chondrosarcoma cells, (2) to determine the in vivo TNC splicing pattern in clinical chondrosarcoma specimens, and (3) to perform survival analysis based on the TNC splicing pattern of the tumor specimens. METHODS: Human articular chondrocytes and chondrosarcoma cells (cell line JJ012) were grown in a three-dimensional alginate bead system and harvested at two time points. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the in vitro TNC splicing pattern for the two cell types. Clinical chondrosarcoma specimens were obtained intra-operatively and underwent RT-PCR to determine the in vivo TNC splicing pattern. Specific immunohistochemical staining for the large TNC splice variant was performed on the clinical specimens. Survival analysis was used to determine the association between the specific TNC splicing pattern and survival. RESULTS: The in vitro mRNA expression pattern of TNC in normal human articular chondrocytes was characterized by a high ratio of the small to the large splice variant (TNC(small):TNC(large)), whereas the in vitro mRNA expression pattern for cultured chondrosarcoma cells was characterized by a low TNC(small):TNC(large) ratio. Clinical chondrosarcoma specimens with a lower TNC(small):TNC(large) ratio showed a trend towards decreased survival. The TNC splicing pattern of these specimens was verified through specific immunohistochemical staining for the large TNC isoform. CONCLUSIONS: The specific TNC splicing pattern may have clinical significance in chondrosarcoma. TNC expression may therefore play a future role in objective tumor grading and novel therapeutic approaches to this malignancy.

Authors
Ghert, MA; Jung, ST; Qi, W; Harrelson, JM; Erickson, HP; Block, JA; Scully, SP
MLA Citation
Ghert, MA, Jung, ST, Qi, W, Harrelson, JM, Erickson, HP, Block, JA, and Scully, SP. "The clinical significance of tenascin-C splice variant expression in chondrosarcoma." Oncology 61.4 (2001): 306-314.
PMID
11721178
Source
pubmed
Published In
Oncology
Volume
61
Issue
4
Publish Date
2001
Start Page
306
End Page
314

Evolution in bacteria

Actin is a major component of the cytoskeleton in yeast, plant and animal cells, but when did it evolve? The discovery of a bacterial protein that forms actin-like filaments suggests an answer.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Evolution in bacteria." Nature 413.6851 (2001): 30--.
Source
scival
Published In
Nature
Volume
413
Issue
6851
Publish Date
2001
Start Page
30-
DOI
10.1038/35092655

Site-specific mutations of FtsZ - Effects on GTPase and in vitro assembly

Background: FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli. Results: The mutants were grouped into three classes. Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro. GTP contact mutations had less than 10% of wild type GTPase activity. They could all assemble in vitro, and several of these mutants could complement ftsZ84. A third, and newly discovered, class of mutations mapped to the sides of the protofilaments. These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84. The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects. Conclusions: Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function. All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division. These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.

Authors
Lu, C; Stricker, J; Erickson, HP
MLA Citation
Lu, C, Stricker, J, and Erickson, HP. "Site-specific mutations of FtsZ - Effects on GTPase and in vitro assembly." BMC Microbiology 1 (2001): 1-12.
Source
scival
Published In
BMC Microbiology
Volume
1
Publish Date
2001
Start Page
1
End Page
12
DOI
10.1186/1471-2180-1-1

XMAP215 is a long thin molecule that does not increase microtubule stiffness

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60±18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4°C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8±1.8 nm compared with 5.6±1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally, sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

Authors
Cassimeris, L; Gard, D; Tran, PT; Erickson, HP
MLA Citation
Cassimeris, L, Gard, D, Tran, PT, and Erickson, HP. "XMAP215 is a long thin molecule that does not increase microtubule stiffness." Journal of Cell Science 114.16 (2001): 3025-3033.
Source
scival
Published In
Journal of Cell Science
Volume
114
Issue
16
Publish Date
2001
Start Page
3025
End Page
3033

Drosophila stretchin-MLCK is a novel member of the Titin/Myosin light chain kinase family.

Members of the titin/myosin light chain kinase family play an essential role in the organization of the actin/myosin cytoskeleton, especially in sarcomere assembly and function. In Drosophila melanogaster, projectin is so far the only member of this family for which a transcription unit has been characterized. The locus of another member of this family, a protein related to Myosin light chain kinase, was also identified. The cDNA and genomic sequences published explain only the shorter transcripts expressed by this locus. Here, we report the complete molecular characterization of this transcription unit, which spans 38 kb, includes 33 exons and accounts for transcripts up to 25 kb in length. This transcription unit contains both the largest exon (12,005 nt) and the largest coding region (25,213 nt) reported so far for Drosophila. This transcription unit features both internal promoters and internal polyadenylation signals, which enable it to express seven different transcripts, ranging from 3.3 to 25 kb in size. The latter encodes a huge, titin-like, 926 kDa kinase that features two large PEVK-rich repeats, 32 immunoglobulin and two fibronectin type-III domains, which we designate stretchin-MLCK. In addition, the 3' end of the stretchin-MLCK transcription unit expresses shorter transcripts that encode 86 to 165 kDa isoforms of stretchin-MLCK that are analogous to vertebrate Myosin light chain kinases. Similarly, the 5' end of the Stretchin-Mlck transcription unit can also express transcripts encoding kettin and Unc-89-like isoforms, which share no sequences with the MLCK-like transcripts. Thus, this locus can be viewed as a single transcription unit, Stretchin-Mlck (genetic abbreviation Strn-Mlck), that expresses large, composite transcripts and protein isoforms (sequences available at http://www.academicpress.com/jmb), as well as a complex of two independent transcription units, the Stretchin and Mlck transcription units (Strn and Mlck, respectively) the result of a "gene fission" event, that encode independent transcripts and proteins with distinct structural and enzymatic functions.

Authors
Champagne, MB; Edwards, KA; Erickson, HP; Kiehart, DP
MLA Citation
Champagne, MB, Edwards, KA, Erickson, HP, and Kiehart, DP. "Drosophila stretchin-MLCK is a novel member of the Titin/Myosin light chain kinase family." J Mol Biol 300.4 (July 21, 2000): 759-777.
PMID
10891286
Source
pubmed
Published In
Journal of Molecular Biology
Volume
300
Issue
4
Publish Date
2000
Start Page
759
End Page
777
DOI
10.1006/jmbi.2000.3802

Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3.

Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.

Authors
Yokoyama, K; Erickson, HP; Ikeda, Y; Takada, Y
MLA Citation
Yokoyama, K, Erickson, HP, Ikeda, Y, and Takada, Y. "Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3." J Biol Chem 275.22 (June 2, 2000): 16891-16898.
PMID
10747940
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
22
Publish Date
2000
Start Page
16891
End Page
16898
DOI
10.1074/jbc.M000610200

Gamma-tubulin nucleation: template or protofilament?

Gamma-tubulin is known to nucleate microtubule assembly from alpha/beta-tubulin, but the molecular mechanism by which this process occurs is the subject of some controversy. Four recent papers have provided new structural and biochemical constraints on the models proposed for nucleation. These have refined, but not yet resolved, the debate.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Gamma-tubulin nucleation: template or protofilament?." Nat Cell Biol 2.6 (June 2000): E93-E96.
PMID
10854332
Source
pubmed
Published In
Nature Cell Biology
Volume
2
Issue
6
Publish Date
2000
Start Page
E93
End Page
E96
DOI
10.1038/35014084

Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.

Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.

Authors
Redick, SD; Settles, DL; Briscoe, G; Erickson, HP
MLA Citation
Redick, SD, Settles, DL, Briscoe, G, and Erickson, HP. "Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis." J Cell Biol 149.2 (April 17, 2000): 521-527.
PMID
10769040
Source
pubmed
Published In
The Journal of Cell Biology
Volume
149
Issue
2
Publish Date
2000
Start Page
521
End Page
527

Dynamin and FtsZ. Missing links in mitochondrial and bacterial division.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Dynamin and FtsZ. Missing links in mitochondrial and bacterial division." J Cell Biol 148.6 (March 20, 2000): 1103-1105. (Review)
PMID
10725322
Source
pubmed
Published In
The Journal of Cell Biology
Volume
148
Issue
6
Publish Date
2000
Start Page
1103
End Page
1105

Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation.

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.

Authors
Fong, AM; Erickson, HP; Zachariah, JP; Poon, S; Schamberg, NJ; Imai, T; Patel, DD
MLA Citation
Fong, AM, Erickson, HP, Zachariah, JP, Poon, S, Schamberg, NJ, Imai, T, and Patel, DD. "Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation." J Biol Chem 275.6 (February 11, 2000): 3781-3786.
PMID
10660527
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
6
Publish Date
2000
Start Page
3781
End Page
3786

Straight and curved conformations of FtsZ are regulated by GTP hydrolysis.

FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24 degrees. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.

Authors
Lu, C; Reedy, M; Erickson, HP
MLA Citation
Lu, C, Reedy, M, and Erickson, HP. "Straight and curved conformations of FtsZ are regulated by GTP hydrolysis." J Bacteriol 182.1 (January 2000): 164-170.
PMID
10613876
Source
pubmed
Published In
Journal of bacteriology
Volume
182
Issue
1
Publish Date
2000
Start Page
164
End Page
170

The straight and curved conformation of FtsZ protofilaments-evidence for rapid exchange of GTP into the curved protofilament.

Bacterial cell division protein FtsZ assembles into protofilaments, which can adopt a straight or curved conformation, similar to its eukaryotic homolog, tubulin. The straight protofilaments can assemble into sheets with a lattice similar to the microtubule wall. The curved protofilaments can form rings when adsorbed to a lipid monolayer, but in solution they form helices. 4 helices assemble together to make a tube, the characteristic polymer of the curved protofilament. GTP favors the straight conformation, while GDP favors the curved. We show here that addition of EDTA and GTP to tubes causes a rapid transformation to straight protofilament sheets. Apparently when the magnesium is chelated the GDP in the curved protofilaments dissociates rapidly and is replaced with GTP, and this GTP induces the transition to straight protofilaments.

Authors
Lu, C; Erickson, HP
MLA Citation
Lu, C, and Erickson, HP. "The straight and curved conformation of FtsZ protofilaments-evidence for rapid exchange of GTP into the curved protofilament." Cell Struct Funct 24.5 (October 1999): 285-290.
PMID
15216884
Source
pubmed
Published In
Cell structure and function
Volume
24
Issue
5
Publish Date
1999
Start Page
285
End Page
290

The compact conformation of fibronectin is determined by intramolecular ionic interactions.

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

Authors
Johnson, KJ; Sage, H; Briscoe, G; Erickson, HP
MLA Citation
Johnson, KJ, Sage, H, Briscoe, G, and Erickson, HP. "The compact conformation of fibronectin is determined by intramolecular ionic interactions." J Biol Chem 274.22 (May 28, 1999): 15473-15479.
PMID
10336438
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
22
Publish Date
1999
Start Page
15473
End Page
15479

Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein.

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.

Authors
Ohashi, T; Kiehart, DP; Erickson, HP
MLA Citation
Ohashi, T, Kiehart, DP, and Erickson, HP. "Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein." Proc Natl Acad Sci U S A 96.5 (March 2, 1999): 2153-2158.
PMID
10051610
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
5
Publish Date
1999
Start Page
2153
End Page
2158

Macramolecular assemblages. Editorial overview

Authors
Fuller, SD; Erickson, HP
MLA Citation
Fuller, SD, and Erickson, HP. "Macramolecular assemblages. Editorial overview." Current Opinion in Structural Biology 9.2 (1999): 213-214.
Source
scival
Published In
Current Opinion in Structural Biology
Volume
9
Issue
2
Publish Date
1999
Start Page
213
End Page
214
DOI
10.1016/S0959-440X(99)80030-X

Oligomeric structure and tissue distribution of ficolins from mouse, pig and human.

Mouse plasma ficolin was purified by GlcNAc affinity and anion-exchange chromatography. Gel-filtration chromatography and gradient sedimentation indicated that mouse plasma ficolin is a 12-mer of approximately 35 kDa subunits, and electron microscopy showed the same parachute-like structure previously characterized for the pig ficolin 12-mer. Whereas the predominant form in pig plasma is a 24-mer, mouse and human plasma ficolin showed only the 12-mer form. We conclude that mouse plasma ficolin corresponds to the recently described ficolin A. We have identified a second mouse ficolin gene, ficolin B, which means that pig and mouse each have two ficolin genes, and human has three. One ficolin gene in all species is expressed in liver and is the primary source of plasma ficolin. Expression of this gene in other tissues, and expression of the second ficolin gene, appears to vary in different species.

Authors
Ohashi, T; Erickson, HP
MLA Citation
Ohashi, T, and Erickson, HP. "Oligomeric structure and tissue distribution of ficolins from mouse, pig and human." Arch Biochem Biophys 360.2 (December 15, 1998): 223-232.
PMID
9851834
Source
pubmed
Published In
Archives of Biochemistry and Biophysics
Volume
360
Issue
2
Publish Date
1998
Start Page
223
End Page
232
DOI
10.1006/abbi.1998.0957

Ultrastructural and biochemical properties of the 120-kDa form of chick kinectin.

Kinectin, an integral membrane protein (160 kDa), was identified as a kinesin-binding protein. Analysis of the predicted amino acid sequence of kinectin cDNA indicated an alpha-helical coiled-coil structure from amino acid 320 to 1310. A 120-kDa kinectin has been observed consistently, and N-terminal sequencing showed that 232 amino acids were missing from the N terminus of full-length kinectin. 120-kDa kinectin was distributed in the supernatant and a low density fraction of vesicles, whereas both forms were in the high density fraction of vesicles. In the electron microscope, the 120-kDa form appeared as a linear molecule of 133 nm in length. In hydrodynamic studies, the cytosolic 120-kDa kinectin was a dimer. Monoclonal antibody molecules (anti-kinectin KR160.9) bound asymmetrically to kinectin often with two antibodies/kinectin, indicative of a parallel coiled-coil. Metabolic labeling with [3H]myristic acid showed that both the 120- and 160-kDa kinectin are myristoylated in chick embryo fibroblasts. The myristoylation of 120-kDa kinectin may provide a mechanism for linking it to a low density fraction of vesicles. Immunoprecipitation with a 160-kDa kinectin-specific antibody brought down the 120-kDa kinectin. Thus, we suggest that kinectin is an extended parallel coiled-coil dimer, often a heterodimer.

Authors
Kumar, J; Erickson, HP; Sheetz, MP
MLA Citation
Kumar, J, Erickson, HP, and Sheetz, MP. "Ultrastructural and biochemical properties of the 120-kDa form of chick kinectin." J Biol Chem 273.48 (November 27, 1998): 31738-31743.
PMID
9822636
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
48
Publish Date
1998
Start Page
31738
End Page
31743

The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge.

Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH2-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180 degrees, separating the terminal domains by 100 nm, or close to near 0 degrees, bringing the terminal globular domains together. A surprising observation is that the approximately 300-amino acid-long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35-45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH2- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.

Authors
Melby, TE; Ciampaglio, CN; Briscoe, G; Erickson, HP
MLA Citation
Melby, TE, Ciampaglio, CN, Briscoe, G, and Erickson, HP. "The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge." J Cell Biol 142.6 (September 21, 1998): 1595-1604.
PMID
9744887
Source
pubmed
Published In
The Journal of Cell Biology
Volume
142
Issue
6
Publish Date
1998
Start Page
1595
End Page
1604

The molecular elasticity of the extracellular matrix protein tenascin.

Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.

Authors
Oberhauser, AF; Marszalek, PE; Erickson, HP; Fernandez, JM
MLA Citation
Oberhauser, AF, Marszalek, PE, Erickson, HP, and Fernandez, JM. "The molecular elasticity of the extracellular matrix protein tenascin." Nature 393.6681 (May 14, 1998): 181-185.
PMID
9603523
Source
pubmed
Published In
Nature
Volume
393
Issue
6681
Publish Date
1998
Start Page
181
End Page
185
DOI
10.1038/30270

Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.

The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.

Authors
Denda, S; Müller, U; Crossin, KL; Erickson, HP; Reichardt, LF
MLA Citation
Denda, S, Müller, U, Crossin, KL, Erickson, HP, and Reichardt, LF. "Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C." Biochemistry 37.16 (April 21, 1998): 5464-5474.
PMID
9548928
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
16
Publish Date
1998
Start Page
5464
End Page
5474
DOI
10.1021/bi9727489

Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains.

In the adult organism, the extracellular matrix molecule tenascin-C is prominently expressed in the bone marrow. Bone marrow mononuclear cells can adhere to plastic-immobilized tenascin-C, and in the present study we have used bacterial expression proteins to map the domains of tenascin-C responsible for binding of hematopoietic cells. A strong binding site was found to be located within the fibrinogen-like domain, and this binding could be inhibited by heparin, suggesting interactions with membrane-bound heparan sulfate proteoglycans. A second strong binding site was identified within the fibronectin type III-like repeats 6-8, and was also inhibitable by heparin. Adhesion to both attachment sites could not be blocked by various anti-integrin antibodies. A third hematopoietic cell binding site is located in the fibronectin type III-like repeats 1-5, which harbor an RGD sequence in the third fibronectin type III-like repeat. Binding to this domain, however, seems to be RGD-independent, since RGD-containing peptides could not inhibit cell binding; the addition of heparin also did not block adhesion to this domain. Since contradictory results had been reported on a proliferative effect of soluble tenascin-C, we also analyzed its activity on hematopoietic cells. The heterogeneous bone marrow mononuclear cells show a striking proliferative response in the presence of tenascin-C which is concentration-dependent. This result indicates a strong mitogenic activity of tenascin-C on primary hematopoietic cells. Using recombinant fragments of human tenascin-C, we identified several mitogenic domains within the tenascin-C molecule. These adhesive and mitogenic effects of tenascin-C suggest a direct functional association with proliferation and differentiation of hematopoietic cells within the bone marrow microenvironment.

Authors
Seiffert, M; Beck, SC; Schermutzki, F; Müller, CA; Erickson, HP; Klein, G
MLA Citation
Seiffert, M, Beck, SC, Schermutzki, F, Müller, CA, Erickson, HP, and Klein, G. "Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains." Matrix Biol 17.1 (April 1998): 47-63.
PMID
9628252
Source
pubmed
Published In
Matrix Biology
Volume
17
Issue
1
Publish Date
1998
Start Page
47
End Page
63

Atomic structures of tubulin and FtsZ.

The recently published atomic structures of tubulin and FtsZ are a research milestone. The N-terminal GTP-binding domains of tubulin and FtsZ are virtually identical in structure, as expected from the substantial sequence identity. Sequence identity is absent from the C-terminal domains, but they also have virtually identical structures. A surprising finding is that the N-terminal GTP-binding domain is structurally homologous to that of Ras and other G proteins, despite the completely different GTP-binding sequence motifs. This article discusses these findings and the molecular mechanisms that can now be addressed with the atomic structures.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Atomic structures of tubulin and FtsZ." Trends Cell Biol 8.4 (April 1998): 133-137. (Review)
PMID
9695825
Source
pubmed
Published In
Trends in Cell Biology
Volume
8
Issue
4
Publish Date
1998
Start Page
133
End Page
137

Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain.

A novel off-resonance rotating-frame 15N NMR spin relaxation experiment is used to characterize conformational fluctuations with correlation times between 32 and 175 microseconds in the third fibronectin type III domain of human tenascin-C. Conformational fluctuations of contiguous regions of the beta-sandwich structure of the type III domain may represent collective motions, such as transient twisting or breathing of the beta-sheets. Flexibility of the loop containing the Arg-Gly-Asp (RGD) tripeptide may affect the accessibility of this motif in protein-protein interactions.

Authors
Akke, M; Liu, J; Cavanagh, J; Erickson, HP; Palmer, AG
MLA Citation
Akke, M, Liu, J, Cavanagh, J, Erickson, HP, and Palmer, AG. "Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain." Nat Struct Biol 5.1 (January 1998): 55-59.
PMID
9437430
Source
pubmed
Published In
Nature Structural Biology
Volume
5
Issue
1
Publish Date
1998
Start Page
55
End Page
59

Purification and assembly of FtsZ.

Authors
Lu, C; Erickson, HP
MLA Citation
Lu, C, and Erickson, HP. "Purification and assembly of FtsZ." Methods Enzymol 298 (1998): 305-313.
PMID
9751890
Source
pubmed
Published In
Methods in Enzymology
Volume
298
Publish Date
1998
Start Page
305
End Page
313

FtsZ From Escherichia coli, Azotobacter vinelandii and Thermotoga maritima - Quantitation, GTP Hydrolysis and Assembly

Authors
Lu, C; Stricker, J; Erickson, HP
MLA Citation
Lu, C, Stricker, J, and Erickson, HP. "FtsZ From Escherichia coli, Azotobacter vinelandii and Thermotoga maritima - Quantitation, GTP Hydrolysis and Assembly." Cell Motility and the Cytoskeleton 40.1 (1998): 71-. (Academic Article)
Source
manual
Published In
Cell Motility and the Cytoskeleton
Volume
40
Issue
1
Publish Date
1998
Start Page
71
DOI
10.1002/(SICI)1097-0169(1998)40:13.0.CO;2-I

FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly.

We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ (and some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro.

