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Lew, Daniel Julio

Overview:

Our research interests encompass questions on cell cycle control, the control of cell polarity, and the specification of distinct cortical domains within cells. We are also trying to understand how cells can monitor their shape and react to environmental influences that affect cytoskeletal behavior.
One focus is the study of how the Cyclin Dependent Kinases (CDKs) that control cell cycle progression act to promote specific changes in cell polarity. A ras-related G protein, Cdc42p, is key for enacting changes in cell polarity involving reorganization of both actin and septins (a poorly understood filamentous system that specializes specific cortical domains) in response to CDK activity. We are tracing the links between the CDK and Cdc42p to understand how polarity is established, and the links between Cdc42p and the cytoskeleton to determine how polarized behavior is executed.
A second focus involves investigation of a cell cycle checkpoint control that monitors cell shape. When environmental insults disrupt cytoskeletal organization, this checkpoint delays entry into mitosis through inhibition of CDK/cyclin kinases. A tyrosine kinase, Swe1p, is responsible for the cell cycle block, and we have found that the degradation of Swe1p is regulated both by cell shape and by perturbation of the actin cytoskeleton. Recently, we discovered that the septin cytoskeleton is directly affected by local cell shape, and that proteins controlling Swe1p degradation can monitor this septin change.
The biological problems we address are universal, and the proteins that we study are widely conserved. We have chosen the experimentally tractable budding yeast as our experimental system and are using genetic, cell biological, and biochemical approaches to study these pathways.

Positions:

James B. Duke Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Professor of Cell Biology

Cell Biology
School of Medicine

Professor in Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1990

Ph.D. — Rockefeller University

Grants:

Genetics Training Grant

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 1979
End Date
June 30, 2020

Organization and Function of Cellular Structure

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

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Program to Support Student Development and Diversity in Duke Biosciences

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 2013
End Date
April 30, 2018

Gradient Tracking and Chemotropism

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2013
End Date
April 30, 2018

Training Program in Developmental and Stem Cell Biology

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2001
End Date
October 31, 2017

Spatiotemporal modeling of signal transduction in yeast

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
September 01, 2016
End Date
August 31, 2017

Polarity Establishment in Yeast

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2001
End Date
June 30, 2017

Duke BioCoRE: Increasing Diversity through Student and Faculty Engagement

Administered By
School of Medicine
AwardedBy
Merck Foundation
Role
Co-Principal Investigator
Start Date
September 01, 2015
End Date
August 31, 2016

Bioinformatics and Computational Biology Training Program

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2005
End Date
June 30, 2016

Cancer Biology Training Grant

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

Instrumentation for Quantitative Phosphoproteomics and Acetylomics

Administered By
Duke Center for Genomic and Computational Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2014
End Date
May 14, 2015

Cell Cycle Checkpoint that Monitors Morphogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1995
End Date
October 30, 2013

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

PREDOCTORAL FELLOWSHIPS FOR STUDENTS WITH DISABILITIES

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2004
End Date
December 31, 2005

Genomw APA-A Tool Biochemical Screening

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 20, 1997
End Date
July 31, 1999

. Cell Cycle Checkpoint That Monitors Morphogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1995
End Date
July 31, 1999

Cell Cycle Checkpoint That Monitors Morpohgenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1995
End Date
July 30, 1999

The Morphogenisis Cell Cycle Checkpoint In Building Yeast

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 20, 1996
End Date
September 19, 1998
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Awards:

AAAS Fellows. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2009

Scholars. Searle Scholars.

Type
National
Awarded By
Searle Scholars
Date
January 01, 1995

Publications:

How do cells know what shape they are?

Studies on a yeast cell cycle checkpoint that can delay mitosis depending on whether cells have built a bud have identified a "sensor" that seems to recognize the organization of filament-forming septin proteins. Innovative work applying correlative light and platinum replica electron microscopy suggests that the informative septin organization involves parallel alignment of septin filaments, and another striking study shows that septin filaments prefer to populate membranes that have positive micron-scale curvature. Together, these findings suggest a model for how cells may monitor aspects of their own shape to influence cell behavior.

Authors
Kang, H; Lew, DJ
MLA Citation
Kang, H, and Lew, DJ. "How do cells know what shape they are?." Current genetics 63.1 (February 2017): 75-77.
PMID
27313005
Source
epmc
Published In
Current Genetics
Volume
63
Issue
1
Publish Date
2017
Start Page
75
End Page
77
DOI
10.1007/s00294-016-0623-1

Parallel Actin-Independent Recycling Pathways Polarize Cdc42 in Budding Yeast.

The highly conserved Rho-family GTPase Cdc42 is an essential regulator of polarity in many different cell types. During polarity establishment, Cdc42 becomes concentrated at a cortical site, where it interacts with downstream effectors to orient the cytoskeleton along the front-back axis. To concentrate Cdc42, loss of Cdc42 by diffusion must be balanced by recycling to the front. In Saccharomyces cerevisiae, the guanine nucleotide dissociation inhibitor (GDI) Rdi1 recycles Cdc42 through the cytoplasm. Loss of Rdi1 slowed but did not eliminate Cdc42 accumulation at the front, suggesting the existence of other recycling pathways. One proposed pathway involves actin-directed trafficking of vesicles carrying Cdc42 to the front. However, we found no role for F-actin in Cdc42 concentration, even in rdi1Δ cells. Instead, Cdc42 was still able to exchange between the membrane and cytoplasm in rdi1Δ cells, albeit at a reduced rate. Membrane-cytoplasm exchange of GDP-Cdc42 was faster than that of GTP-Cdc42, and computational modeling indicated that such exchange would suffice to promote polarization. We also uncovered a novel role for the Cdc42-directed GTPase-activating protein (GAP) Bem2 in Cdc42 polarization. Bem2 was known to act in series with Rdi1 to promote recycling of Cdc42, but we found that rdi1Δ bem2Δ mutants were synthetically lethal, suggesting that they also act in parallel. We suggest that GAP activity cooperates with the GDI to counteract the dissipative effect of a previously unappreciated pathway whereby GTP-Cdc42 escapes from the polarity site through the cytoplasm.

Authors
Woods, B; Lai, H; Wu, C-F; Zyla, TR; Savage, NS; Lew, DJ
MLA Citation
Woods, B, Lai, H, Wu, C-F, Zyla, TR, Savage, NS, and Lew, DJ. "Parallel Actin-Independent Recycling Pathways Polarize Cdc42 in Budding Yeast." Current biology : CB 26.16 (August 2016): 2114-2126.
Website
http://hdl.handle.net/10161/13035
PMID
27476596
Source
epmc
Published In
Current Biology
Volume
26
Issue
16
Publish Date
2016
Start Page
2114
End Page
2126
DOI
10.1016/j.cub.2016.06.047

Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1.

Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a "bud sensor." Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.

Authors
Kang, H; Tsygankov, D; Lew, DJ
MLA Citation
Kang, H, Tsygankov, D, and Lew, DJ. "Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1." Molecular biology of the cell 27.11 (June 2016): 1764-1775.
PMID
27053666
Source
epmc
Published In
Molecular Biology of the Cell
Volume
27
Issue
11
Publish Date
2016
Start Page
1764
End Page
1775
DOI
10.1091/mbc.e16-01-0014

Imaging Polarization in Budding Yeast.

We describe methods for live-cell imaging of yeast cells that we have exploited to image yeast polarity establishment. As a rare event occurring on a fast time-scale, imaging polarization involves a trade-off between spatiotemporal resolution