Authors
Lu, C; Stricker, J; Erickson, HP
MLA Citation
Lu, C, Stricker, J, and Erickson, HP. "FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly." Cell Motil Cytoskeleton 40.1 (1998): 71-86.
PMID
9605973
Source
pubmed
Published In
Cell Motility and the Cytoskeleton
Volume
40
Issue
1
Publish Date
1998
Start Page
71
End Page
86
DOI
10.1002/(SICI)1097-0169(1998)40:1<71::AID-CM7>3.0.CO;2-I

[25] Purification and asseembly of FtsZ

Authors
Lu, C; Erickson, HP
MLA Citation
Lu, C, and Erickson, HP. "[25] Purification and asseembly of FtsZ." Methods in Enzymology 298 (1998): 305-313.
Source
scival
Published In
Methods in Enzymology
Volume
298
Publish Date
1998
Start Page
305
End Page
313
DOI
10.1016/S0076-6879(98)98027-2

Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus.

Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) amplification. Previously the protein has been purified from P. furiosus bacterial cultures, and a recombinant form has been produced in a baculovirus system. We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC, Accession No. 87496) and found that this plasmid is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction. However, the plasmid was stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90-kDa protein was expressed upon addition of isopropyl beta-D-thiogalactopyranoside. The protein was purified by heating (to denature E. coli proteins), followed by chromatography on P11 phosphocellulose and mono Q columns. The purified protein had the same activity as the commercially obtained baculovirus-expressed Pfu in both DNA polymerase and PCR reactions. This bacterial expression system appears to be the method of choice for production of Pfu.

Authors
Lu, C; Erickson, HP
MLA Citation
Lu, C, and Erickson, HP. "Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus." Protein Expr Purif 11.2 (November 1997): 179-184.
PMID
9367814
Source
pubmed
Published In
Protein Expression and Purification
Volume
11
Issue
2
Publish Date
1997
Start Page
179
End Page
184
DOI
10.1006/prep.1997.0775

Extracellular annexin II.

Annexin II belongs to a family of calcium-dependent, phospholipid binding proteins. Annexin II was first identified as an intracellular protein and attributed intracellular functions. Although it lacks a signal peptide and its mechanism of secretion is unknown, extracellular annexin II has recently been found in several tissues as both soluble and membrane-bound protein. Cell-surface annexin II has been identified as a receptor for a number of polypeptide ligands. Extracellular annexin II may be important in several biological processes, such as fibrinolysis, cell adhesion, ligand-mediated cell signaling and virus infection. These processes provide several possibilities for therapeutic approaches targeting extracellular annexin II, and future research should further illuminate the biology of this molecule.

Authors
Siever, DA; Erickson, HP
MLA Citation
Siever, DA, and Erickson, HP. "Extracellular annexin II." Int J Biochem Cell Biol 29.11 (November 1997): 1219-1223. (Review)
PMID
9451817
Source
pubmed
Published In
The International Journal of Biochemistry & Cell Biology
Volume
29
Issue
11
Publish Date
1997
Start Page
1219
End Page
1223

Soluble monomeric P-selectin containing only the lectin and epidermal growth factor domains binds to P-selectin glycoprotein ligand-1 on leukocytes.

Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.

Authors
Mehta, P; Patel, KD; Laue, TM; Erickson, HP; McEver, RP
MLA Citation
Mehta, P, Patel, KD, Laue, TM, Erickson, HP, and McEver, RP. "Soluble monomeric P-selectin containing only the lectin and epidermal growth factor domains binds to P-selectin glycoprotein ligand-1 on leukocytes." Blood 90.6 (September 15, 1997): 2381-2389.
PMID
9310489
Source
pubmed
Published In
Blood
Volume
90
Issue
6
Publish Date
1997
Start Page
2381
End Page
2389

A tenascin knockout with a phenotype.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "A tenascin knockout with a phenotype." Nat Genet 17.1 (September 1997): 5-7.
PMID
9288085
Source
pubmed
Published In
Nature Genetics
Volume
17
Issue
1
Publish Date
1997
Start Page
5
End Page
7
DOI
10.1038/ng0997-5

FtsZ, a tubulin homologue in prokaryote cell division.

Sequence alignments convincingly demonstrate that FtsZ is a prokaryotic homologue of eukaryotic tubulins. FtsZ appears to be universal in eubacteria and archaebacteria and has also been identified in chloroplasts. Like tubulin, it appears to have a cytoskeletal role as both proteins assemble into two types of characteristic polymers in vitro - protofilament sheets and mini-rings. Recent advances in immunofluorescence and the use of green fluorescent protein have provided clear images of FtsZ localized in a ring at the septation site in bacteria, and new insights into assembly of this Z-ring.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "FtsZ, a tubulin homologue in prokaryote cell division." Trends Cell Biol 7.9 (September 1997): 362-367.
PMID
17708981
Source
pubmed
Published In
Trends in Cell Biology
Volume
7
Issue
9
Publish Date
1997
Start Page
362
End Page
367
DOI
10.1016/S0962-8924(97)01108-2

Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin.

BACKGROUND: Fibronectin type III domains are found as autonomously-folded domains in a large variety of multidomain proteins, including extracellular matrix proteins. A subset of these domains employ an Arg-Gly-Asp (RGD) tripeptide motif to mediate contact with cell-surface receptors (integrins). This motif mediates protein-protein interactions in a diverse range of biological processes, such as in tissue development, would healing and metastasis. The molecular basis for affinity and specificity of cell adhesion via type III domains has not been clearly established. The tenth type III domain from fibronectin (FNfn10) and the third type III domain from tenascin-C (TNfn3) have 27% sequence identity and share the same overall protein fold, but present the RGD motifs in different structural contexts. The dynamical properties of the RGD motifs may affect the specificity and affinity of the FNfn10 and TNfn3 domains. Structure-dynamics correlations for these structurally homologous proteins may reveal common molecular features which are important to the dynamical properties of proteins. RESULTS: The intramolecular dynamics of the protein backbones of FNfn10 and TNfn3 have been studied by 15N nuclear spin relaxation. The FG loop in FNfn10, which contains the RGD motif, exhibits extensive flexibility on picosecond to nanosecond timescales, but motions on microsecond to millisecond timescales are not observed. The equivalent region in TNfn3 is as rigid as regular elements of secondary structure. The CC' loop also is more flexible on picosecond-nanosecond timescales in FNfn10 than in TNfn3. Conformational exchange, reflecting flexibility on microsecond-millisecond timescales, is observed in beta strands A and B of both FNfn10 and TNfn3. CONCLUSIONS: Comparison of the structures of the FNfn10 and TNfn3 reveals several features related to their different dynamical properties. The larger amplitude motions of loops in FNfn10 are consistent with the hypothesis that flexibility of these regions facilitates induced-fit recognition of fibronectin by multiple receptors. Similarly, the more rigid loops of TNfn3 may reflect greater specificity for particular integrins. The correlations observed between structural features and dynamical properties of the homologous type III domains indicate the influence of hydrogen bonding and hydrophobic packing on dynamical fluctuations in proteins.

Authors
Carr, PA; Erickson, HP; Palmer, AG
MLA Citation
Carr, PA, Erickson, HP, and Palmer, AG. "Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin." Structure 5.7 (July 15, 1997): 949-959.
PMID
9261088
Source
pubmed
Published In
Structure
Volume
5
Issue
7
Publish Date
1997
Start Page
949
End Page
959
DOI
10.1016/S0969-2126(97)00248-7

Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C.

We have investigated the role of glycosaminoglycans in fibronectin matrix assembly and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production failed to assemble a fibronectin matrix, and incorporated no tenascin-C. Another mutant with reduced heparan sulfate produced a normal fibronectin matrix but failed to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibited the binding of tenascin-C to purified fibronectin in ELISA, and completely blocked incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocked their ability to incorporate tenascin-C into matrix fibrils. We conclude that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. We examined more closely the large heparan sulfate proteoglycan, perlecan, and found that it co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C was mapped to the fibronectin type III domains 3-5, but this binding was strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently when the alternative splice segment is inserted after domain 5 it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils.

Authors
Chung, CY; Erickson, HP
MLA Citation
Chung, CY, and Erickson, HP. "Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C." J Cell Sci 110 ( Pt 12) (June 1997): 1413-1419.
PMID
9217327
Source
pubmed
Published In
Journal of cell science
Volume
110 ( Pt 12)
Publish Date
1997
Start Page
1413
End Page
1419

Two oligomeric forms of plasma ficolin have differential lectin activity.

Ficolins are plasma proteins with binding activity for carbohydrates, elastin, and corticosteroids. The ficolin polypeptide has a collagen-like domain that presumably brings three subunits together in a triple helical rod, a C-terminal fibrinogen-like domain (fbg) similar to that of tenascin, which presumably has the binding activities, and a small N-terminal domain that we find to be the primary site for forming the ficolin oligomer. By sedimentation equilibrium we determined that the main plasma form, which we call big ficolin, had mass of 827,000 Da, consistent with 24 subunits. Little ficolin, about half this size, was obtained after binding to a GlcNAc affinity column. Electron microscopy of little ficolin showed a parachute-like structure, with a small globe at one end, corresponding to the 12 N-terminal domains, and the fbg domains clustered together at the ends of the collagen rods. Big ficolin was formed by the face to face fusion of the fbg domains of two little ficolins, leaving the rods and N-terminal domains projecting at opposite ends. Little ficolin maintained a high affinity for the GlcNAc column, and big ficolin had a low affinity or none. The binding sites for ligands may be obscured in this big ficolin oligomer, providing a regulation of their activity.

Authors
Ohashi, T; Erickson, HP
MLA Citation
Ohashi, T, and Erickson, HP. "Two oligomeric forms of plasma ficolin have differential lectin activity." J Biol Chem 272.22 (May 30, 1997): 14220-14226.
PMID
9162054
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
22
Publish Date
1997
Start Page
14220
End Page
14226

Stretching single protein molecules: titin is a weird spring.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Stretching single protein molecules: titin is a weird spring." Science 276.5315 (May 16, 1997): 1090-1092.
PMID
9173540
Source
pubmed
Published In
Science
Volume
276
Issue
5315
Publish Date
1997
Start Page
1090
End Page
1092

Tenascin supports lymphocyte rolling.

Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.

Authors
Clark, RA; Erickson, HP; Springer, TA
MLA Citation
Clark, RA, Erickson, HP, and Springer, TA. "Tenascin supports lymphocyte rolling." J Cell Biol 137.3 (May 5, 1997): 755-765.
PMID
9151679
Source
pubmed
Published In
The Journal of Cell Biology
Volume
137
Issue
3
Publish Date
1997
Start Page
755
End Page
765

Tenascin-C knockout mouse has no detectable tenascin-C protein.

A recent study by Mitrovic and Schachner (J Neurosci Res 42:710-717, 1995) reported the detection of a small amount of truncated tensacin-C (TN-C) in the nervous system of the TN-C knockout mice created by Saga et al. (Genes Dev 6:1821-1831, 1992). The authors suggested that the truncated protein might be responsible for the failure to detect any phenotypic abnormalities in the knockout mice. We have reexamined the knockout mice in our laboratories by Western blot and immunocytochemistry, and have not detected any full-length or truncated TN-C protein. In addition, we note that the construction of the knockout gene deleted the signal sequence, so if any residual truncated protein were produced it would be trapped in the cytoplasm, and therefore inaccessible to extracellular ligands or receptors. We therefore conclude that the TN-C knockout created by Saga et al. is a valid TN-C null.

Authors
Settles, DL; Kusakabe, M; Steindler, DA; Fillmore, H; Erickson, HP
MLA Citation
Settles, DL, Kusakabe, M, Steindler, DA, Fillmore, H, and Erickson, HP. "Tenascin-C knockout mouse has no detectable tenascin-C protein." J Neurosci Res 47.1 (January 1, 1997): 109-117.
PMID
8981244
Source
pubmed
Published In
Journal of Neuroscience Research
Volume
47
Issue
1
Publish Date
1997
Start Page
109
End Page
117

How Calicum Causes Microtubule Depolymerization

Authors
O'Brien, ET; Salmon, ED; Erickson, HP
MLA Citation
O'Brien, ET, Salmon, ED, and Erickson, HP. "How Calicum Causes Microtubule Depolymerization." Cell Motility and the Cytoskeleton 36.2 (1997): 125-. (Academic Article)
Source
manual
Published In
Cell Motility and the Cytoskeleton
Volume
36
Issue
2
Publish Date
1997
Start Page
125
DOI
10.1002/(SICI)1097-0169(1997)36:23.0.CO;2-8

How calcium causes microtubule depolymerization.

The effects of calcium (Ca) were assessed using video-enhanced differential interference contrast light microscopy on individual microtubules in vitro. Phosphocellulose-purified (PC) and microtubule associated protein (MAP)-containing preparations of porcine brain tubulin were assembled in a flow chamber onto sperm axoneme fragments and the pattern of growth and shortening of the microtubules was observed. Tubulin plus Ca was then added to the chamber and observation continued. Ca promoted the disassembly of microtubules by specifically promoting the catastrophe reaction in both PC- and MAP-containing microtubules, without an appreciable change in elongation rate. The effect on catastrophe frequency increased very rapidly above 0.5 mM free Ca, implying a possible cooperative effect. The rescue rate remained very high after Ca addition in MAP-containing microtubules, and the shortening rate was unchanged, while in phosphocellulose-purified microtubules, rescue appeared to be decreased by Ca addition and shortening rates increased 4 to 6-fold. These results illustrate that Ca can directly destabilize growing microtubule ends without changing the effective concentration of free tubulin, and that this effect can be seen even against the background of the profound differences in dynamics conferred by the microtubule-associated proteins. Considered within models of the GTP cap, the results imply that high Ca may act to increase the rate of GTP hydrolysis within the cap.

Authors
O'Brien, ET; Salmon, ED; Erickson, HP
MLA Citation
O'Brien, ET, Salmon, ED, and Erickson, HP. "How calcium causes microtubule depolymerization." Cell Motil Cytoskeleton 36.2 (1997): 125-135.
PMID
9015201
Source
pubmed
Published In
Cell Motility and the Cytoskeleton
Volume
36
Issue
2
Publish Date
1997
Start Page
125
End Page
135
DOI
10.1002/(SICI)1097-0169(1997)36:2<125::AID-CM3>3.0.CO;2-8

Protofilaments and rings, two conformations of the tubulin family conserved from bacterial FtsZ to alpha/beta and gamma tubulin.

Authors
Erickson, HP; Stoffler, D
MLA Citation
Erickson, HP, and Stoffler, D. "Protofilaments and rings, two conformations of the tubulin family conserved from bacterial FtsZ to alpha/beta and gamma tubulin." J Cell Biol 135.1 (October 1996): 5-8. (Review)
PMID
8858158
Source
pubmed
Published In
The Journal of Cell Biology
Volume
135
Issue
1
Publish Date
1996
Start Page
5
End Page
8

Structural analysis of a human glial variant laminin.

Astrocytes secrete laminin-like molecules in culture and may represent a major source of laminin in the developing central nervous system, yet these laminins have not been extensively characterized. We previously reported the presence of an astrocyte-derived variant laminin in media conditioned by human U251 MG astrocytoma cells. This laminin was partially purified in a highly anionic Mono Q fraction with strong adhesion activity for fibroblasts and glial cells (Aukhil et al. (1990) Matrix 10: 98-111). We now show that glial laminin could be dissociated from an anionic species, perhaps an approximately 400-kDa keratan sulfate proteoglycan present in the preparation, by a second round of Mono Q anion exchange chromatography in the presence of 6 M urea. Cell adhesion activity remained tightly associated with laminin-containing fractions, suggesting that glial laminin was responsible for the adhesion activity in the original preparation. Immunochemical and SDS-PAGE gel analyses of laminin heterotrimers demonstrated that glial laminin contained the beta 2 and gamma 1 chains in disulfide-bonded heterotrimeric complexes with a 360-kDa chain, a 320-kDa chain, or a postulated approximately 200-kDa chain. While these chains were not recognized by antibodies directed against the alpha 1-, alpha 2-, or alpha 3-related laminin chains, rotary shadowed glial laminin molecules appeared to contain alpha chains, as judged by the presence of an apparent G-domain terminating the long arm of each laminin molecule. These findings suggest that glial laminin contains one or more variant alpha chains, perhaps related to one of the more recently described alpha chains, alpha 3B, alpha 4, or alpha 5. Together our results implicate human U251 MG glial laminin as a previously uncharacterized laminin isoform with strong adhesive activity for fibroblasts and glial cells.

Authors
LeMosy, EK; Lightner, VA; Erickson, HP
MLA Citation
LeMosy, EK, Lightner, VA, and Erickson, HP. "Structural analysis of a human glial variant laminin." Exp Cell Res 227.1 (August 25, 1996): 80-88.
PMID
8806454
Source
pubmed
Published In
Experimental Cell Research
Volume
227
Issue
1
Publish Date
1996
Start Page
80
End Page
88
DOI
10.1006/excr.1996.0252

Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II.

In a previous study we demonstrated that the alternatively spliced region of tenascin-C, TNfnA-D, bound with high affinity to a cell surface receptor, annexin II. In the present study we demonstrate three changes in cellular activity that are produced by adding intact tenascin-C or TNfnA-D to cells, and we show that all three activities are blocked by antibodies against annexin II. 1) TNfnA-D added to confluent endothelial cells induced loss of focal adhesions. 2) TNfnA-D produced a mitogenic response of confluent, growth-arrested endothelial cells in 1% serum. TNfnA-D stimulated mitogenesis only when it was added to cells before or during exposure to other mitogens, such as basic fibroblast growth factor or serum. Thus the effect of TNfnA-D seems to be to facilitate the subsequent response to growth factors. 3) TNfnA-D enhanced cell migration in a cell culture wound assay. Antibodies to annexin II blocked all three cellular responses to TNfnA-D. These data show that annexin II receptors on endothelial cells mediate several cell regulatory functions attributed to tenascin-C, potentially through modulation of intracellular signalling pathways.

Authors
Chung, CY; Murphy-Ullrich, JE; Erickson, HP
MLA Citation
Chung, CY, Murphy-Ullrich, JE, and Erickson, HP. "Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II." Mol Biol Cell 7.6 (June 1996): 883-892.
PMID
8816995
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
7
Issue
6
Publish Date
1996
Start Page
883
End Page
892

Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies.

P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human leukocytes, mediates rolling of leukocytes on P-selectin expressed by activated platelets or endothelial cells under shear forces. PSGL-1 requires both tyrosine sulfate and O-linked glycans to bind P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is -50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. Using bacterially expressed fusion proteins and synthetic peptides derived from the extracellular domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound to a region within residues 188-235 that is located in a series of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to P-selectin, recognized an epitope spanning residues 49-62. This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSGL-1 to P-selectin. Our results demonstrate that PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress.

Authors
Li, F; Erickson, HP; James, JA; Moore, KL; Cummings, RD; McEver, RP
MLA Citation
Li, F, Erickson, HP, James, JA, Moore, KL, Cummings, RD, and McEver, RP. "Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies." J Biol Chem 271.11 (March 15, 1996): 6342-6348.
PMID
8626430
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
11
Publish Date
1996
Start Page
6342
End Page
6348

Tenascin-C expression in dystrophin-related muscular dystrophy.

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas, but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration.

Authors
Settles, DL; Cihak, RA; Erickson, HP
MLA Citation
Settles, DL, Cihak, RA, and Erickson, HP. "Tenascin-C expression in dystrophin-related muscular dystrophy." Muscle Nerve 19.2 (February 1996): 147-154.
PMID
8559162
Source
pubmed
Published In
Muscle and Nerve
Volume
19
Issue
2
Publish Date
1996
Start Page
147
End Page
154
DOI
10.1002/(SICI)1097-4598(199602)19:2<147::AID-MUS4>3.0.CO;2-E

2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region.

We have determined the 2.0 A crystal structure of a fragment of human fibronectin encompassing the seventh through the RGD-containing tenth type III repeats (FN7-10). The structure reveals an extended rod-like molecule with a long axis of approximately 140 A and highly variable relationships between adjacent domains. An unusually small rotation between domains 9 and 10 creates a distinctive binding site, in which the RGD loop from domain 10 and the "synergy" region from domain 9 are on the same face of FN7-10 and thus easily accessible to a single integrin molecule. The cell-binding RGD loop is well-ordered in this structure and extends approximately 10 A away from the FN7-10 core.

Authors
Leahy, DJ; Aukhil, I; Erickson, HP
MLA Citation
Leahy, DJ, Aukhil, I, and Erickson, HP. "2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region." Cell 84.1 (January 12, 1996): 155-164.
PMID
8548820
Source
pubmed
Published In
Cell
Volume
84
Issue
1
Publish Date
1996
Start Page
155
End Page
164

Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.

The bacterial cell division protein FtsZ is a homolog of tubulin, but it has not been determined whether FtsZ polymers are structurally related to the microtubule lattice. In the present study, we have obtained high-resolution electron micrographs of two FtsZ polymers that show remarkable similarity to tubulin polymers. The first is a two-dimensional sheet of protofilaments with a lattice very similar to that of the microtubule wall. The second is a miniring, consisting of a single protofilament in a sharply curved, planar conformation. FtsZ minirings are very similar to tubulin rings that are formed upon disassembly of microtubules but are about half the diameter. This suggests that the curved conformation occurs at every FtsZ subunit, but in tubulin rings the conformation occurs at either beta- or alpha-tubulin subunits but not both. We conclude that the functional polymer of FtsZ in bacterial cell division is a long thin sheet of protofilaments. There is sufficient FtsZ in Escherichia coli to form a protofilament that encircles the cell 20 times. The similarity of polymers formed by FtsZ and tubulin implies that the protofilament sheet is an ancient cytoskeletal system, originally functioning in bacterial cell division and later modified to make microtubules.

Authors
Erickson, HP; Taylor, DW; Taylor, KA; Bramhill, D
MLA Citation
Erickson, HP, Taylor, DW, Taylor, KA, and Bramhill, D. "Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers." Proc Natl Acad Sci U S A 93.1 (January 9, 1996): 519-523.
PMID
8552673
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
1
Publish Date
1996
Start Page
519
End Page
523

Articular chondrocyte tenascin-C production and assembly into de novo extracellular matrix

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that is expressed in a variety of processes including development, tissue remodeling, wound healing, cell adhesion/antiadhesion, and cell/matrix interactions. Tenascin has recently been acknowledged as a component of the extracellular matrix of articular cartilage, but its function remains unclear. In this study, bovine articular chondrocytes were grown in alginate beads for 35 days to examine the kinetics of tenascin synthesis and incorporation into de novo extracellular matrix. During the culture period, 6 harvest days were established in which culture medium was recovered, alginate beads were dissociated with an EDTA solution, and chondrocytes were collected and lysed by sonication. Total DNA determination performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the medium, EDTA/alginate, and lysate samples demonstrated the production of both the 220 and 320 kDa tenascin size variants and their differential compartmentalization within the culture system. Tenascin was incorporated into the alginate bead matrix at a constant rate of 3.8 μg/day. The 320 kDa variant was produced in higher quantity, but the 220 kDa fragment was twice as likely to be incorporated into the de novo matrix. Methylene blue/acid fuchsin staining and tenascin immunohistochemistry demonstrated the incorporation of tenascin into a progressively expanding matrix surrounding the chondrocytes. The results suggest a role for tenascin in the assembly of the chondrocyte matrix and as a soluble mediator of chondrocytes with possible diverse functions for the tenascin size variants.

Authors
Savarese, JJ; Erickson, H; Scully, SP
MLA Citation
Savarese, JJ, Erickson, H, and Scully, SP. "Articular chondrocyte tenascin-C production and assembly into de novo extracellular matrix." Journal of Orthopaedic Research 14.2 (1996): 273-281.
PMID
8648506
Source
scival
Published In
Journal of Orthopaedic Research
Volume
14
Issue
2
Publish Date
1996
Start Page
273
End Page
281
DOI
10.1002/jor.1100140216

Binding of tenascin-C to soluble fibronectin and matrix fibrils.

The small splice variant of tenascin-C (TN) has eight fibronectin type III (FN3) domains. The major large splice variant has three (in chicken) or seven (in human) additional FN3 domains inserted between domains five and six. Chiquet-Ehrismann et al. (Chiquet-Ehrismann, R., Matsuoka, Y., Hofer, U., Spring, J., Bernasconi, C., and Chiquet, M. (1991, Cell Regul. 2, 927-938) demonstrated that the small variant bound preferentially to fibronectin in enzyme-linked immunosorbent assay, and only the small variant was incorporated into the matrix by cultures of chicken fibroblasts. Here we have studied human TN, and confirmed that the small variant binds preferentially to purified fibronectin and to fibronectin-containing extracellular matrix. Thus this differential binding appears to be conserved across vertebrate species. Using bacterial expression proteins, we mapped the major binding site to the third FN3 domain of TN. Consistent with this mapping, a monoclonal antibody against an epitope in this domain did not stain TN segments bound to cell culture matrix fibrils. The enhanced binding of the small TN variant suggests the existence of another, weak binding site probably in FN3 domains 6-8, which is only positioned to bind fibronectin in the small splice variant. This binding of domains 6-8 may involve a third molecule present in matrix fibrils, as the enhanced binding of small TN was much more prominent to matrix fibrils than to purified fibronectin.

Authors
Chung, CY; Zardi, L; Erickson, HP
MLA Citation
Chung, CY, Zardi, L, and Erickson, HP. "Binding of tenascin-C to soluble fibronectin and matrix fibrils." J Biol Chem 270.48 (December 1, 1995): 29012-29017.
PMID
7499434
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
48
Publish Date
1995
Start Page
29012
End Page
29017

A novel alternative splice domain in zebrafish tenascin-C.

We have identified and sequenced clones from a zebrafish library that correspond to tenascin-C (TN-C). The 2036-bp sequence covers the C-terminal segment of the protein. Comparison of this sequence to TN-C from other vertebrates indicates that our sequence starts in FN-III domain 5, continues with one alternative splice domain, then with FN-III domains 6-8 and, finally, the complete fibrinogen-like domain. The alternative splice domain, designated TNfnQ, is not closely identified to any of the domains in other species, and may represent a novel descendent of the alternative splice-domain precursor.

Authors
Qiao, T; Maddox, BK; Erickson, HP
MLA Citation
Qiao, T, Maddox, BK, and Erickson, HP. "A novel alternative splice domain in zebrafish tenascin-C." Gene 156.2 (April 24, 1995): 307-308.
PMID
7538963
Source
pubmed
Published In
Gene
Volume
156
Issue
2
Publish Date
1995
Start Page
307
End Page
308

Tenascin knockout mice: barrels, boundary molecules, and glial scars.

In light of a previous report suggesting that the brains of tenascin-deficient animals are grossly normal, we have studied the somatosensory cortical barrel field and injured cerebral cortex in postnatal homozygous tenascin knockout, heterozygote, and normal wild-type mice. Nissl staining, cytochrome oxidase, and Dil axonal tracing of thalamocortical axonal projections to the somatosensory cortex, all reveal the formation of normal barrels in the first postnatal week in homozygous knockout mice that cannot be distinguished from heterozygote or normal wild-type barrels. In addition to confirming the absence of tenascin in knockout animals, and reporting apparently reduced levels of the glycoprotein in barrel boundaries of heterozygote animals using well-characterized antibodies and immunocytochemistry, we also studied the DSD-1-PG proteoglycan, another developmentally regulated molecule known to be associated with transient glial/glycoconjugate boundaries that surround developing barrels; DSD-1-PG was also found to be expressed in barrel boundaries in apparently normal time frames in tenascin knockout mice. Peanut agglutinin (PNA) binding of galactosyl-containing glycoconjugates also revealed barrel boundaries in all three genotypes. We also examined the expression of tenascin-R, a paralog of tenascin-C (referred to here simply as tenascin). As previously reported, tenascin-R is prominently expressed in subcortical white matter, and we found it was not expressed in the barrel boundaries in any of the genotypes. Thus, the absence of tenascin does not result in a compensatory expression of tenascin-R in the barrel boundaries. Finally, we studied wounds of the cerebral cortex in the late postnatal mouse. The astroglial scar formed, for the most part, in the same time course and spatial distribution in the wild-type and tenascin knockout mice. However, there may be some differences in the extent of gliosis between the knockout and the wild type that warrant further study. Roles for boundary molecules like tenascin during brain pattern formation and injury are reconsidered in light of these findings on barrel development and cortical lesions in tenascin-deficient mice.

Authors
Steindler, DA; Settles, D; Erickson, HP; Laywell, ED; Yoshiki, A; Faissner, A; Kusakabe, M
MLA Citation
Steindler, DA, Settles, D, Erickson, HP, Laywell, ED, Yoshiki, A, Faissner, A, and Kusakabe, M. "Tenascin knockout mice: barrels, boundary molecules, and glial scars." J Neurosci 15.3 Pt 1 (March 1995): 1971-1983.
PMID
7534342
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
15
Issue
3 Pt 1
Publish Date
1995
Start Page
1971
End Page
1983

FtsZ, a prokaryotic homolog of tubulin?

Authors
Erickson, HP
MLA Citation
Erickson, HP. "FtsZ, a prokaryotic homolog of tubulin?." Cell 80.3 (February 10, 1995): 367-370. (Review)
PMID
7859278
Source
pubmed
Published In
Cell
Volume
80
Issue
3
Publish Date
1995
Start Page
367
End Page
370

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments.

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

Authors
Jones, PL; Boudreau, N; Myers, CA; Erickson, HP; Bissell, MJ
MLA Citation
Jones, PL, Boudreau, N, Myers, CA, Erickson, HP, and Bissell, MJ. "Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments." J Cell Sci 108 ( Pt 2) (February 1995): 519-527.
PMID
7539436
Source
pubmed
Published In
Journal of cell science
Volume
108 ( Pt 2)
Publish Date
1995
Start Page
519
End Page
527

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

Authors
Burns, CG; Larochelle, DA; Erickson, H; Reedy, M; Lozanne, AD
MLA Citation
Burns, CG, Larochelle, DA, Erickson, H, Reedy, M, and Lozanne, AD. "Single-headed myosin II acts as a dominant negative mutation in Dictyostelium." Proceedings of the National Academy of Sciences of the United States of America 92.18 (1995): 8244-8248.
PMID
7667276
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
92
Issue
18
Publish Date
1995
Start Page
8244
End Page
8248
DOI
10.1073/pnas.92.18.8244

Tenascin knockout mice: Barrels, boundary molecules, and glial scars

In light of a previous report suggesting that the brains of tenascin- deficient animals are grossly normal, we have studied the somatosensory cortical barrel field and injured cerebral cortex in postnatal homozygous tenascin knockout, heterozygote, and normal wild-type mice. Nissl staining, cytochrome oxidase, and Dil axonal tracing of thalamocortical axonal projections to the somatosensory cortex, all reveal the formation of normal barrels in the first postnatal week in homozygous knockout mice that cannot be distinguished from heterozygote or normal wild-type barrels. In addition to confirming the absence of tenascin in knockout animals, and reporting apparently reduced levels of the glycoprotein in barrel boundaries of heterozygote animals using well-characterized antibodies and immunocytochemistry, we also studied the DSD-1-PG proteoglycan, another developmentally regulated molecule known to be associated with transient glial/glycoconjugate boundaries that surround developing barrels; DSD-1-PG was also found to be expressed in barrel boundaries in apparently normal time frames in tenascin knockout mice. Peanut agglutinin (PNA) binding of galactosyl-containing glycoconjugates also revealed barrel boundaries in all three genotypes. We also examined the expression of tenascin-R, a paralog of tenascin-C (referred to here simply as tenascin). As previously reported, tenascin-R is prominently expressed in subcortical white matter, and we found it was not expressed in the barrel boundaries in any of the genotypes. Thus, the absence of tenascin does not result in a compensatory expression of tenascin-R in the barrel boundaries. Finally, we studied wounds of the cerebral cortex in the late postnatal mouse. The astroglial scar formed, for the most part, in the same time course and spatial distribution in the wild- type and tenascin knockout mice. However, there may be some differences in the extent of gliosis between the knockout and the wild type that warrant further study. Roles for boundary molecules like tenascin during brain pattern formation and injury are reconsidered in light of these findings on barrel development and cortical lesions in tenascin-deficient mice.

Authors
Steindler, DA; Settles, D; Erickson, HP; Laywell, ED; Yoshiki, A; Faissner, A; Kusakabe, M
MLA Citation
Steindler, DA, Settles, D, Erickson, HP, Laywell, ED, Yoshiki, A, Faissner, A, and Kusakabe, M. "Tenascin knockout mice: Barrels, boundary molecules, and glial scars." Journal of Neuroscience 15.3 I (1995): 1971-1983.
Source
scival
Published In
Journal of Neuroscience
Volume
15
Issue
3 I
Publish Date
1995
Start Page
1971
End Page
1983

Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin.

The elastic protein titin comprises a tandem array of fibronectin type III and immunoglobulin domains, which are structurally similar 7-strand beta-sandwiches. A proposed mechanism for stretching titin, by sequential denaturation of individual fibronectin type III-immunoglobulin domains in response to applied tension, is analyzed here quantitatively. The folded domain is approximately 4 nm long, and the unraveled polypeptide can extend to 29 nm, providing a 7-fold stretch over the relaxed length. Elastic recoil is achieved by refolding of the denatured domains when the force is released. The critical force required to denature a domain is calculated to be 3.5-5 pN, based on a net free energy for denaturation of 7-14 kcal/mol, plus 5 kcal/mol to extend the polypeptide (1 cal = 4.184 J). This force is comparable to the 2- to 7-pN force generated by single myosin or kinesin molecules. The force needed to pull apart a noncovalent protein-protein interface is estimated here to be 10-30 pN, implying that titin will stretch internally before the molecule is pulled from its attachment at the Z band. Many extracellular matrix and cell adhesion molecules, such as fibronectin, contain tandem arrays of fibronectin type III domains. Both single molecules and matrix fibers should have elastic properties similar to titin.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin." Proc Natl Acad Sci U S A 91.21 (October 11, 1994): 10114-10118.
PMID
7937847
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
91
Issue
21
Publish Date
1994
Start Page
10114
End Page
10118

LFA-1 binding site in ICAM-3 contains a conserved motif and non-contiguous amino acids.

The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-3 and VCAM-1 and hence many form a common site of contact in all CAMs of this type. Other ICAM-3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggest an evolutionary relationship between ICAMs and fibronectin interactions with integrins.

Authors
Sadhu, C; Lipsky, B; Erickson, HP; Hayflick, J; Dick, KO; Gallatin, WM; Staunton, DE
MLA Citation
Sadhu, C, Lipsky, B, Erickson, HP, Hayflick, J, Dick, KO, Gallatin, WM, and Staunton, DE. "LFA-1 binding site in ICAM-3 contains a conserved motif and non-contiguous amino acids." Cell Adhes Commun 2.5 (October 1994): 429-440.
PMID
7531103
Source
pubmed
Published In
Cell adhesion & communication
Volume
2
Issue
5
Publish Date
1994
Start Page
429
End Page
440

Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C.

We have investigated the binding of soluble tenascin-C (TN-C) to several cell lines using a radioligand binding assay. Specific binding was demonstrated to U-251MG human glioma cells and to a line of bovine aortic endothelial cells, but hamster fibroblasts showed no specific binding. Recombinant proteins corresponding to specific domains of TN-C were used to map the binding site(s) in TN-C. The alternatively spliced segment (TNfnA-D) inhibited the binding of native TN-C most strongly, and itself bound to glioma and endothelial cells. Scatchard analysis of TNfnA-D binding indicated 2-5 x 10(5) binding sites per cell, with an apparent 2 nM dissociation constant. The cell surface receptor for TNfnA-D was identified as a 35-kD protein on the basis of blot binding assays and affinity chromatography of membrane extracts on native TN-C and TNfnA-D columns. Protein sequencing indicated that this 35-kD receptor was annexin II. Annexin II is well characterized as a cytoplasmic protein, so it was surprising to find it as a presumably extracellular receptor for TN-C. To confirm that it was the 35-kD receptor, we obtained purified annexin II and demonstrated its binding to TNfnA-D and TN-C at nM concentrations. Antibodies to annexin II prominently stained the external surface of live endothelial cells and blocked the binding of TNfnA-D to the cells. Thus annexin II appears to be a receptor for the alternatively spliced segment of TN-C, and may mediate cellular responses to soluble TN-C in the extracellular matrix.

Authors
Chung, CY; Erickson, HP
MLA Citation
Chung, CY, and Erickson, HP. "Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C." J Cell Biol 126.2 (July 1994): 539-548.
PMID
7518469
Source
pubmed
Published In
The Journal of Cell Biology
Volume
126
Issue
2
Publish Date
1994
Start Page
539
End Page
548

Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.

Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.

Authors
Leahy, DJ; Erickson, HP; Aukhil, I; Joshi, P; Hendrickson, WA
MLA Citation
Leahy, DJ, Erickson, HP, Aukhil, I, Joshi, P, and Hendrickson, WA. "Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine." Proteins 19.1 (May 1994): 48-54.
PMID
8066086
Source
pubmed
Published In
Proteins: Structure, Function and Bioinformatics
Volume
19
Issue
1
Publish Date
1994
Start Page
48
End Page
54
DOI
10.1002/prot.340190107

Tenascin is induced at implantation sites in the mouse uterus and interferes with epithelial cell adhesion.

Expression of tenascin, an extracellular matrix protein associated with morphogenetic events and altered states of cellular adhesion, was examined in mouse uterus during the peri-implantation period. A uniform low level expression of tenascin was detected in stromal extracellular matrix during the estrous cycle and days 1 through 4 of early pregnancy. During the period of blastocyst attachment (day 4.5), an intense deposition of tenascin fibrils was located in the extracellular matrix of stroma immediately subjacent to the uterine epithelium surrounding the attaching blastocyst. This localized intensity of tenascin expression was both spatially and temporally restricted. By day 5.5, differentiation of stroma in the immediate area around the embryo to form the primary decidual zone was accompanied by a reduced amount of tenascin expression in the form of fragmented fibrils. Tenascin also could be induced by an artificial stimulus in uterine stroma of mice that had been hormonally prepared for implantation. The ability of artificial stimuli to induce tenascin expression suggested that the tenascin-inducing signals were derived from uterine cells, presumably lumenal epithelium, rather than embryonic cells. Consistent with this, conditioned medium from primary cultures of uterine epithelium was found to induce tenascin expression (2- to 4-fold) in isolated uterine stroma. Artificial stimuli generated a temporal pattern of tenascin expression similar to that observed during early pregnancy; however, in the artificially induced model, tenascin was induced in stroma immediately subjacent to lumenal epithelium along the entire length of the uterus. Purified tenascin and a recombinant tenascin fragment consisting of alternatively spliced fibronectin type III repeats, interfered with maintenance of uterine epithelial cell adhesion to Matrigel. In contrast, other recombinant tenascin fragments or fibronectin had no effect in this regard. Tenascin had no effect on adhesion of uterine stroma. Collectively, these results suggest that stimulation of TN expression in stromal extracellular matrix in vivo occurs via hormonally regulated, epithelial-mesenchymal interactions and serves as an early marker for uterine receptivity and the attachment phase of implantation. Furthermore, tenascin may facilitate embryo penetration by disrupting uterine epithelial cell adhesion to underlying basal lamina.

Authors
Julian, J; Chiquet-Ehrismann, R; Erickson, HP; Carson, DD
MLA Citation
Julian, J, Chiquet-Ehrismann, R, Erickson, HP, and Carson, DD. "Tenascin is induced at implantation sites in the mouse uterus and interferes with epithelial cell adhesion." Development 120.3 (March 1994): 661-671.
PMID
7512897
Source
pubmed
Published In
Development (Cambridge)
Volume
120
Issue
3
Publish Date
1994
Start Page
661
End Page
671

Tenascin-C in rat lung: distribution, ontogeny and role in branching morphogenesis.

Extracellular matrix is important to organogenesis and may function by modifying cellular adhesion, motility, proliferation, and differentiation. Tenascin-C (TN-C) is a matrix molecule reported to bind some cell lines and to inhibit adhesion of some cell types to fibronectin. This report describes the ontogeny and possible functions of TN-C expression in fetal and newborn rat lung. There was a moderate concentration of TN-C protein at the epithelial-mesenchymal interface during fetal lung development in the period of branching morphogenesis. There was a remarkable accumulation of TN-C during the first postnatal week when alveolarization peaked, followed by a decline to barely detectable levels after the third postnatal week when alveolarization was essentially completed. Loss of TN-C protein followed quickly the loss of TN-C mRNA, suggesting a rapid turnover of TN-C in the extracellular matrix. By light microscopy, immunoreactive TN-C was present in early postnatal lung at the epithelial-mesenchymal interface and was distributed throughout lung mesenchyme. Electron microscopic immunocytochemistry showed TN-C was not a part of the basal lamina and that its lung localization was punctate and different from the uniform distribution of laminin. Antiserum to TN-C significantly inhibited branching morphogenesis of fetal lung explants but did not block their growth. Three bacterially expressed segments of TN-C comprising different fibronectin type III domains inhibited branching morphogenesis as effectively as did antiserum, but an expression protein of the carboxyterminal fibrinogen-like segment had no effect. We conclude that TN-C is expressed in a spatio-temporal pattern consistent with a role in lung development and our in vitro studies indicated a functional role for TN-C during lung branching morphogenesis.

Authors
Young, SL; Chang, LY; Erickson, HP
MLA Citation
Young, SL, Chang, LY, and Erickson, HP. "Tenascin-C in rat lung: distribution, ontogeny and role in branching morphogenesis." Dev Biol 161.2 (February 1994): 615-625.
PMID
7508872
Source
pubmed
Published In
Developmental Biology
Volume
161
Issue
2
Publish Date
1994
Start Page
615
End Page
625
DOI
10.1006/dbio.1994.1057

Induction of human tenascin (neuronectin) by growth factors and cytokines: cell type-specific signals and signalling pathways.

The extracellular matrix protein tenascin (TN) is expressed with precise temporo-spatial patterns during embryonic and fetal development and is induced in healing wounds, inflammatory lesions and solid tumors. These tissue patterns suggest that TN synthesis may be modulated by soluble factors present in developing tissues or released from injured, inflammatory or neoplastic cells. To characterize the extrinsic control of human TN we examined the effects of several signalling molecules on cultured neural, melanocytic and fibroblastic cells. Results obtained with alpha TN antibodies in enzyme-linked immunosorbent and immunoprecipitation assays indicate that TN expression is tightly regulated in a cell type-specific manner: (1) Primitive neuroectodermal tumor (PNET) cells grown in chemically defined, serum-free media show up to > 100-fold TN induction in response to fibroblast growth factors (aFGF, bFGF, K-FGF) and phorbol ester, independent of changes in cell proliferation or total protein synthesis; no induction is seen in PNET cultures stimulated with serum or other growth and differentiation factors. (2) Normal melanocytes, which require FGF and phorbol ester for survival in vitro, fail to express TN; however, they produce TN following oncogenic transformation. (3) Fibroblasts derived from disparate tissues differ up to 100-fold in basal TN production; for example, fetal lung fibroblasts are TNhigh, but conjunctival fibroblasts derived from the same donors and fetal leptomeningeal cells are TNlow. (4) TNlow fibroblasts treated with interleukin-1, tumor necrosis factor-alpha, and interleukin-4 show up to > 100-fold increased TN secretion and TN incorporation into their extracellular matrix. Transforming growth factor-beta, which acts as an inducer of fibronectin, collagen, and integrin-type matrix receptors, has variable effects on fibroblast TN, ranging from increased deposition in the extracellular matrix of fetal conjunctival fibroblasts to reduced secretion in newborn foreskin fibroblasts. In contrast, FGFs (which are potent fibroblast mitogens), phorbol ester, bone morphogenetic proteins, and several other factors tested produced no discernible effects on fibroblast TN expression. These findings suggest that discrete sets of extrinsic signals modify TN expression in specific cell types, with the effects of a given ligand/receptor system determined by cell type-specific signalling pathways that may be linked to unique cis-regulatory elements of the TN gene. As a result, a limited set of regulatory peptides may produce highly diversified TN distribution patterns in developing and lesional tissues.

Authors
Rettig, WJ; Erickson, HP; Albino, AP; Garin-Chesa, P
MLA Citation
Rettig, WJ, Erickson, HP, Albino, AP, and Garin-Chesa, P. "Induction of human tenascin (neuronectin) by growth factors and cytokines: cell type-specific signals and signalling pathways." J Cell Sci 107 ( Pt 2) (February 1994): 487-497.
PMID
7515896
Source
pubmed
Published In
Journal of cell science
Volume
107 ( Pt 2)
Publish Date
1994
Start Page
487
End Page
497

Evolution of the tenascin family--implications for function of the C-terminal fibrinogen-like domain.

The three members of the tenascin (TN) family, TN-C, TN-R, and TN-X, are apparently conserved in all vertebrates and therefore must have functions that contribute to survival. One specific domain of tenascins, the fibrinogen-like terminal knob, can be argued to have an essential function. Its position at the C-terminus makes it most vulnerable to loss through mutation or deletion, and it should have been eliminated in evolution if there were no selective pressure to maintain it. The epidermal growth factor and fibronectin III domains probably play an important role as spacers, placing the terminal knob at the end of the tribrachion or hexabrachion arms. In addition to functioning as spacers, at least some of these domains may have additional functional interactions. The conservation of these domains in evolution is comparable to that of some growth factors, consistent with this possibility. A phylogenetic tree of all known fibrinogen-related domains, including those in tenascins, is presented.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Evolution of the tenascin family--implications for function of the C-terminal fibrinogen-like domain." Perspect Dev Neurobiol 2.1 (1994): 9-19. (Review)
PMID
7530148
Source
pubmed
Published In
Perspectives on Developmental Neurobiology
Volume
2
Issue
1
Publish Date
1994
Start Page
9
End Page
19

Colocalization of tenascin with versican, a hyaluronate-binding chondroitin sulfate proteoglycan.

Rabbit antisera against tenascin, a large extracellular matrix protein, in conjunction with monoclonal antibodies of mouse origin against versican, a large hyaluronate-binding proteoglycan, were used to make a comparative study of the distribution of the two antigens in the same cryostat sections by double immunofluorescence. In the central nervous system, tenascin was invariably associated with versican, but the reverse was not true, in that versican was also found where tenascin was not detectable, particularly in gray matter. There were major species differences in the distribution of tenascin in the central nervous system. In the cow, tenascin was found in cerebral and spinal cord white matter and in the granule cell layer of the cerebellum. In the human brain, tenascin was found in cerebral white matter but not in the cerebellum. In the rat, tenascin was mainly confined to brain periventricular layer and spinal cord white matter. During the development of the cerebellum of the rat, the tenascin immunoreactivity decreased, and a lower molecular weight band appeared (J1-160/180/restrictin?) and persisted throughout adulthood. Tenascin expression was a relatively late event in the development of the rat central nervous system, immunoreactivity being first observed after birth. In the rat embryo, tenascin was found to co-localize with versican in precartilaginous mesenchyme and in connective tissue underlying epithelia. The colocalization of versican with tenascin suggests that versican may be the tenascin (cytotactin)-associated proteoglycan reported in the literature.

Authors
Perides, G; Erickson, HP; Rahemtulla, F; Bignami, A
MLA Citation
Perides, G, Erickson, HP, Rahemtulla, F, and Bignami, A. "Colocalization of tenascin with versican, a hyaluronate-binding chondroitin sulfate proteoglycan." Anat Embryol (Berl) 188.5 (November 1993): 467-479.
PMID
7508696
Source
pubmed
Published In
Anatomy and embryology
Volume
188
Issue
5
Publish Date
1993
Start Page
467
End Page
479

Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions.

"Mice develop normally without tenascin", was a shock to biologists studying the extracellular matrix. Could tenascin be a useless protein? This seems most improbable, as it is conserved in every vertebrate species. Moreover, two new proteins have been discovered in the last year that are closely related to the original tenascin, providing evidence for a tenascin family. Speculations on functions are ripe for re-evaluation.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions." Curr Opin Cell Biol 5.5 (October 1993): 869-876. (Review)
PMID
7694605
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
5
Issue
5
Publish Date
1993
Start Page
869
End Page
876

Endothelial cells adhere to the RGD domain and the fibrinogen-like terminal knob of tenascin.

We have found that endothelial cells adhere much more strongly than fibroblasts to domains of tenascin and fibronectin. Endothelial cells adhered weakly, without spreading, to bacterial expression proteins corresponding to the tenth fibronectin type III (FN-III) domain of fibronectin, which contains the RGD. A larger fibronectin protein, containing this domain and the three amino-terminal 'synergy' domains gave strong adhesion and spreading. Two widely separated domains of tenascin gave adhesion. The third FN-III domain, TNfn3, which contains an RGD sequence in human and chicken tenascin, gave very strong adhesion and spreading of endothelial cells when tested as an isolated domain. Larger segments containing TNfn3 and the adjacent TNfn2 gave weaker adhesion, probably because the RGD sequence is partially blocked. Adhesion to this domain required divalent cations, was exquisitely sensitive to soluble GRGDSP peptide, and was blocked by antisera to the integrin alpha v beta 3. The second tenascin adhesion domain was the fibrinogen-like C-terminal knob, TNfbg. Cells adhered to but did not spread on this domain. This adhesion required divalent cations and was also sensitive to GRGDSP peptide, so it may be mediated by an integrin receptor. We have explored a range of conditions for preparing the adhesion substratum, and our results may resolve the controversy over whether tenascin can act as a substratum adhesion molecule. When coated for short times (1-2 hours) on plastic, tenascin had no adhesion activity, in contrast to fibronectin and the expression proteins, which gave strong adhesion under these conditions. When coated for longer times (12-24 hours) on plastic, the tenascin substratum supported good adhesion, but not spreading, of endothelial cells. Tenascin coated on nitrocellulose gave substantially stronger adhesion than on plastic, but still required long coating times for maximal activity. Adhesion of endothelial cells to native TN was inhibited by GRDGSP peptide. The cell adhesion activity demonstrates the presence on endothelial cells of tenascin receptors, which may play a supportive role in angiogenesis, in the structure of blood vessels, or in binding tenascin to the cell surface to elicit or enhance a signalling function.

Authors
Joshi, P; Chung, CY; Aukhil, I; Erickson, HP
MLA Citation
Joshi, P, Chung, CY, Aukhil, I, and Erickson, HP. "Endothelial cells adhere to the RGD domain and the fibrinogen-like terminal knob of tenascin." J Cell Sci 106 ( Pt 1) (September 1993): 389-400.
PMID
7505785
Source
pubmed
Published In
Journal of cell science
Volume
106 ( Pt 1)
Publish Date
1993
Start Page
389
End Page
400

Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin.

P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.

Authors
Ushiyama, S; Laue, TM; Moore, KL; Erickson, HP; McEver, RP
MLA Citation
Ushiyama, S, Laue, TM, Moore, KL, Erickson, HP, and McEver, RP. "Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin." J Biol Chem 268.20 (July 15, 1993): 15229-15237.
PMID
7686912
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
20
Publish Date
1993
Start Page
15229
End Page
15237

Structural and functional domains of the Drosophila ncd microtubule motor protein.

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein that is required for proper chromosome distribution in Drosophila. Despite its sequence similarity to kinesin heavy chain, ncd translocates with the opposite polarity as kinesin, toward microtubule minus ends. We have expressed different regions of the protein in bacteria and analyzed the proteins for function. Results indicate that ncd consists of three domains: a basic, proline-rich N-terminal "tail," a central alpha-helical coiled-coil stalk, and a C-terminal motor domain. The ncd N terminus proteins bundle microtubules in motility assays and show ATP-independent binding to microtubules in solution. Truncated proteins, lacking the tail but containing the predicted motor domain and differing lengths of the stalk, did not support microtubule gliding in in vitro assays but showed microtubule-stimulated MgATPase activity in solution. Addition of a nonspecific N terminus to two of the truncated proteins restored directional gliding and rotation of microtubules in motility assays, demonstrating that these properties map to the predicted mechanochemical domain of ncd. Physical properties of the C terminus proteins indicate that the stalk region is important for dimerization and that the ncd protein probably exists and functions as a dimer.

Authors
Chandra, R; Salmon, ED; Erickson, HP; Lockhart, A; Endow, SA
MLA Citation
Chandra, R, Salmon, ED, Erickson, HP, Lockhart, A, and Endow, SA. "Structural and functional domains of the Drosophila ncd microtubule motor protein." J Biol Chem 268.12 (April 25, 1993): 9005-9013.
PMID
8473343
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
12
Publish Date
1993
Start Page
9005
End Page
9013

Editorial overview

Authors
Erickson, H; Holmes, K
MLA Citation
Erickson, H, and Holmes, K. "Editorial overview." Current Opinion in Structural Biology 3.2 (April 1, 1993): 165-166.
Source
scopus
Published In
Current Opinion in Structural Biology
Volume
3
Issue
2
Publish Date
1993
Start Page
165
End Page
166

Gene knockouts of c-src, transforming growth factor beta 1, and tenascin suggest superfluous, nonfunctional expression of proteins.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Gene knockouts of c-src, transforming growth factor beta 1, and tenascin suggest superfluous, nonfunctional expression of proteins." J Cell Biol 120.5 (March 1993): 1079-1081. (Review)
PMID
7679670
Source
pubmed
Published In
The Journal of Cell Biology
Volume
120
Issue
5
Publish Date
1993
Start Page
1079
End Page
1081

Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins.

We have produced a set of bacterial expression proteins corresponding to 10 segments of tenascin and two of fibronectin and tested them for heparin binding and cell adhesion. We used polymerase chain reaction cloning to terminate the segments precisely at domain boundaries. Heparin binding activity was mapped to two different tenascin segments: one comprising the fourth and fifth fibronectin type III domains, and to TNfbg, the fibrinogen-like terminal knob. TNfbg, but none of the other tanascin segments, also supported adhesion of primary rat embryo skin fibroblasts. The fibroblasts did not spread on TNfbg but remained rounded. Cell binding to TNfbg occurred in the presence or absence of divalent cations and was not inhibited by RGD peptides, suggesting that integrins are not involved. Fibroblast binding to TNfbg was strongly inhibited by soluble heparin, by treating the cells with heparitinase, or by culture conditions that cause undersulfation of proteoglycans. These observations suggest that cell attachment to TNfbg is mediated by cell surface proteoglycans. We have also made full-length cDNA constructs for the largest and smallest splice variants of human tenascin, as well as one truncated after the 14th epidermal growth factor-like domain, in the pNUT mammalian cell expression vector. Stably transfected baby hamster kidney cell lines secreted large quantities of tenascin, and this was assembled into normal hexabrachions, the arm length corresponding to the construct.

Authors
Aukhil, I; Joshi, P; Yan, Y; Erickson, HP
MLA Citation
Aukhil, I, Joshi, P, Yan, Y, and Erickson, HP. "Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins." J Biol Chem 268.4 (February 5, 1993): 2542-2553.
PMID
7679097
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
4
Publish Date
1993
Start Page
2542
End Page
2553

Machinery of life

Authors
Goodsell, DS; Erickson Harold, P
MLA Citation
Goodsell, DS, and Erickson Harold, P. "Machinery of life." Nature 365.6444 (1993): 306-. (Academic Article)
Source
manual
Published In
Nature
Volume
365
Issue
6444
Publish Date
1993
Start Page
306

Localization of tenascin in uterine sarcomas and partially transformed endometrial stromal cells.

Normal mesenchymal cells within developing embryonic organs and transformed stromal cells in organs undergoing spontaneous carcinogenesis have the capacity for normal or altered expression of the extracellular matrix glycoprotein tenascin (Tn). Mesenchymal cell constituents of normal adult organs show only a very limited tendency to deposit Tn in their extracellular matrix. In the present study, we investigated whether malignant human mesenchymal cells derived from uterine sarcomas or normal human endometrial stromal cells partially transformed via transfection with selected oncogenes have the capacity to produce and deposit Tn. We reached the following conclusions: (1) compared with normal endometrial tissues, uterine sarcomas show heterogeneous, but increased, immunoreactive staining patterns exclusively within the extracellular compartment, regardless of the histologic subtype of the tumor; (2) in vitro, all normal and transfected stromal cells and cell lines examined secreted Tn into the tissue culture medium; (3) this secretory ability was not translated into morphologic uniformity, since immunoreactivity detected by confocal laser scanning microscopy was observed in only selected cell populations; (4) also, the deposition and the incorporation of Tn depended upon the density of transfected cells, and (5) double-staining experiments revealed that Tn could always be localized in close proximity to fibronectin. In summary, the production of Tn is increased in most cases of human uterine sarcoma. The capacity of stromal cells to deposit Tn in a matrix-like structure in vitro, rather than increase production of Tn, is correlated with the degree of neoplastic progression.

Authors
Vollmer, G; Lightner, VA; Carter, CA; Siegal, GP; Erickson, HP; Knuppen, R; Kaufman, DG
MLA Citation
Vollmer, G, Lightner, VA, Carter, CA, Siegal, GP, Erickson, HP, Knuppen, R, and Kaufman, DG. "Localization of tenascin in uterine sarcomas and partially transformed endometrial stromal cells." Pathobiology 61.2 (1993): 67-76.
PMID
7692874
Source
pubmed
Published In
Pathobiology : journal of immunopathology, molecular and cellular biology
Volume
61
Issue
2
Publish Date
1993
Start Page
67
End Page
76

Macromolecular assemblages

Authors
Erickson, H; Holmes, K
MLA Citation
Erickson, H, and Holmes, K. "Macromolecular assemblages." Current Opinion in Structural Biology 3.2 (1993): 165-166.
Source
scival
Published In
Current Opinion in Structural Biology
Volume
3
Issue
2
Publish Date
1993
Start Page
165
End Page
166

Structural organization of the human insulin receptor ectodomain.

To provide an experimental system amenable to a detailed biochemical and structural investigation of the extracellular (ligand binding) domain of the insulin receptor, we developed a mammalian heterologous cell expression system from which tens of milligrams of the soluble secreted ectodomain (the IR921 protein) can be routinely purified using methods that do not require harsh elution conditions. The purified IR921 protein has a Stokes radius of 6.8 nm and a sedimentation coefficient of 9.8 S, from which we calculate a hydro-dynamic mass of 281 kDa. Electron microscopic images, using both rotary shadowing and negative staining techniques, demonstrate a characteristic substructure for the IR921 protein consisting of two elongated arms, with a globular domain at each end, connected to each other at a point somewhat off-center to form a Y structure. Analysis using circular dichroism and fluorescence spectroscopy illustrate that insulin binding results in conformational changes in the ectodomain. Furthermore, fluorescence anisotropy decay data reveal segmental mobility within the IR921 protein that is successively frozen as a result of insulin binding, in contrast to results obtained in a previous study of the epidermal growth factor receptor ectodomain. This result suggests a divergence in hormone-induced signaling mechanisms used by the insulin and epidermal growth factor receptors.

Authors
Schaefer, EM; Erickson, HP; Federwisch, M; Wollmer, A; Ellis, L
MLA Citation
Schaefer, EM, Erickson, HP, Federwisch, M, Wollmer, A, and Ellis, L. "Structural organization of the human insulin receptor ectodomain." J Biol Chem 267.32 (November 15, 1992): 23393-23402.
PMID
1385419
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
32
Publish Date
1992
Start Page
23393
End Page
23402

Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein.

Fibronectin type III domains are found in many different proteins including cell surface receptors and cell adhesion molecules. The crystal structure of one such domain from the extracellular matrix protein tenascin was determined. The structure was solved by multiwavelength anomalous diffraction (MAD) phasing of the selenomethionyl protein and has been refined to 1.8 angstrom resolution. The folding topology of this domain is identical to that of the extracellular domains of the human growth hormone receptor, the second domain of CD4, and PapD. Although distinct, this topology is similar to that of immunoglobulin constant domains. An Arg-Gly-Asp (RGD) sequence that can function for cell adhesion is found in a tight turn on an exposed loop.

Authors
Leahy, DJ; Hendrickson, WA; Aukhil, I; Erickson, HP
MLA Citation
Leahy, DJ, Hendrickson, WA, Aukhil, I, and Erickson, HP. "Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein." Science 258.5084 (November 6, 1992): 987-991.
PMID
1279805
Source
pubmed
Published In
Science
Volume
258
Issue
5084
Publish Date
1992
Start Page
987
End Page
991

Kinetics of protein-protein association explained by Brownian dynamics computer simulation.

Protein-protein bond formations, such as antibody-antigen complexation or aggregation of protein monomers into dimers and larger aggregates, occur with bimolecular rate constants on the order of 10(6) M-1.s-1, which is only 3 orders of magnitude slower than the diffusion-limited Smoluchowski rate. However, since the protein-protein bond requires rotational alignment to within a few angstroms of tolerance, purely geometric estimates would suggest that the observed rates might be 6 orders of magnitude below the Smoluchowski rate. Previous theoretical treatments have not been solved for the highly specific docking criteria of protein-protein association--the entire subunit interface must be aligned within 2 A of the correct position. Several studies have suggested that diffusion alone could not produce the rapid association kinetics and have postulated "lengthy collisions" and/or the operation of electrostatic or hydrophobic steering forces to accelerate the association. In the present study, the Brownian dynamics simulation method is used to compute the rate of association of neutral spherical model proteins with the stated docking criteria. The Brownian simulation predicts a rate of 2 x 10(6) M-1.s-1 for this generic protein-protein association, a rate that is 2000 times faster than that predicted by the simplest geometric calculation and is essentially equal to the rates observed for protein-protein association in aqueous solution. This high rate is obtained by simple diffusive processes and does not require any attractive or steering forces beyond those achieved for a partially formed bond. The rate enhancement is attributed to a diffusive entrapment effect, in which a protein pair surrounded and trapped by water undergoes multiple collisions with rotational reorientation during each encounter.

Authors
Northrup, SH; Erickson, HP
MLA Citation
Northrup, SH, and Erickson, HP. "Kinetics of protein-protein association explained by Brownian dynamics computer simulation." Proc Natl Acad Sci U S A 89.8 (April 15, 1992): 3338-3342.
PMID
1565624
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
8
Publish Date
1992
Start Page
3338
End Page
3342

Visualization of purified fibronectin-transglutaminase complexes.

It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.

Authors
LeMosy, EK; Erickson, HP; Beyer, WF; Radek, JT; Jeong, JM; Murthy, SN; Lorand, L
MLA Citation
LeMosy, EK, Erickson, HP, Beyer, WF, Radek, JT, Jeong, JM, Murthy, SN, and Lorand, L. "Visualization of purified fibronectin-transglutaminase complexes." J Biol Chem 267.11 (April 15, 1992): 7880-7885.
PMID
1348509
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
11
Publish Date
1992
Start Page
7880
End Page
7885

Microtubule dynamic instability and GTP hydrolysis.

Authors
Erickson, HP; O'Brien, ET
MLA Citation
Erickson, HP, and O'Brien, ET. "Microtubule dynamic instability and GTP hydrolysis." Annu Rev Biophys Biomol Struct 21 (1992): 145-166. (Review)
PMID
1525467
Source
pubmed
Published In
Annual Review of Biophysics and Biomolecular Structure
Volume
21
Publish Date
1992
Start Page
145
End Page
166
DOI
10.1146/annurev.bb.21.060192.001045

Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin.

Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.

Authors
Murphy-Ullrich, JE; Lightner, VA; Aukhil, I; Yan, YZ; Erickson, HP; Höök, M
MLA Citation
Murphy-Ullrich, JE, Lightner, VA, Aukhil, I, Yan, YZ, Erickson, HP, and Höök, M. "Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin." J Cell Biol 115.4 (November 1991): 1127-1136.
PMID
1720121
Source
pubmed
Published In
The Journal of Cell Biology
Volume
115
Issue
4
Publish Date
1991
Start Page
1127
End Page
1136

Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap.

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubule of 60 microns/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437-1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.

Authors
Voter, WA; O'Brien, ET; Erickson, HP
MLA Citation
Voter, WA, O'Brien, ET, and Erickson, HP. "Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap." Cell Motil Cytoskeleton 18.1 (1991): 55-62.
PMID
2004433
Source
pubmed
Published In
Cell Motility and the Cytoskeleton
Volume
18
Issue
1
Publish Date
1991
Start Page
55
End Page
62
DOI
10.1002/cm.970180106

Effects of magnesium on the dynamic instability of individual microtubules.

We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.

Authors
O'Brien, ET; Salmon, ED; Walker, RA; Erickson, HP
MLA Citation
O'Brien, ET, Salmon, ED, Walker, RA, and Erickson, HP. "Effects of magnesium on the dynamic instability of individual microtubules." Biochemistry 29.28 (July 17, 1990): 6648-6656.
PMID
2397205
Source
pubmed
Published In
Biochemistry
Volume
29
Issue
28
Publish Date
1990
Start Page
6648
End Page
6656

HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits.

The ultrastructure and biochemical characteristics of HMW-2, the Sertoli cell cytoplasmic dynein isolated from rat testes, were analyzed. Electron microscopic studies revealed a two-headed two-stem structure with dimensions very similar to other dyneins. We found that, like other cytoplasmic dyneins, both heads have an approximately spherical shape with a central cavity. Heavy chain analysis suggested the presence of only one type of heavy chain, a finding that was supported by the simple Michaelis-Menten kinetics displayed by the HMW-2-associated ATPase activity. In addition, dissociation of the HMW-2 complex resulted in a single type of dynein subunit sedimenting at 11.8 S. This fraction contained all the polypeptides present in the undissociated HMW-2. Ultrastructurally the HMW-2 subunits were composed of one globular domain with a tail. The simplest interpretation is that HMW-2 is a dimer of nearly identical subunits, each containing one heavy chain, one 90-kDa intermediate chain, and two light chains.

Authors
Neely, MD; Erickson, HP; Boekelheide, K
MLA Citation
Neely, MD, Erickson, HP, and Boekelheide, K. "HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits." J Biol Chem 265.15 (May 25, 1990): 8691-8698.
PMID
2140361
Source
pubmed
Published In
The Journal of biological chemistry
Volume
265
Issue
15
Publish Date
1990
Start Page
8691
End Page
8698

Purification of hexabrachion (tenascin) from cell culture conditioned medium, and separation from a cell adhesion factor.

We describe a protocol for purifying hexabrachion from conditioned medium of cell cultures, using gel filtration chromatography on Sephacryl 500, followed by anion-exchange chromatography on a Mono Q column, followed optionally by a second gel filtration or zone sedimentation on glycerol gradients. The protocol has several advantages over previous procedures based on affinity chromatography on monoclonal antibodies. Perhaps foremost, the protein is never exposed to the denaturing solvents that are required for elution from the antibody column. The Mono Q column also separated hexabrachion from a prominent cell adhesion activity that eluted with the hexabrachion on the first gel filtration, and co-sedimented with hexabrachions on glycerol gradients. The cell adhesion fractions showed several bands between 190 and 400 kDa. A single band at 220 kDa stained prominently with a polyclonal antibody against mouse EHS laminin, and a band at 190 kDa stained with a monoclonal antibody against s-laminin. The purification protocol gave hexabrachion at high concentration and with no detectable contamination by fibronectin or laminin. The highest yield of hexabrachion (1-4 mg from 400 ml of conditioned medium) was from human glioblastoma cell cultures, but the same procedure allowed us to purify and characterize the rat hexabrachion. Protein purified from primary cultures of rat embryo fibroblasts showed approximately equal amounts of three subunit sizes: 280, 230, and 220 kDa. These different subunits, presumably derived from alternative RNA splicing, appeared to be segregated into large and small hexabrachions, which could be separated on glycerol gradients.

Authors
Aukhil, I; Slemp, CC; Lightner, VA; Nishimura, K; Briscoe, G; Erickson, HP
MLA Citation
Aukhil, I, Slemp, CC, Lightner, VA, Nishimura, K, Briscoe, G, and Erickson, HP. "Purification of hexabrachion (tenascin) from cell culture conditioned medium, and separation from a cell adhesion factor." Matrix 10.2 (May 1990): 98-111.
PMID
1695709
Source
pubmed
Published In
Matrix Biology
Volume
10
Issue
2
Publish Date
1990
Start Page
98
End Page
111

The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus.

Intercellular adhesion molecule 1 (ICAM-1, CD54) binds to the integrin LFA-1 (CD11a/CD18), promoting cell adhesion in immune and inflammatory reactions. ICAM-1 is also subverted as a receptor by the major group of rhinoviruses. Electron micrographs show that ICAM-1 is a bent rod, 18.7 nm long, suggesting a model in which the five immunoglobulin-like domains are oriented head to tail at a small angle to the rod axis. ICAM-1 sequences important to binding LFA-1, rhinovirus, and four monoclonal antibodies were identified through the characterization of chimeric ICAM-1 molecules and mutants. The amino-terminal two immunoglobulin-like domains of ICAM-1 appear to interact conformationally. Domain 1 of ICAM-1 contains the primary site of contact for both LFA-1 and rhinovirus; the presence of domains 3-5 markedly affects the accessibility of the binding site for rhinovirus and less so for LFA-1. The binding sites appear to be distinct but overlapping; rhinovirus binding also differs from LFA-1 binding in its lack of divalent cation dependence. Our analysis suggests that rhinoviruses mimic LFA-1 in binding to the most membrane-distal, and thus most accessible, site of ICAM-1.

Authors
Staunton, DE; Dustin, ML; Erickson, HP; Springer, TA
MLA Citation
Staunton, DE, Dustin, ML, Erickson, HP, and Springer, TA. "The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus." Cell 61.2 (April 20, 1990): 243-254.
PMID
1970514
Source
pubmed
Published In
Cell
Volume
61
Issue
2
Publish Date
1990
Start Page
243
End Page
254

Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion.

Hexabrachion is a large glycoprotein of the extracellular matrix (ECM) that is prominent in embryogenesis, wound healing and tumorigenesis. Because of the role of extracellular matrix proteins in the regulation of cell differentiation and migration, the interaction of hexabrachion with cells as well as with other components of the ECM is of great interest. Early reports suggested that hexabrachion does not bind to fibronectin or gelatin but does bind to chondroitin sulfate proteoglycans. However, more recent reports have suggested that hexabrachion binds to fibronectin and inhibits cell adhesion as well as cell migration on fibronectin. We have found no evidence of strong hexabrachion-fibronectin binding on either a solid-phase ELISA assay or in a fluid-phase sedimentation assay in which the reactants were allowed to dissociate. However, hexabrachion sedimentation was accelerated in a gradient containing fibronectin throughout. This demonstrates an association between hexabrachions and fibronectin, but the complex is apparently weak and readily reversible. The solid-phase ELISA also shows no evidence of hexabrachion binding to gelatin, laminin or types I, III, IV or V collagen. Hexabrachion does not support strong cell attachment of the cell lines tested. Moreover, hexabrachion can inhibit cell attachment to fibronectin. We demonstrate here that this inhibition requires the hexabrachion to be able to bind to the plastic substratum. The results suggest that hexabrachion inhibition is via a steric inhibition. When the hexabrachion molecules bind to the plastic, they cover up a significant fraction of the underlying fibronectin molecules. Antibody studies are presented that show that hexabrachion can nonspecifically block access of immunoglobulin G molecules to the underlying matrix. This steric blocking is not unique to hexabrachion.

Authors
Lightner, VA; Erickson, HP
MLA Citation
Lightner, VA, and Erickson, HP. "Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion." J Cell Sci 95 ( Pt 2) (February 1990): 263-277.
PMID
1695220
Source
pubmed
Published In
Journal of cell science
Volume
95 ( Pt 2)
Publish Date
1990
Start Page
263
End Page
277

Localization and quantitation of hexabrachion (tenascin) in skin, embryonic brain, tumors, and plasma.

Authors
Lightner, VA; Slemp, CA; Erickson, HP
MLA Citation
Lightner, VA, Slemp, CA, and Erickson, HP. "Localization and quantitation of hexabrachion (tenascin) in skin, embryonic brain, tumors, and plasma." Ann N Y Acad Sci 580 (1990): 260-275.
PMID
1692456
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
580
Publish Date
1990
Start Page
260
End Page
275

Biochemical and structural studies of tenascin/hexabrachion proteins.

Tenascin is a large, disulfide-bonded glycoprotein of the extracellular matrix. The predominant form of tenascin observed by electron microscopy is a six-armed oligomer, termed a hexabrachion. We have determined the molecular mass of the native human hexabrachion to be 1.9 x 10(6) Da by sedimentation equilibrium analysis and by electrophoresis on non-reducing agarose gels. On reducing polyacrylamide gel electrophoresis (SDS-PAGE), human tenascin showed a single prominent band at 320 kDa and minor bands of 220 and 230 kDa. The molecular weight of the native human hexabrachion is thus consistent with a disulfide-bonded hexamer of the 320 kDa subunits. Upon treatment with neuraminidase, the apparent molecular weights of all human and chicken tenascin subunits on reducing SDS-PAGE were decreased by about 10 kDa. Prolonged incubation with alpha-mannosidase, however, caused no apparent change in the apparent molecular weight of tenascin subunits. Sedimentation in a cesium chloride gradient gave a higher buoyant density for human tenascin than for fibronectin, suggesting that it has a higher degree of glycosylation. The far-UV circular dichroism spectrum indicates a predominance of beta-structure and a lack of collagen-like or alpha-helical structure. When human hexabrachions were reduced and acetylated, the resulting fragments were single arms which sedimented at 6 S in glycerol gradients and migrated at 320 kDa on non-reducing gels. Treatment of tenascin with trypsin and alpha-chymotrypsin also produced large fragments which were fractionated by gradient sedimentation and analyzed by non-reducing SDS-PAGE and electron microscopy. We present a structural model for the assembly of the observed fragments into the elaborate native hexabrachion.

Authors
Taylor, HC; Lightner, VA; Beyer, WF; McCaslin, D; Briscoe, G; Erickson, HP
MLA Citation
Taylor, HC, Lightner, VA, Beyer, WF, McCaslin, D, Briscoe, G, and Erickson, HP. "Biochemical and structural studies of tenascin/hexabrachion proteins." J Cell Biochem 41.2 (October 1989): 71-90.
PMID
2482292
Source
pubmed
Published In
Journal of Cellular Biochemistry
Volume
41
Issue
2
Publish Date
1989
Start Page
71
End Page
90
DOI
10.1002/jcb.240410204

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response.

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.

Authors
Lotz, MM; Burdsal, CA; Erickson, HP; McClay, DR
MLA Citation
Lotz, MM, Burdsal, CA, Erickson, HP, and McClay, DR. "Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response." J Cell Biol 109.4 Pt 1 (October 1989): 1795-1805.
PMID
2477381
Source
pubmed
Published In
The Journal of Cell Biology
Volume
109
Issue
4 Pt 1
Publish Date
1989
Start Page
1795
End Page
1805

Tenascin/hexabrachion in human skin: biochemical identification and localization by light and electron microscopy.

Tenascin/hexabrachion is a large glycoprotein of the extracellular matrix. Previous reports have demonstrated that tenascin is associated with epithelial-mesenchymal interfaces during embryogenesis and is prominent in the matrix of many tumors. However, the distribution of tenascin is more restricted in adult tissues. We have found tenascin to be present in normal human skin in a distribution distinct from other matrix proteins. Immunohistochemical studies showed staining of the papillary dermis immediately beneath the basal lamina. Examination of skin that had been split within the lamina lucida of the basement membrane suggested a localization of tenascin beneath the lamina lucida. In addition, there was finely localized staining within the walls of blood vessels and in the smooth muscle bundles of the arrectori pilorem. Very prominent staining was seen around the cuboidal cells that formed the basal layer of sweat gland ducts. The sweat glands themselves did not stain. The distribution of tenascin in the papillary dermis was studied at high resolution by immunoelectron microscopy. Staining was concentrated in small amorphous patches scattered amongst the collagen fibers beneath the basal lamina. These patches were not associated with cell structures, collagen, or elastic fibers. Tenascin could be partially extracted from the papillary dermis by urea, guanidine hydrochloride, or high pH solution. The extracted protein showed a 320-kD subunit similar to that purified from fibroblast or glioma cell cultures. We have developed a sensitive ELISA assay that can quantitate tenascin at concentrations as low as 5 ng/ml. Tests on extracts of the papillary dermis showed tenascin constituted about 0.02-0.05% of the protein extracted.

Authors
Lightner, VA; Gumkowski, F; Bigner, DD; Erickson, HP
MLA Citation
Lightner, VA, Gumkowski, F, Bigner, DD, and Erickson, HP. "Tenascin/hexabrachion in human skin: biochemical identification and localization by light and electron microscopy." J Cell Biol 108.6 (June 1989): 2483-2493.
PMID
2472409
Source
pubmed
Published In
The Journal of Cell Biology
Volume
108
Issue
6
Publish Date
1989
Start Page
2483
End Page
2493

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

Co-operativity in protein-protein association. The structure and stability of the actin filament.

Co-operative association, in which a protein subunit is held simultaneously by two bonds, is enormously more favorable than association forming either bond alone. A theoretical framework for calculating the effect of co-operativity is developed here, which should have a broad application to protein-protein and protein-DNA associations. The theory is applied in detail to actin. Fragmentation of an actin filament is extremely unfavorable: the association constant for annealing-fragmentation is estimated here to be at least 10(13) M-1. In contrast to these very strong bonds within the filament, subunits are loosely attached at the end, with an association constant of 2 x 10(5) M-1. The eight orders of magnitude difference between annealing-fragmentation and end association can be attributed to the co-operative formation of one additional protein-protein bond in the annealing reaction. This observation, and a quantitative analysis of the co-operativity, lead to an important conclusion: the longitudinal bond, which connects subunits in the long-pitch helix, must be substantially stronger than the diagonal bond, which connect subunits between these helices. This conclusion contradicts some recent models based on Fourier construction, in which the longitudinal bond is weak or absent. Prominent longitudinal bonds also require a rigidity of the actin filament that must be reconciled with previous reports of torsional flexibility. A hinge within the actin subunit is suggested, separating it into two flexibly attached domains. In one possible model the two domains are oriented radially: the inner domains are connected by longitudinal and diagonal bonds to form a relatively rigid helical backbone, and the outer domains are attached to this backbone by flexible hinges, permitting them to move through angles of 10 degrees to 20 degrees or more. Flexibility of the outer, myosin-binding domain should be functionally important, permitting attachment of myosin cross-bridges over a range of angles.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Co-operativity in protein-protein association. The structure and stability of the actin filament." J Mol Biol 206.3 (April 5, 1989): 465-474.
PMID
2716058
Source
pubmed
Published In
Journal of Molecular Biology
Volume
206
Issue
3
Publish Date
1989
Start Page
465
End Page
474

Synthesis of alpha 2-macroglobulin by bovine adrenal cortical cell cultures.

Primary cultures of bovine adrenal medullary cells synthesize and secrete a high-molecular-weight protein into the culture medium. The protein was purified from the serum-free medium of cultured cells and was identified as alpha 2-macroglobulin by gel electrophoresis, sedimentation velocity, electron microscopy, immunoprecipitation, immunodiffusion, and autoradiography. Antisera directed against the protein were prepared and used to determine the cell types that synthesize the protein. Immunohistofluorescence studies show that adrenal cortical cells present in the adrenal medullary cell cultures reacted with the antisera to the protein purified from the medium, but adrenal medullary chromaffin cells did not. Cell cultures prepared from bovine adrenal cortex also synthesize and secrete alpha 2-macroglobulin and react with the antisera.

Authors
Kirshner, N; Corcoran, JJ; Erickson, HP
MLA Citation
Kirshner, N, Corcoran, JJ, and Erickson, HP. "Synthesis of alpha 2-macroglobulin by bovine adrenal cortical cell cultures." Am J Physiol 256.4 Pt 1 (April 1989): C779-C785.
PMID
2468288
Source
pubmed
Published In
The American journal of physiology
Volume
256
Issue
4 Pt 1
Publish Date
1989
Start Page
C779
End Page
C785

Domain structure of phytochrome from Avena sativa visualized by electron microscopy.

Highly purified phytochrome from Avena sativa was visualized by electron microscopy after negative staining with uranyl acetate and after rotary shadowing with platinum. The particle shape was variable in both types of specimens, but tripartite structures resembling a 'Y' were consistently observed. The tripartite substructure is composed of three globular domains each having a diameter of 7 to 8 nm and equally spaced in an equilateral triangle. The dimensions of the tripartite particle measured 15 nm between the centers of any two of the three particles. When phytochrome was digested with trypsin in a manner which releases the amino-terminal globular domain from the polypeptide, the tripartite structure was lost and only small globular particles were seen. We propose that the outer particles of this tripartite structure are the amino-terminal domains of the phytochrome dimer, and the central particle comprises the carboxyl domains of the two subunits.

Authors
Jones, AM; Erickson, HP
MLA Citation
Jones, AM, and Erickson, HP. "Domain structure of phytochrome from Avena sativa visualized by electron microscopy." Photochem Photobiol 49.4 (April 1989): 479-483.
PMID
2727087
Source
pubmed
Published In
Photochemistry & Photobiology
Volume
49
Issue
4
Publish Date
1989
Start Page
479
End Page
483

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues.

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.

Authors
O'Brien, ET; Erickson, HP
MLA Citation
O'Brien, ET, and Erickson, HP. "Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues." Biochemistry 28.3 (February 7, 1989): 1413-1422.
PMID
2713372
Source
pubmed
Published In
Biochemistry
Volume
28
Issue
3
Publish Date
1989
Start Page
1413
End Page
1422

Topology of cell adhesion molecules.

The neural cell adhesion molecule (N-CAM) exists in two major forms [ld (large cytoplasmic domain) peptide and sd (small cytoplasmic domain) peptide] that contain transmembrane segments and different cytoplasmic domains and in a third form [ssd (small surface domain) peptide] that lacks transmembrane and cytoplasmic regions. All forms have the same extracellular region of more than 600 amino acid residues, a region also found in a fragment (Fr2) that can be released from cells by proteolysis. The liver cell adhesion molecule (L-CAM) is expressed as a single species that is distinct from N-CAM, but its extracellular region can also be obtained as a proteolytic fragment (Ft1). Examination of the various forms of N-CAM and the Ft1 fragment of L-CAM by electron microscopy of rotary shadowed molecules indicated that they all have rod-shaped structures that contain a hinge region which is apparently flexible. Both the ssd chain and the Fr2 fragment of N-CAM are single rods bent into arms approximately 18 and 10 nm long. The ld and sd chains are longer bent rods that form rosettes comprising two to six branches; detergent treatment disrupts these rosettes into single rods. Specific antibodies that block homophilic N-CAM binding labeled the distal ends of the branches of the ld/sd rosettes and the ends of the longer arm of both the ssd chain and the Fr2 fragment. Antibodies that bind to the sialic acid-rich region of N-CAM bound near the hinge. These data indicate that the N-CAM rosettes are formed by interaction between their transmembrane or cytoplasmic domains and not by interactions involving their homophilic binding sites. The L-CAM Ft1 fragment is also a bent rod with an apparently flexible hinge; like the ssd chain and the Fr2 fragment of N-CAM, it does not form aggregates. The similarities between L-CAM and N-CAM, despite their differences in amino acid sequence, suggest that their general configuration and the presence of a flexible hinge are important elements in assuring effective and specific cell-cell adhesion.

Authors
Becker, JW; Erickson, HP; Hoffman, S; Cunningham, BA; Edelman, GM
MLA Citation
Becker, JW, Erickson, HP, Hoffman, S, Cunningham, BA, and Edelman, GM. "Topology of cell adhesion molecules." Proc Natl Acad Sci U S A 86.3 (February 1989): 1088-1092.
PMID
2915974
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
3
Publish Date
1989
Start Page
1088
End Page
1092

Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex.

Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.

Authors
Anderson, K; Lai, FA; Liu, QY; Rousseau, E; Erickson, HP; Meissner, G
MLA Citation
Anderson, K, Lai, FA, Liu, QY, Rousseau, E, Erickson, HP, and Meissner, G. "Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex." J Biol Chem 264.2 (January 15, 1989): 1329-1335.
PMID
2463249
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
2
Publish Date
1989
Start Page
1329
End Page
1335

Electron microscopy and hydrodynamic properties of factor XIII subunits.

Factor XIII is a transglutaminase important in blood coagulation and fibrinolysis. Its function is to catalyze peptide bond formation between the gamma-carboxamide group of glutamines in one protein and the epsilon-amino group of lysine in another. There are two zymogenic forms of factor XIII: one is a noncovalent, intracellular dimer (A2); the other is a noncovalent, extracellular tetramer (A2B2). The catalytic function resides in the activated A chain (A2.). Purified forms of factor XIII (A2B2, A2, A2.B2, B) were prepared and analyzed by electron microscopy, gel filtration, and gradient centrifugation. Hydrodynamic constants were derived. Electron microscopy of rotary-shadowed molecules showed A2 to consist of two globular particles each about 6 x 9 nm in size. The A2 dimer is significantly elongated, 18 nm long and 6 nm in diameter. Sedimentation and gel filtration of the A2 dimer are consistent with this asymmetric structure. B protein is a filamentous, flexible strand with kinks, with a contour length of 30 nm and a diameter of approximately 2-3 nm. The sedimentation and gel filtration behavior of the B subunit are characteristic of a highly asymmetric molecule. The observed structure of the B subunit, combined with data for its amino acid sequence, suggests a modular structure. The B subunit is a member of a family of proteins composed of tandem, repeating structures (referred to as GP-I domains); the structure seen by electron microscopy for B subunit is probably applicable to all proteins in this family. Plasma and platelet factor XIII zymogens are tetrameric and dimeric, but B protein, in the absence of A protein, appears to be monomeric. Our model for the A2B2 zymogen has the elongated A2 dimer forming the core and the two B strands wrapping around the outside.

Authors
Carrell, NA; Erickson, HP; McDonagh, J
MLA Citation
Carrell, NA, Erickson, HP, and McDonagh, J. "Electron microscopy and hydrodynamic properties of factor XIII subunits." J Biol Chem 264.1 (January 5, 1989): 551-556.
PMID
2491853
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
1
Publish Date
1989
Start Page
551
End Page
556

Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors.

Authors
Erickson, HP; Bourdon, MA
MLA Citation
Erickson, HP, and Bourdon, MA. "Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors." Annu Rev Cell Biol 5 (1989): 71-92. (Review)
PMID
2480799
Source
pubmed
Published In
Annual Review of Cell Biology
Volume
5
Publish Date
1989
Start Page
71
End Page
92
DOI
10.1146/annurev.cb.05.110189.000443

Synthesis of α2-macroglobulin by bovine adrenal cortical cell cultures

Primary cultures of bovine adrenal medullary cells synthesize and secrete a high-molecular-weight protein into the culture medium. The protein was purified from the serum-free medium of cultured cells and was identified as α2-macroglobulin by gel electrophoresis, sedimentation velocity, electron microscopy, immunoprecipitation, immunodiffusion, and autoradiography. Antisera directed against the protein were prepared and used to determine the cell types that synthesize the protein. Immunohistofluorescence studies show that adrenal cortical cells present in the adrenal medullary cell cultures reacted with the antisera to the protein purified from the medium, but adrenal medullary chromaffin cells did not. Cell cultures prepared from bovine adrenal cortex also synthesize and secrete α2-macroglobulin and react with the antisera.

Authors
Kirshner, N; Corcoran, JJ; Erickson, HP
MLA Citation
Kirshner, N, Corcoran, JJ, and Erickson, HP. "Synthesis of α2-macroglobulin by bovine adrenal cortical cell cultures." American Journal of Physiology - Cell Physiology 256.4 (1989): 25/4-.
Source
scival
Published In
American Journal of Physiology - Cell Physiology
Volume
256
Issue
4
Publish Date
1989
Start Page
25/4

Cell adhesion to fibronectin and tenascin: Quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4°C were on the order of 10-5 dynes/cell and did not involve the cytoskeleton. Adhesions to fibronectin after 15 min at 37°C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37°C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37°C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37°C, glioma cells increased their surface area within close contact to the substrate by ~125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin B suggest that the adhesive 'strengthening' in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.

Authors
Lotz, MM; Burdsal, CA; Erickson, HP; McClay, DR
MLA Citation
Lotz, MM, Burdsal, CA, Erickson, HP, and McClay, DR. "Cell adhesion to fibronectin and tenascin: Quantitative measurements of initial binding and subsequent strengthening response." Journal of Cell Biology 109.4 I (1989): 1795-1805.
Source
scival
Published In
Journal of Cell Biology
Volume
109
Issue
4 I
Publish Date
1989
Start Page
1795
End Page
1805

Echinonectin: a new embryonic substrate adhesion protein.

An extracellular matrix molecule has been purified from sea urchin (Lytechinus variegatus) embryos. Based on its functional properties and on its origin, this glycoprotein has been given the name "echinonectin." Echinonectin is a 230-kD dimer with a unique bow tie shape when viewed by electron microscopy. The molecule is 12 nm long, 8 nm wide at the ends, and narrows to approximately 4 nm at the middle. It is composed of two 116-kD U-shaped subunits that are attached to each other by disulfide bonds at their respective apices. Polyclonal antibodies were used to localize echinonectin in paraffin-embedded, sectioned specimens by indirect immunofluorescence. The protein is stored in vesicles or granules in unfertilized eggs, is released after fertilization, and later becomes localized on the apical surface of ectoderm cells in the embryo. When used as a substrate in a quantitative in vitro assay, echinonectin is highly effective as an adhesive substrate for dissociated embryonic cells. Because of the quantity, pattern of appearance, distribution, and adhesive characteristics of this protein, we suggest that echinonectin serves as a substrate adhesion molecule during sea urchin development.

Authors
Alliegro, MC; Ettensohn, CA; Burdsal, CA; Erickson, HP; McClay, DR
MLA Citation
Alliegro, MC, Ettensohn, CA, Burdsal, CA, Erickson, HP, and McClay, DR. "Echinonectin: a new embryonic substrate adhesion protein." J Cell Biol 107.6 Pt 1 (December 1988): 2319-2327.
PMID
3198690
Source
pubmed
Published In
The Journal of Cell Biology
Volume
107
Issue
6 Pt 1
Publish Date
1988
Start Page
2319
End Page
2327

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.

Authors
Walker, RA; O'Brien, ET; Pryer, NK; Soboeiro, MF; Voter, WA; Erickson, HP; Salmon, ED
MLA Citation
Walker, RA, O'Brien, ET, Pryer, NK, Soboeiro, MF, Voter, WA, Erickson, HP, and Salmon, ED. "Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies." J Cell Biol 107.4 (October 1988): 1437-1448.
PMID
3170635
Source
pubmed
Published In
The Journal of Cell Biology
Volume
107
Issue
4
Publish Date
1988
Start Page
1437
End Page
1448

Detection of DNA looping due to simultaneous interaction of a DNA-binding protein with two spatially separated binding sites on DNA.

We describe different and relatively rapid biochemical techniques to detect protein-mediated DNA looping. These techniques, based on enhancement of DNA knotting and that of ligase-catalyzed cyclization, were used to show that the replication initiator protein of plasmid R6K can bring together two intramolecular gamma origin of replication sequences located as far apart as 2 kilobases. The site-site interaction causes looping out of the intervening DNA sequence as visualized by electron microscopy. Because the autoregulatory sequence of the initiator cistron also binds initiator protein, we investigated whether the gamma origin-bound protein can participate in autoregulation by interaction of the two sites through a protein bridge. We discovered that the two sites do not interact in vitro at their natural locations when on opposite faces of the double helix. Moving the two sites to the same face of the double helix by introducing a half turn into the intervening sequence allows protein-mediated site-site interaction to occur.

Authors
Mukherjee, S; Erickson, H; Bastia, D
MLA Citation
Mukherjee, S, Erickson, H, and Bastia, D. "Detection of DNA looping due to simultaneous interaction of a DNA-binding protein with two spatially separated binding sites on DNA." Proc Natl Acad Sci U S A 85.17 (September 1988): 6287-6291.
PMID
3413096
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
85
Issue
17
Publish Date
1988
Start Page
6287
End Page
6291

Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein.

Initiation of DNA replication from ori beta of plasmid R6K requires the presence of the ori gamma sequence in cis. We demonstrate that binding of initiator protein to the seven strong, tandem binding sites in gamma increases binding of the protein at the very weak binding site present in ori beta by cooperativity at a distance. The gamma-beta interaction via the initiator results in a DNA loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease III protection, electron microscopy, and chemical footprinting. The protein-mediated gamma-beta interaction in vitro suggests that the cooperative interaction of gamma-bound protein with the beta sequence by DNA looping is an early step in the initiation of DNA replication at the beta origin of R6K.

Authors
Mukherjee, S; Erickson, H; Bastia, D
MLA Citation
Mukherjee, S, Erickson, H, and Bastia, D. "Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein." Cell 52.3 (February 12, 1988): 375-383.
PMID
3345564
Source
pubmed
Published In
Cell
Volume
52
Issue
3
Publish Date
1988
Start Page
375
End Page
383

Purification and reconstitution of the calcium release channel from skeletal muscle.

The calcium release channel from rabbit muscle sarcoplasmic reticulum (SR) has been purified and reconstituted as a functional unit in lipid bilayers. Electron microscopy reveals the four-leaf clover structure previously described for the 'feet' that span the transverse tubule (T)-SR junction. Ca2+ release from the SR induced by T-system depolarization during excitation-contraction coupling in muscle may thus be effected through a direct association of the T-system with SR Ca2+-release channels.

Authors
Lai, FA; Erickson, HP; Rousseau, E; Liu, QY; Meissner, G
MLA Citation
Lai, FA, Erickson, HP, Rousseau, E, Liu, QY, and Meissner, G. "Purification and reconstitution of the calcium release channel from skeletal muscle." Nature 331.6154 (January 28, 1988): 315-319.
PMID
2448641
Source
pubmed
Published In
Nature
Volume
331
Issue
6154
Publish Date
1988
Start Page
315
End Page
319
DOI
10.1038/331315a0

Echinonectin: A new embryonic substrate adhesion protein

An extracellular matrix molecule has been purified from sea urchin (Lytechinus variegatus) embryos. Based on its functional properties and on its origin, this glycoprotein has been given the name 'echinonectin'. Echinonectin is a 230-kD dimer with a unique bow tie shape when viewed by electron microscopy. The molecule is 12 nm long, 8 nm wide at the ends, and narrows to ~4 nm at the middle. It is composed of two 116-kD U-shaped subunits that are attached to each other by disulfide bonds at their respective apices. Polyclonal antibodies were used to localize echinonectin in paraffin-embedded, sectioned specimens by indirect immunofluorescence. The protein is stored in vesicles or granules in unfertilized eggs, is released after fertilization, and later becomes localized on the apical surface of ectoderm cells in the embryo. When used as a substrate in a quantitative in vitro assay, echinonectin is highly effective as an adhesive substrate for dissociated embryonic cells. Because of the quantity, pattern of appearance, distribution, and adhesive characteristics of this protein, we suggest that echinonectin serves as a substrate adhesion molecule during sea urchin development.

Authors
Alliegro, MC; Ettensohn, CA; Burdsal, CA; Erickson, HP; McClay, DR
MLA Citation
Alliegro, MC, Ettensohn, CA, Burdsal, CA, Erickson, HP, and McClay, DR. "Echinonectin: A new embryonic substrate adhesion protein." Journal of Cell Biology 107.6 I (1988): 2319-2327.
Source
scival
Published In
Journal of Cell Biology
Volume
107
Issue
6 I
Publish Date
1988
Start Page
2319
End Page
2327

Hexabrachion Protein (Tenascin, Cytotactin, Brachionectin) in Connective Tissues, Embryonic Brain, and Tumors

Authors
Erickson, HP; Lightner, VA
MLA Citation
Erickson, HP, and Lightner, VA. "Hexabrachion Protein (Tenascin, Cytotactin, Brachionectin) in Connective Tissues, Embryonic Brain, and Tumors." Advances in Molecular and Cell Biology 2.C (1988): 55-90.
Source
scival
Published In
Advances in Molecular and Cell Biology
Volume
2
Issue
C
Publish Date
1988
Start Page
55
End Page
90
DOI
10.1016/S1569-2558(08)60430-0

Hexabrachion proteins in embryonic chicken tissues and human tumors.

Cell cultures of chicken embryo and human fibroblasts produce a large extracellular matrix molecule with a six-armed structure that we called a hexabrachion (Erickson, H. P., and J. L. Iglesias, 1984, Nature (Lond.), 311:267-269. In the present work we have determined that the myotendinous (M1) antigen described by M. Chiquet and D. M. Fambrough in chicken tissues (1984, J. Cell Biol., 98:1926-1936), and the glioma mesenchymal extracellular matrix protein described by Bourdon et al. in human tumors (Bourdon, M. A., C. J. Wikstrand, H. Furthmayr, T. J. Matthews, and D. D. Bigner, 1983, Cancer Res. 43:2796-2805) have the structure of hexabrachions. We also demonstrate that the M1 antigen is present in embryonic brain, where it was previously reported absent, and have purified hexabrachions from brain homogenates. The recently described cytotactin (Grumet, M., S. Hoffman, K. L. Crossin, and G. M. Edelman, 1985, Proc. Natl. Acad. Sci. USA, 82:8075-8079) now appears to be identical to the chicken hexabrachion protein. In a search for functional roles, we looked for a possible cell attachment activity. A strong, fibronectin-like attachment activity was present in (NH4)2SO4 precipitates of cell supernatant and sedimented with hexabrachions in glycerol gradients. Hexabrachions purified by antibody adsorption, however, had lost this activity, suggesting that it was due to a separate factor associated with hexabrachions in the gradient fractions. The combined information in the several, previously unrelated studies suggests that hexabrachions may play a role in organizing localized regions of extracellular matrix. The protein is prominently expressed at specific times and locations during embryonic development, is retained in certain adult tissues, and is reexpressed in a variety of tumors.

Authors
Erickson, HP; Taylor, HC
MLA Citation
Erickson, HP, and Taylor, HC. "Hexabrachion proteins in embryonic chicken tissues and human tumors." J Cell Biol 105.3 (September 1987): 1387-1394.
PMID
3654758
Source
pubmed
Published In
The Journal of Cell Biology
Volume
105
Issue
3
Publish Date
1987
Start Page
1387
End Page
1394

GTP hydrolysis during microtubule assembly.

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.

Authors
O'Brien, ET; Voter, WA; Erickson, HP
MLA Citation
O'Brien, ET, Voter, WA, and Erickson, HP. "GTP hydrolysis during microtubule assembly." Biochemistry 26.13 (June 30, 1987): 4148-4156.
PMID
3651443
Source
pubmed
Published In
Biochemistry
Volume
26
Issue
13
Publish Date
1987
Start Page
4148
End Page
4156

Evidence for a junctional feet-ryanodine receptor complex from sarcoplasmic reticulum

Heavy sarcoplasmic reticulum vesicles, labelled with the Ca2+ release channel probe [3H]ryanodine, were solubilized in detergent, then centrifuged through sucrose gradients. A single peak of ryanodine binding activity was observed with an apparent sedimentation coefficient of 30S. Electron microscopy of the peak fraction showed disk structures of 25-28 nm diameter and 10 nm thickness. Proteins specifically enriched in the peak fraction were the Mr 160,000 and 260,000 and junctional feet proteins (Mr 320,000 and 300,000). This suggests that the feet proteins and ryanodine receptor may be specifically associated into a large oligomeric complex comprising subunits of Mr 160,000 - 320,000. © 1987.

Authors
Lai, FA; Erickson, H; Block, BA; Meissner, G
MLA Citation
Lai, FA, Erickson, H, Block, BA, and Meissner, G. "Evidence for a junctional feet-ryanodine receptor complex from sarcoplasmic reticulum." Biochemical and Biophysical Research Communications 143.2 (1987): 704-709.
PMID
2436616
Source
scival
Published In
Biochemical and Biophysical Research Communications
Volume
143
Issue
2
Publish Date
1987
Start Page
704
End Page
709

Lateral packing of protofibrils in fibrin fibers and fibrinogen polymers.

Authors
Voter, WA; Lucaveche, C; Blaurock, AE; Erickson, HP
MLA Citation
Voter, WA, Lucaveche, C, Blaurock, AE, and Erickson, HP. "Lateral packing of protofibrils in fibrin fibers and fibrinogen polymers." Biopolymers 25.12 (December 1986): 2359-2373.
PMID
3801588
Source
pubmed
Published In
Biopolymers
Volume
25
Issue
12
Publish Date
1986
Start Page
2359
End Page
2373
DOI
10.1002/bip.360251213

Concentration of protein in fibrin fibers and fibrinogen polymers determined by refractive index matching.

Authors
Voter, WA; Lucaveche, C; Erickson, HP
MLA Citation
Voter, WA, Lucaveche, C, and Erickson, HP. "Concentration of protein in fibrin fibers and fibrinogen polymers determined by refractive index matching." Biopolymers 25.12 (December 1986): 2375-2384.
PMID
3801589
Source
pubmed
Published In
Biopolymers
Volume
25
Issue
12
Publish Date
1986
Start Page
2375
End Page
2384
DOI
10.1002/bip.360251214

Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

Authors
Fretto, LJ; Fowler, WE; McCaslin, DR; Erickson, HP; McKee, PA
MLA Citation
Fretto, LJ, Fowler, WE, McCaslin, DR, Erickson, HP, and McKee, PA. "Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains." J Biol Chem 261.33 (November 25, 1986): 15679-15689.
PMID
3536910
Source
pubmed
Published In
The Journal of biological chemistry
Volume
261
Issue
33
Publish Date
1986
Start Page
15679
End Page
15689

Nucleosome structure.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Nucleosome structure." Science 233.4771 (September 26, 1986): 1429-1431. (Letter)
PMID
3749888
Source
pubmed
Published In
Science
Volume
233
Issue
4771
Publish Date
1986
Start Page
1429
End Page
1431

Nucleation of microtubule assembly. Experimental kinetics, computer fitting of models, and observations on tubulin rings.

Authors
Erickson, HP; Voter, WA
MLA Citation
Erickson, HP, and Voter, WA. "Nucleation of microtubule assembly. Experimental kinetics, computer fitting of models, and observations on tubulin rings." Ann N Y Acad Sci 466 (1986): 552-565.
PMID
3460431
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
466
Publish Date
1986
Start Page
552
End Page
565

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.

Authors
Fowler, WE; Fretto, LJ; Hamilton, KK; Erickson, HP; McKee, PA
MLA Citation
Fowler, WE, Fretto, LJ, Hamilton, KK, Erickson, HP, and McKee, PA. "Substructure of human von Willebrand factor." J Clin Invest 76.4 (October 1985): 1491-1500.
PMID
2932468
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
76
Issue
4
Publish Date
1985
Start Page
1491
End Page
1500
DOI
10.1172/JCI112129

A structural comparison of tryptic fragments of three types of intermediate filaments.

We have compared tryptic fragments of three types of intermediate filaments, emphasizing structural characteristics as seen in the electron microscope. Variable, long alpha-helical rod fragments were found to be similar for keratin, neurofilaments and desmin filaments. Short rod fragments from keratin and neurofilaments appeared similar when observed by electron microscopy. Short rod fragments were not seen in desmin filament digests. In addition to these elongated particles, globular fragments, which have not been described previously, were obtained from all three types of intermediate filaments. These globular fragments were characterized by gel filtration and electron microscopy, and compared to globular proteins of known size using both methods. The diameter was about 6 nm and the molecular weight was estimated to be 50 000-60 000. These globular particles may comprise the short, nonhelical regions from several IF protein subunits, which are clustered into an interface in the intact filament or protofilaments.

Authors
Milam, LM; Erickson, HP
MLA Citation
Milam, LM, and Erickson, HP. "A structural comparison of tryptic fragments of three types of intermediate filaments." J Ultrastruct Res 90.3 (March 1985): 251-260.
PMID
2416948
Source
pubmed
Published In
Journal of Structural Biology
Volume
90
Issue
3
Publish Date
1985
Start Page
251
End Page
260

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy.

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.

Authors
Carrell, NA; Fitzgerald, LA; Steiner, B; Erickson, HP; Phillips, DR
MLA Citation
Carrell, NA, Fitzgerald, LA, Steiner, B, Erickson, HP, and Phillips, DR. "Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy." J Biol Chem 260.3 (February 10, 1985): 1743-1749.
PMID
3155738
Source
pubmed
Published In
The Journal of biological chemistry
Volume
260
Issue
3
Publish Date
1985
Start Page
1743
End Page
1749

Structural characteristics of the desmin protofilament.

Biochemical investigations of intermediate filaments in soluble or partially assembled forms are often difficult to perform due to the unusual insolubility of most types of intermediate filaments. However, desmin is soluble in 10 mM Tris. The structure of partially soluble native desmin was studied by gel-filtration chromatography and electron microscopy. The lowest molecular weight species of soluble desmin is a flexible rod averaging 53 nm in length. Calculations of f/fmin values from a previously published sedimentation value allowed comparisons with other elongated proteins. These values and the dimensions obtained from electron microscopy suggest that the desmin protofilament contains three or four protein subunits.

Authors
Milam, L; Erickson, HP
MLA Citation
Milam, L, and Erickson, HP. "Structural characteristics of the desmin protofilament." J Ultrastruct Res 89.2 (November 1984): 179-186.
PMID
6544885
Source
pubmed
Published In
Journal of Structural Biology
Volume
89
Issue
2
Publish Date
1984
Start Page
179
End Page
186

A six-armed oligomer isolated from cell surface fibronectin preparations.

Fibronectins are adhesive glycoproteins thought to mediate the attachment of cells to various substrates. Plasma fibronectin (PFN) is a dimer comprising subunits of molecular weight 220,000, connected by one or two disulphide bonds. Electron microscopy shows that PFN is a long, flexible strand, 2-3 nm in diameter and 140 nm long. Many cells in tissue culture elaborate an extracellular matrix of insoluble (highly cross-linked by disulphide bonds) fibronectin, and a variable amount of 'cell surface fibronectin' (CSFN) that can be extracted by mild urea treatment. This CSFN, soluble in 1 M urea and at high pH, is a mixture of dimers and disulphide-bonded oligomers. In the present study we have examined the structure of these molecules by electron microscopy. Oligomers were separated from dimers and contaminating proteins by zone sedimentation through glycerol gradients. We report that the CSFN dimers are identical in structure to PFN. In contrast, the oligomers have an elaborate and well defined structure that we call a 'hexabrachion': six arms emanating from a central globular particle. The arms are similar to PFN in being long, thin and flexible, but have several distinctly different features.

Authors
Erickson, HP; Inglesias, JL
MLA Citation
Erickson, HP, and Inglesias, JL. "A six-armed oligomer isolated from cell surface fibronectin preparations." Nature 311.5983 (September 20, 1984): 267-269.
PMID
6482952
Source
pubmed
Published In
Nature
Volume
311
Issue
5983
Publish Date
1984
Start Page
267
End Page
269

The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism.

A model describing the nucleation and assembly of purified tubulin has been developed. The novel feature of this model is a two stage nucleation process to allow the explicit inclusion of the two-dimensional nature of the early stages of microtubule assembly. In actin assembly the small starting nucleus has only one site for subunit addition as the two-stranded helix is formed. In contrast, microtubule assembly begins with the formation of a small two-dimensional section of microtubule wall. The model we propose is a modification of the work of Wegner and Engel (Wegner, A., and Engel, J. (1975) Biophys. Chem. 3, 215-225) wherein we add a second stage of nucleation to directly account for lateral growth, i.e. the addition of a small number of subunits to the side of an existing sheet structure. Subsequent elongation of the sheets is treated in the usual way. The experimental system used to test this model was the Mg2+/glycerol induced assembly of purified tubulin. The computer simulation of the polymerization time courses gave a fairly good fit to experimental kinetics for our model, where the primary nucleus comprises two protofilaments, of four and three subunits, and lateral growth requires a three-subunit nucleus to initiate a new protofilament.

Authors
Voter, WA; Erickson, HP
MLA Citation
Voter, WA, and Erickson, HP. "The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism." J Biol Chem 259.16 (August 25, 1984): 10430-10438.
PMID
6469971
Source
pubmed
Published In
The Journal of biological chemistry
Volume
259
Issue
16
Publish Date
1984
Start Page
10430
End Page
10438

Fibronectin in extended and compact conformations. Electron microscopy and sedimentation analysis.

We have studied the ionic strength-dependent change in conformation of fibronectin, half-molecules of fibronectin produced by reduction and carboxyamidomethylation, and proteolytic fragments. In zone sedimentation through glycerol gradients, intact fibronectin sedimented at 13.5 and 10 S in 0.02 and 0.2 M NaCl, respectively, in agreement with previous studies. Half-molecules sedimented at 11.5 and 7.5 S in the two salt concentrations, demonstrating that the change in conformation occurs independently within each half-molecule. Gelatin-binding plasmic fragments of 165-215 kDa showed a similar large shift in sedimentation coefficient, and one of 60 kDa showed a small shift. We conclude that the change in conformation is effected by short range electrostatic interactions along the strand, rather than by attraction of distant segments of the molecule. Electron microscopy showed that both intact fibronectin and half-molecules exist as extended strands at high ionic strength. At low ionic strength the strands are more strongly curved or bent to produce an irregularly coiled compact structure. No regular points of folding or crossover were seen, suggesting that the compact conformation is produced by increased bending over most or all of the strand.

Authors
Erickson, HP; Carrell, NA
MLA Citation
Erickson, HP, and Carrell, NA. "Fibronectin in extended and compact conformations. Electron microscopy and sedimentation analysis." J Biol Chem 258.23 (December 10, 1983): 14539-14544.
PMID
6643500
Source
pubmed
Published In
The Journal of biological chemistry
Volume
258
Issue
23
Publish Date
1983
Start Page
14539
End Page
14544

Electron microscopy of fibrinogen, its plasmic fragments and small polymers.

Authors
Erickson, HP; Fowler, WE
MLA Citation
Erickson, HP, and Fowler, WE. "Electron microscopy of fibrinogen, its plasmic fragments and small polymers." Ann N Y Acad Sci 408 (June 27, 1983): 146-163.
PMID
6575682
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
408
Publish Date
1983
Start Page
146
End Page
163

Assembly of proteolytically cleaved tubulin.

Conditions have been found for limited proteolysis of purified tubulin, in which 70-90% of the molecules are cleaved at one or two sites. Thermolysin and chymotrypsin cleave the alpha and beta subunits, respectively, at single sites. Trypsin cleaves the alpha subunit at two sites. The chymotrypsin site and one of the trypsin sites are apparently inaccessible on assembled microtubules. The different samples of proteolyzed tubulin were all fully competent to assemble in a buffer containing 1 M sodium glutamate. In another buffer (50 mM morpholinoethanesulfonic acid, 3.4 M glycerol) tubulin digested by thermolysin assembled as well as native tubulin, but samples digested by chymotrypsin or trypsin would not assemble even at high protein concentrations.

Authors
Brown, HR; Erickson, HP
MLA Citation
Brown, HR, and Erickson, HP. "Assembly of proteolytically cleaved tubulin." Arch Biochem Biophys 220.1 (January 1983): 46-51.
PMID
6338834
Source
pubmed
Published In
Archives of Biochemistry and Biophysics
Volume
220
Issue
1
Publish Date
1983
Start Page
46
End Page
51

Electron microscopy of human factor VIII/Von Willebrand glycoprotein: effect of reducing reagents on structure and function.

The structure of native and progressively reduced human factor VIII/von Willebrand factor (FVIII/vWF) was examined by electron microscopy and SDS gel electrophoresis and then correlated with its biological activities. Highly resolved electron micrographs of well-spaced, rotary-shadowed FVIII/vWF molecules showed their structure to consist of a very flexible filament that contains irregularly spaced small nodules. Filaments ranged from 50 to 1,150 nm with a mean length of 478 nm and lacked fixed, large globular domains as seen in fibrinogen and IgM. A population of multimeric FVIII/vWF species ranging in molecular weight from 1 to 5 million daltons and differing in size alternately by one and two subunits was observed on SDS-2% polyacrylamide-0.5% agarose gel electrophoresis. With progressive reduction of disulfide bonds by dithiothreitol (DTT), the electron microscopic size of FVIII/vWF decreased in parallel with increased electrophoretic mobility on SDS-agarose gels; between 0.1 and 0.5 mM DTT its structure changed from predominantly fibrillar species to large nodular forms. A 50% loss of vWF specific activity and FVIII procoagulant activity occurred at 0.4 mM DTT and 1 mM DTT, respectively, corresponding to the reduction of 4 and 12 disulfide bonds of the 62 disulfides per 200,000-dalton subunit. We conclude that reduction of a few critical disulfide bonds results in a major structural change by electron microscopy and a concomitant loss of approximately 50% of the vWF function.

Authors
Ohmori, K; Fretto, LJ; Harrison, RL; Switzer, ME; Erickson, HP; McKee, PA
MLA Citation
Ohmori, K, Fretto, LJ, Harrison, RL, Switzer, ME, Erickson, HP, and McKee, PA. "Electron microscopy of human factor VIII/Von Willebrand glycoprotein: effect of reducing reagents on structure and function." J Cell Biol 95.2 Pt 1 (November 1982): 632-640.
PMID
6815213
Source
pubmed
Published In
The Journal of Cell Biology
Volume
95
Issue
2 Pt 1
Publish Date
1982
Start Page
632
End Page
640

Electron microscopy of MAP 2 (microtubule-associated protein 2).

Authors
Voter, WA; Erickson, HP
MLA Citation
Voter, WA, and Erickson, HP. "Electron microscopy of MAP 2 (microtubule-associated protein 2)." J Ultrastruct Res 80.3 (September 1982): 374-382.
PMID
7131650
Source
pubmed
Published In
Journal of Structural Biology
Volume
80
Issue
3
Publish Date
1982
Start Page
374
End Page
382

Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments.

Unidirectional and rotary shadowing techniques have been applied in studying the surface structure of two types of intermediate filaments. Keratin filaments and neurofilaments demonstrate a approximately 21-nm axial periodicity which probably indicates the helical pitch of the outer shell of the filament. Analysis of unidirectionally shadowed keratin showed that the helix is left-handed. The observation of a left-handed helix of 21-nm pitch supports the three-stranded protofilament model of Fraser, Macrae, and Suzuki (1976, J. Mol. Biol. 108:435-452), and indicates that keratin filaments probably consist of 10 three-stranded protofilaments surrounding a core of three such protofilaments, as predicted by models based on x-ray diffraction of hard keratin filaments. Neurofilaments do not demonstrate an easily identifiable hand, so their consistency with the model is, as yet, uncertain.

Authors
Milam, L; Erickson, HP
MLA Citation
Milam, L, and Erickson, HP. "Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments." J Cell Biol 94.3 (September 1982): 592-596.
PMID
6182147
Source
pubmed
Published In
The Journal of Cell Biology
Volume
94
Issue
3
Publish Date
1982
Start Page
592
End Page
596

ELECTRON-MICROSCOPY OF HUMAN FACTOR-VIII VON WILLEBRAND GLYCOPROTEIN - EFFECT OF REDUCING REAGENTS ON STRUCTURE AND FUNCTION

Authors
OHMORI, K; FRETTO, LJ; HARRISON, RL; SWITZER, MEP; ERICKSON, HP; MCKEE, PA
MLA Citation
OHMORI, K, FRETTO, LJ, HARRISON, RL, SWITZER, MEP, ERICKSON, HP, and MCKEE, PA. "ELECTRON-MICROSCOPY OF HUMAN FACTOR-VIII VON WILLEBRAND GLYCOPROTEIN - EFFECT OF REDUCING REAGENTS ON STRUCTURE AND FUNCTION." JOURNAL OF CELL BIOLOGY 95.2 (1982): 632-640.
Source
wos-lite
Published In
The Journal of Cell Biology
Volume
95
Issue
2
Publish Date
1982
Start Page
632
End Page
640
DOI
10.1083/jcb.95.2.632

Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand.

We have determined the structure of plasma fibronectin by electron microscopy of shadowed specimens. the 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand. The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. the large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends. This model is supported by images of fibronectin-fibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand.

Authors
Erickson, HP; Carrell, N; McDonagh, J
MLA Citation
Erickson, HP, Carrell, N, and McDonagh, J. "Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand." J Cell Biol 91.3 Pt 1 (December 1981): 673-678.
PMID
7328116
Source
pubmed
Published In
The Journal of Cell Biology
Volume
91
Issue
3 Pt 1
Publish Date
1981
Start Page
673
End Page
678

Structural properties of an active form of rabbit muscle phosphofructokinase.

The quaternary structure of an active form of rabbit muscle phosphofructokinase was studied by sedimentation and electron microscopy. Active enzyme centrifugation studies at pH 7.0 and 23 +/- 1 degrees C showed that phosphofructokinase sediments as a single component with a sedimentation coefficient of 12.2 +/- 0.5 S. Identical results were obtained in two assay and three solvent systems. Boundary sedimentation studies of phosphofructokinase in the presence of 1.0 mM fructose 6-phosphate, 0.1 mM adenylyl imidodiphosphate at pH 7.0 and 23 +/- 1 degrees C were performed. The results showed that the sedimentation coefficient of phosphofructokinase remains constant within the range of protein concentration studied and assumes a value of 12.4 S. The molecular weights of the subunit and the 12.4 S component were measured by sedimentation equilibrium yielding values of 83,000 and 330,000 for the monomeric and polymeric species, respectively. It is, therefore, concluded that the active form of phosphofructokinase is indeed the tetrameric species. The structure of the phosphofructokinase tetramer was also studied by electron microscopy of negatively stained specimens. Particles identified as tetramers measured approximately 9 nm in diameter by 14 nm in length. The observed size and shape are consistent with the hydrodynamic measurements. Structural features within the tetramer were interpreted as due to the four individual subunits, each one approximately 4 X 6 X 6 nm in size, arranged with D2 symmetry.

Authors
Hesterberg, LK; Lee, JC; Erickson, HP
MLA Citation
Hesterberg, LK, Lee, JC, and Erickson, HP. "Structural properties of an active form of rabbit muscle phosphofructokinase." J Biol Chem 256.18 (September 25, 1981): 9724-9730.
PMID
6457038
Source
pubmed
Published In
The Journal of biological chemistry
Volume
256
Issue
18
Publish Date
1981
Start Page
9724
End Page
9730

Structure of the fibrin protofibril.

We identified the two-stranded fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and on considerations of symmetry, we constructed a model of the protofibril in which the two strands of trinodular fibrin molecules are related by a two-fold screw axis between the strands and two-fold axes perpendicular to them. The two strands are held together by staggered lateral contacts between the central nodules of one strand and outer nodules of the other. The molecules within a strand are joined by longitudinal contacts between outer nodules. This interpretation of the structure of protofibrils is supported by images of trimer complexes whose preparation and structure are described here, in which the central nodule of a fibrin monomer is attached to the crosslinked outer nodules of two other molecules. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules of adjacent, in-register strands. In total, we identify three intermolecular contacts involved in the polymerization of fibrin.

Authors
Fowler, WE; Hantgan, RR; Hermans, J; Erickson, HP
MLA Citation
Fowler, WE, Hantgan, RR, Hermans, J, and Erickson, HP. "Structure of the fibrin protofibril." Proc Natl Acad Sci U S A 78.8 (August 1981): 4872-4876.
PMID
6946434
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
78
Issue
8
Publish Date
1981
Start Page
4872
End Page
4876

The role of subunit entropy in cooperative assembly. Nucleation of microtubules and other two-dimensional polymers.

The self-assembly and nucleation of two-dimensional polymers is described by a theory based on a model of rigid subunits and bonds and simple principles of thermodynamics. The key point in the theory is to separate as an explicit parameter the free energy, primarily attributed to the entropy of the free subunit, that is required to immobilize a subunit in the polymer. Quantitative relations for the association of a subunit forming a longitudinal bond, a lateral bone, or both together are obtained, which demonstrate the basis and magnitude of cooperativity. The same formalism leads to a quantitative estimate for th concentration of the small polymers that are important intermediates in nucleation. It is shown that, if the concentration of free subunits is below a certain "critical supersaturation," the concentration of some essential intermediates is too low to support any significant assembly and nucleation is blocked. If the subunit concentration is above the critical supersaturation, all of the small intermediates are sufficiently stable to form and grow spontaneously. The theory predicts a critical supersaturation of 3.5 to 7 (the ratio of subunit concentration to the equilibrium solubility) for parameters appropriate to assembly of the microtubule wall. Experimentally, nucleation and assembly of microtubules is obtained at somewhat lower concentrations, 1.5 to 3 times the equilibrium solubility. Special mechanisms that could stabilize small polymers and facilitate nucleation of microtubule assembly are suggested.

Authors
Erickson, HP; Pantaloni, D
MLA Citation
Erickson, HP, and Pantaloni, D. "The role of subunit entropy in cooperative assembly. Nucleation of microtubules and other two-dimensional polymers." Biophys J 34.2 (May 1981): 293-309.
PMID
7236853
Source
pubmed
Published In
Biophysical Journal
Volume
34
Issue
2
Publish Date
1981
Start Page
293
End Page
309
DOI
10.1016/S0006-3495(81)84850-3

Cross-linked fibrinogen dimers demonstrate a feature of the molecular packing in fibrin fibers.

Authors
Fowler, WE; Erickson, HP; Hantgan, RR; McDonagh, J; Hermans, J
MLA Citation
Fowler, WE, Erickson, HP, Hantgan, RR, McDonagh, J, and Hermans, J. "Cross-linked fibrinogen dimers demonstrate a feature of the molecular packing in fibrin fibers." Science 211.4479 (January 16, 1981): 287-289.
PMID
6108612
Source
pubmed
Published In
Science
Volume
211
Issue
4479
Publish Date
1981
Start Page
287
End Page
289

Structure of the fibrin protofibril

We identified the two-stranded fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and on considerations of symmetry, we constructed a model of the protofibril in which the two strands of trinodular fibrin molecules are related by a two-fold screw axis between the strands and two-fold axes perpendicular to them. The two strands are held together by staggered lateral contacts between the central nodules of one strand and outer nodules of the other. The molecules within a strand are joined by longitudinal contacts between outer nodules. This interpretation of the structure of protofibrils is supported by images of trimer complexes whose preparation and structure are described here, in which the central nodule of a fibrin monomer is attached to the crosslinked outer nodules of two other molecules. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules of adjacent, in-register strands. In total, we identify three intermolecular contacts involved in the polymerization of fibrin.

Authors
Fowler, WE; Hantgan, RR; Hermans, J; Erickson, HP
MLA Citation
Fowler, WE, Hantgan, RR, Hermans, J, and Erickson, HP. "Structure of the fibrin protofibril." Proceedings of the National Academy of Sciences of the United States of America 78.8 I (1981): 4872-4876.
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
78
Issue
8 I
Publish Date
1981
Start Page
4872
End Page
4876

Electron microsocpy of plasmic fragments of human fibrinogen as related to trinodular structure of the intact molecule.

We have examined rotary shadowed, purified plasmic fragments of human fibrinogen with the electron microscope and have determined the relation of these fragments to the intact fibrinogen molecule. Both intact fibrinogen and its earliest cleavage product, fragment X, are trinodular. The next largest product, fragment Y, consists of two linked nodules. The two terminal products, fragments D and E, are single nodules. From measurements of simultaneously shadowed specimens of these different species, we conclude that the outer nodules of the trinodular fibrinogen molecule are the fragment D-containing regions and the central nodule is the fragment E-containing region.

Authors
Fowler, WE; Fretto, LJ; Erickson, HP; McKee, PA
MLA Citation
Fowler, WE, Fretto, LJ, Erickson, HP, and McKee, PA. "Electron microsocpy of plasmic fragments of human fibrinogen as related to trinodular structure of the intact molecule." J Clin Invest 66.1 (July 1980): 50-56.
PMID
7400308
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
66
Issue
1
Publish Date
1980
Start Page
50
End Page
56
DOI
10.1172/JCI109834

Thermodynamic and kinetic aspects of assembly.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Thermodynamic and kinetic aspects of assembly." Prog Clin Biol Res 40 (1980): 327-330.
PMID
7454727
Source
pubmed
Published In
Progress in Clinical and Biological Research
Volume
40
Publish Date
1980
Start Page
327
End Page
330

Fibrin assembly: A comparison of electron microscopic and light scattering results

To test the validity of a proposed two step fibrin assembly mechanism and directly visualise the molecular species present at various stages of fibrin formation, we have carried out an electron microscopic investigation. Assembly conditions duplicated those of a recent light scattering study and specimens were prepared at different time points with the use of a negative staining technique recently employed to visualise the trinodular structure of fibrinogen. Under near-physiological buffer conditions, protofibrils structurally similar to those postulated by Ferry have been found at early stages of fibrin assembly. In parallel with the light scattering results, a dramatic increase in fiber diameter was found in specimens prepared during the postulated lateral association stage of gelation. Light scattering and electron microscopic results both showed that high ionic strength reduces the rate and extent of fiber formation. Reptilase cleavage is shown to result in typical cross striated fibrin.

Authors
Hantgan, R; Fowler, W; Erickson, H; Hermans, J
MLA Citation
Hantgan, R, Fowler, W, Erickson, H, and Hermans, J. "Fibrin assembly: A comparison of electron microscopic and light scattering results." Thrombosis and Haemostasis 44.3 (1980): 119-124.
PMID
6162207
Source
scival
Published In
Thrombosis and Haemostasis
Volume
44
Issue
3
Publish Date
1980
Start Page
119
End Page
124

Trinodular structure of fibrinogen. Confirmation by both shadowing and negative stain electron microscopy.

Authors
Fowler, WE; Erickson, HP
MLA Citation
Fowler, WE, and Erickson, HP. "Trinodular structure of fibrinogen. Confirmation by both shadowing and negative stain electron microscopy." J Mol Biol 134.2 (October 25, 1979): 241-249.
PMID
537064
Source
pubmed
Published In
Journal of Molecular Biology
Volume
134
Issue
2
Publish Date
1979
Start Page
241
End Page
249

Tubulin rings: curved filaments with limited flexibility and two modes of association.

Tubulin rings have been previously identified as composed of linear polymers of tubulin subunits, equivalent to a protofilament in the microtubule wall but in a curved rather than a straight conformation. We have examined and measured a number of different ring structures obtained under different conditions. The preferred curvature is indicated by a single ring of 380 A outside diameter. Radially double rings consist of two coplanar rings of 460 A and 350 A outside diameter, held together by a pattern of eight identical contacts between the 40 A subunits in the inner and outer rings. In some circumstances a larger ring, 570 A diameter, can be added to the outside, or a smaller ring, 240 A diameter, may be added to the inside of the radially double ring, in both cases repeating the pattern of eight radial contacts. The distortion of the filament from its relaxed 380 A diameter curvature apparently can be made without disrupting the longitudinal bond between subunits in the filament, but must be stabilized by the energy of the radial contact. All of these rings (single and radially double and triple) are observed to associate axially to form pairs or in some cases larger stacks. The radially double rings or an axially associated pair of these (quadruple ring) may also associate to form crystals. These are thin plates, up to 100 micrometers in extent and several micrometers thick which have been of limited use so far in diffraction studies because of irregularities in the packing of adjacent rings.

Authors
Voter, WA; Erickson, HP
MLA Citation
Voter, WA, and Erickson, HP. "Tubulin rings: curved filaments with limited flexibility and two modes of association." J Supramol Struct 10.4 (1979): 419-431.
PMID
513771
Source
pubmed
Published In
Journal of supramolecular structure
Volume
10
Issue
4
Publish Date
1979
Start Page
419
End Page
431
DOI
10.1002/jss.400100405

Image reconstruction in electron microscopy: enhancement of periodic structure by optical filtering.

Authors
Erickson, HP; Voter, WA; Leonard, K
MLA Citation
Erickson, HP, Voter, WA, and Leonard, K. "Image reconstruction in electron microscopy: enhancement of periodic structure by optical filtering." Methods Enzymol 49 (1978): 39-63.
PMID
651674
Source
pubmed
Published In
Methods in Enzymology
Volume
49
Publish Date
1978
Start Page
39
End Page
63

[4] Image reconstruction in electron microscopy: Enhancement of periodic structure by optical filtering

Authors
Erickson, HP; Voter, WA; Leonard, K
MLA Citation
Erickson, HP, Voter, WA, and Leonard, K. "[4] Image reconstruction in electron microscopy: Enhancement of periodic structure by optical filtering." Methods in Enzymology 49.C (1978): 39-63.
Source
scival
Published In
Methods in Enzymology
Volume
49
Issue
C
Publish Date
1978
Start Page
39
End Page
63
DOI
10.1016/S0076-6879(78)49006-8

Guanosinetriphosphatase activity of tubulin associated with microtubule assembly.

Tubulin, purified by cycles of assembly followed by phosphocellulose chromatography, exhibits a characteristic GTPase activity that is polymerization dependent and can be attributed to the tubulin itself. This activity has been observed, in a standard reassembly buffer containing low Mg2+, under three conditions that induce microtubule assembly: in the presence of microtubule-associated proteins, in the presence of DEAE-dextran, or after addition of high Mg2+ and glycerol. The phosphocellulose-purified tubulin showed no GTPase activity under the following nonpolymerizing conditions: in buffer with low Mg2+ in the absence of microtubule-associated proteins or DEAE-dextran, in buffer with high Mg2+ and glycerol at tubulin concentrations below the critical concentration, or when microtubule assembly was inhibited by vinblastine. Colchicine, on the other hand, while blocking microtubule assembly, induced a significant GTPase activity in the phosphocellulose-purified tubulin. During the process of assembly, GTP appears to be hydrolyzed as a free tubulin dimer polymerizes into a microtubule. A constant GTPase activity when polymerization equilibrium is reached apparently reflects the cyclic polymerization-depolymerization of tubulin dimers at the ends of the microtubules.

Authors
David-Pfeuty, T; Erickson, HP; Pantaloni, D
MLA Citation
David-Pfeuty, T, Erickson, HP, and Pantaloni, D. "Guanosinetriphosphatase activity of tubulin associated with microtubule assembly." Proc Natl Acad Sci U S A 74.12 (December 1977): 5372-5376.
PMID
202954
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
74
Issue
12
Publish Date
1977
Start Page
5372
End Page
5376

Polycation-induced assembly of purified tubulin.

Several different polycations have been found that can substitute for the microtubule-associated proteins, or tau factor, in facilitating assembly of tubulin that has been purified by ion exchange chromatography. In low concentrations of the polycation diethylaminoethyl-dextran, 7 mg of tubulin is pelleted per 1 mg of polycation added. Under conditions favorable to microtubule assembly the entire pellet is seen by electron microscopy to consist of "double wall microtubules", which are essentially identical to normal microtubules in subunit structure and arrangement. When assembly is inhibited approximately the same amount of tubulin is pelleted, but it is in the form of clusters of curved sheets or filaments apparently related to tubulin rings. When conditions are changed to favor assembly, the tubulin within these clusters appears to reassemble to form the double wall microtubules.

Authors
Erickson, HP; Voter, WA
MLA Citation
Erickson, HP, and Voter, WA. "Polycation-induced assembly of purified tubulin." Proc Natl Acad Sci U S A 73.8 (August 1976): 2813-2817.
PMID
1066692
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
73
Issue
8
Publish Date
1976
Start Page
2813
End Page
2817

The structure and assembly of microtubules.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "The structure and assembly of microtubules." Ann N Y Acad Sci 253 (June 30, 1975): 60-77. (Review)
PMID
1096724
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
253
Publish Date
1975
Start Page
60
End Page
77

Negatively stained vinblastine aggregates.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Negatively stained vinblastine aggregates." Ann N Y Acad Sci 253 (June 30, 1975): 51-52.
PMID
1056755
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
253
Publish Date
1975
Start Page
51
End Page
52

Microtubule surface lattice and subunit structure and observations on reassembly.

Neuronal microtubules have been reassembled from brain tissue homogenates and purified. In reassembly from purified preparations, one of the first structures formed was a flat sheet, consisting of up to 13 longitudinal filaments, which was identified as an incomplete microtubule wall. Electron micrographs of these flat sheets and intact microtubules were analyzed by optical diffraction, and the surface lattice on which the subunits are arranged was determined to be a 13 filament, 3-start helix. A similar, and probably identical, lattice was found for outer-doublet microtubules. Finally, a 2-D image of the structure and arrangement of the microtubule subunits was obtained by processing selected images with a computer filtering and averaging system. The 40 x 50 A morphological subunit, which has previously been seen only as a globular particle and identified as the 55,000-dalton tubulin monomer, is seen in this higher resolution reconstructed image to be elongated, and split symmetrically by a longitudinal cleft into two lobes.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Microtubule surface lattice and subunit structure and observations on reassembly." J Cell Biol 60.1 (January 1974): 153-167.
PMID
4855592
Source
pubmed
Published In
The Journal of Cell Biology
Volume
60
Issue
1
Publish Date
1974
Start Page
153
End Page
167

Assembly of microtubules from preformed, ring-shaped protofilaments and 6-S tubulin.

Authors
Erickson, HP
MLA Citation
Erickson, HP. "Assembly of microtubules from preformed, ring-shaped protofilaments and 6-S tubulin." J Supramol Struct 2.2-4 (1974): 393-411.
PMID
4474573
Source
pubmed
Published In
Journal of supramolecular structure
Volume
2
Issue
2-4
Publish Date
1974
Start Page
393
End Page
411
DOI
10.1002/jss.400020228

Electron microscopic study of base sequence in nucleic acids VI. Guanine sites in yeast alanine transfer RNA

Yeast alanine t-RNA was reacted with 2-diazo-p-benzene-disulphonic acid, which is known to attach with high specificity to the guanosine monophosphate nucleotides. The guanine marked RNA molecules were extended on thin carbon film, stained with uranyl acetate, and examined in the electron microscope. In high resolution micrographs most of the markers made visible by the uranyl acetate staining, could be distinguished from the background noise on the basis of contrast and size. They were found in linear arrays with a length not exceeding that of an extended t-RNA molecule and their number not exceeding that expected from chemical reactivity data as well as from the known number of guanosine residues in ala t-RNA molecule. © 1970.

Authors
Bartl, P; Erickson, H; Beer, M
MLA Citation
Bartl, P, Erickson, H, and Beer, M. "Electron microscopic study of base sequence in nucleic acids VI. Guanine sites in yeast alanine transfer RNA." Micron (1969) 1.4 (1970): 374-392.
Source
scival
Published In
Micron (1969)
Volume
1
Issue
4
Publish Date
1970
Start Page
374
End Page
392

Electron microscopic study of the base sequence in nucleic acids. VI. Preparation of ribonucleic acid with marked guanosine monophosphate nucleotides

It has been previously shown that the compound 2-diazo-p-benzenedisulfonic acid can be treated with deoxyribonucleic acid (DNA) at pH 9 to form an addition product specifically with the guanosine monophosphate (GMP) nucleotides. These can then be stained with uranyl acetate for identification of the GMP nucleotide sites by electron microscopy. Further investigaton of the chemical reaction, reported here, has shown that the reaction at pH 9 also causes an acid-labile attachment of the diazonium marker to adenosine monophosphate (AMP) and cytidine monophosphate (CMP) nucleotides. This attachment is reversed by the acid precipitation which had been used for purifying the marked DNA. A much milder acid treatment, involving incubation at pH 4, has been developed to reverse this attachment with less damage to the nucleic acid and the marked GMP. The selectivity of the reaction with ribonucleic acid (RNA) was found to be slightly less than with DNA. When 60% of the GMP's have been marked, about 10% of each of the other nucleotides is also marked. The RNA molecules are somewhat fragmented during the reaction as found by sedimentation analysis and by measuring molecular length by electron microscopy. Using sucrose gradient sedimentation to fractionate the molecules according to size it has been possible to purify small amounts of marked MS-2 RNA molecules which are more than one-half their native length. The preparation of specimens for electron microscopic analysis is described.

Authors
Erickson, H; Beer, M
MLA Citation
Erickson, H, and Beer, M. "Electron microscopic study of the base sequence in nucleic acids. VI. Preparation of ribonucleic acid with marked guanosine monophosphate nucleotides." Biochemistry 6.9 (1967): 2694-2701.
PMID
4861594
Source
scival
Published In
Biochemistry
Volume
6
Issue
9
Publish Date
1967
Start Page
2694
End Page
2701

Rapid nucleotide separation by chromatography on cation-exchange columns

Authors
Blattner, FR; Erickson, HP
MLA Citation
Blattner, FR, and Erickson, HP. "Rapid nucleotide separation by chromatography on cation-exchange columns." Analytical Biochemistry 18.2 (1967): 220-227.
Source
scival
Published In
Analytical Biochemistry
Volume
18
Issue
2
Publish Date
1967
Start Page
220
End Page
227
Show More

Research Areas:

  • Acquired Immunodeficiency Syndrome
  • Acrylamide
  • Actin Cytoskeleton
  • Actins
  • Adenosine Triphosphatases
  • Adenosine Triphosphate
  • Age Factors
  • Alkaline Phosphatase
  • Allosteric Regulation
  • Alternative Splicing
  • Amidohydrolases
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Anion Exchange Resins
  • Annexin A2
  • Antibodies
  • Antibodies, Monoclonal
  • Antigens, CD
  • Antigens, CD18
  • Antigens, CD29
  • Antigens, Differentiation
  • Antigens, Nuclear
  • Antigens, Surface
  • Aorta
  • Archaea
  • Archaeal Proteins
  • Artificial Gene Fusion
  • Aspartic Acid
  • Astrocytes
  • Astrocytoma
  • Azotobacter vinelandii
  • Bacillus anthracis
  • Bacillus subtilis
  • Bacillus thuringiensis
  • Bacteria
  • Bacterial Physiological Phenomena
  • Bacterial Proteins
  • Base Sequence
  • Binding Sites
  • Binding Sites, Antibody
  • Binding, Competitive
  • Biochemistry
  • Biological Evolution
  • Biophysical Phenomena
  • Biophysics
  • Biopolymers
  • Blood Coagulation Factors
  • Blood Platelets
  • Blotting, Northern
  • Blotting, Western
  • Bone Marrow
  • Bone Neoplasms
  • Brain
  • Brain Chemistry
  • Brain Injuries
  • Buffers
  • CHO Cells
  • Caenorhabditis elegans Proteins
  • Calcium
  • Calmodulin-Binding Proteins
  • Calorimetry
  • Carrier Proteins
  • Catalytic Domain
  • Cations
  • Cations, Divalent
  • Cattle
  • Caulobacter crescentus
  • Cell Adhesion
  • Cell Adhesion Molecules
  • Cell Adhesion Molecules, Neuronal
  • Cell Cycle Proteins
  • Cell Differentiation
  • Cell Division
  • Cell Line
  • Cell Line, Transformed
  • Cell Membrane
  • Cell Movement
  • Cell Separation
  • Cell Survival
  • Cell Transformation, Neoplastic
  • Cells, Cultured
  • Central Nervous System
  • Centrifugation
  • Centrifugation, Density Gradient
  • Cereals
  • Chelating Agents
  • Chemical Fractionation
  • Chemical Phenomena
  • Chemical Precipitation
  • Chemistry
  • Chemistry, Physical
  • Chemokine CX3CL1
  • Chemokines, CX3C
  • Chemokines, CXC
  • Chick Embryo
  • Chickens
  • Child
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  • Chondrocytes
  • Chondroitin Sulfate Proteoglycans
  • Chondroitin Sulfates
  • Chromaffin System
  • Chromatography
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
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  • Chromatography, Liquid
  • Chromosomal Proteins, Non-Histone
  • Chromosome Deletion
  • Chromosome Segregation
  • Chromosomes
  • Chromosomes, Bacterial
  • Cloning, Molecular
  • Colchicine
  • Cold Temperature
  • Collagen
  • Computer Simulation
  • Connective Tissue Diseases
  • Cricetinae
  • Cross-Linking Reagents
  • Crystallization
  • Crystallography
  • Crystallography, X-Ray
  • Culture Media
  • Culture Media, Conditioned
  • Culture Techniques
  • Cycloheximide
  • Cysteine
  • Cytokines
  • Cytokinesis
  • Cytoplasm
  • Cytoskeletal Proteins
  • Cytoskeleton
  • DEAE-Dextran
  • DNA
  • DNA Helicases
  • DNA Ligases
  • DNA Repair
  • DNA Replication
  • DNA, Bacterial
  • DNA, Circular
  • DNA, Complementary
  • DNA-Binding Proteins
  • Dactinomycin
  • Detergents
  • Dextrans
  • Diffusion
  • Dimerization
  • Disulfides
  • Dithiothreitol
  • Dogs
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Drosophila
  • Drosophila Proteins
  • Drosophila melanogaster
  • Drug Interactions
  • Drug Stability
  • E-Selectin
  • Edetic Acid
  • Elasticity
  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Embryo Implantation
  • Embryo, Mammalian
  • Embryonic and Fetal Development
  • Endopeptidases
  • Endosomal Sorting Complexes Required for Transport
  • Endothelial Cells
  • Endothelium
  • Endothelium, Vascular
  • Energy Metabolism
  • Entropy
  • Enzyme Activation
  • Epidermal Growth Factor
  • Epidermis
  • Epithelium
  • Epitope Mapping
  • Epitopes
  • Erythrocytes
  • Escherichia coli
  • Escherichia coli Proteins
  • Estrus
  • Eukaryotic Cells
  • Evolution, Molecular
  • Exercise
  • Exodeoxyribonucleases
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  • Extracellular Matrix Proteins
  • Factor VIII
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  • Female
  • Fetus
  • Fibrinogen
  • Fibroblasts
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  • Flow Cytometry
  • Fluorescein
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  • Fluorescence Resonance Energy Transfer
  • Fluorescent Antibody Technique
  • Fluorescent Dyes
  • Focal Adhesions
  • Fourier Analysis
  • Fungal Proteins
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • Gene Deletion
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  • Gene Expression Regulation
  • Gene Library
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  • Genetic Variation
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  • Glycerol
  • Glycoproteins
  • Glycosaminoglycans
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  • Gram-Negative Anaerobic Bacteria
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  • Green Fluorescent Proteins
  • Growth Substances
  • Guanine Nucleotides
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  • Guinea Pigs
  • HEK293 Cells
  • Half-Life
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  • Heart Ventricles
  • Helminth Proteins
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  • Hemocyanin
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  • Humans
  • Hydrogen Bonding
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  • Hydrolysis
  • Image Processing, Computer-Assisted
  • Immune Sera
  • Immunoglobulin Constant Regions
  • Immunoglobulins
  • Immunohistochemistry
  • Immunologic Techniques
  • Immunoprecipitation
  • Inhibitory Concentration 50
  • Insect Proteins
  • Integrin alpha Chains
  • Integrin alphaVbeta3
  • Integrins
  • Intercellular Adhesion Molecule-1
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  • Kinesin
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  • Light
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  • Liposomes
  • Liver
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  • Lung
  • Lymphocyte Function-Associated Antigen-1
  • Lymphocytes
  • Macromolecular Substances
  • Magnesium
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  • Membrane Proteins
  • Membranes
  • Mercaptoethanol
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  • Microspheres
  • Microtubule Proteins
  • Microtubules
  • Milk, Human
  • Mitogens
  • Models, Biological
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  • Models, Molecular
  • Models, Structural
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  • Molecular Conformation
  • Molecular Sequence Data
  • Molecular Structure
  • Molecular Weight
  • Monte Carlo Method
  • Morphogenesis
  • Motion
  • Mucins
  • Multigene Family
  • Multiprotein Complexes
  • Muscle Proteins
  • Muscle, Skeletal
  • Muscles
  • Muscular Dystrophy, Animal
  • Mutagenesis
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  • Mutation
  • Mycobacterium tuberculosis
  • Myocardium
  • Myosin-Light-Chain Kinase
  • Myosins
  • Negative Staining
  • Neoplasm Proteins
  • Neoplasms
  • Nerve Growth Factors
  • Nerve Tissue Proteins
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  • Neural Cell Adhesion Molecules
  • Neurons
  • Neutrophils
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins
  • Nucleic Acid Conformation
  • Octoxynol
  • Oligonucleotide Probes
  • Oligopeptides
  • Organ Specificity
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  • Oxygen
  • P-Selectin
  • Paclitaxel
  • Palatine Tonsil
  • Peptide Fragments
  • Peptide Mapping
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Peptides
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  • Phylogeny
  • Physicochemical Phenomena
  • Physicochemical Processes
  • Phytochrome
  • Plant Proteins
  • Plasmids
  • Plastics
  • Platelet Aggregation
  • Platelet Membrane Glycoproteins
  • Platelet-Derived Growth Factor
  • Pleurodeles
  • Pliability
  • Poly A
  • Polyethylene Glycols
  • Polymerase Chain Reaction
  • Polymers
  • Potassium
  • Precipitin Tests
  • Pregnancy
  • Proline
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Conformation
  • Protein Denaturation
  • Protein Engineering
  • Protein Folding
  • Protein Interaction Mapping
  • Protein Isoforms
  • Protein Kinases
  • Protein Multimerization
  • Protein Precursors
  • Protein Sorting Signals
  • Protein Stability
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Subunits
  • Protein Transport
  • Proteins
  • Proteoglycans
  • Proto-Oncogene Proteins pp60(c-src)
  • Pseudomonas aeruginosa
  • R Factors
  • RNA Splicing
  • RNA, Messenger
  • Rabbits
  • Radioligand Assay
  • Rats
  • Rats, Sprague-Dawley
  • Receptors
  • Receptors, Cell Surface
  • Receptors, Cholinergic
  • Receptors, Fibronectin
  • Receptors, Leukocyte-Adhesion
  • Receptors, Somatotropin
  • Receptors, Virus
  • Receptors, Vitronectin
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Recombination, Genetic
  • Repetitive Sequences, Amino Acid
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  • Resins, Synthetic
  • Reticulin
  • Rheology
  • Rhinovirus
  • Rhodamines
  • Ristocetin
  • Rubidium
  • Ryanodine
  • Ryanodine Receptor Calcium Release Channel
  • Salts
  • Sarcoplasmic Reticulum
  • Scattering, Radiation
  • Sea Urchins
  • Selenomethionine
  • Sensitivity and Specificity
  • Sequence Alignment
  • Sequence Analysis
  • Sequence Analysis, DNA
  • Sequence Homology
  • Sequence Homology, Amino Acid
  • Serine Endopeptidases
  • Sertoli Cells
  • Signal Transduction
  • Silver Staining
  • Skin
  • Sodium
  • Solubility
  • Solutions
  • Species Specificity
  • Spectrometry, Fluorescence
  • Spectrophotometry
  • Spectrum Analysis
  • Spinal Cord
  • Static Electricity
  • Structural Homology, Protein
  • Structure-Activity Relationship
  • Succinimides
  • Sulfuric Acid Esters
  • Surface Plasmon Resonance
  • Surface Properties
  • Swine
  • Temperature
  • Tenascin
  • Tensile Strength
  • Thermodynamics
  • Thermolysin
  • Time Factors
  • Tissue Scaffolds
  • Tongue
  • Torsion, Mechanical
  • Trans-Activators
  • Transfection
  • Transforming Growth Factor beta
  • Transglutaminases
  • Trypsin
  • Tryptophan
  • Tubulin
  • Tumor Cells, Cultured
  • Ultracentrifugation
  • Unilamellar Liposomes
  • Urea
  • Uterus
  • Versicans
  • Vibrissae
  • Video Recording
  • Vinculin
  • Viral Proteins
  • Wound Healing
  • X-Ray Diffraction
  • X-ray crystallography
  • Xenopus
  • Xenopus Proteins
  • Yeasts
  • Zebrafish
  • von Willebrand Factor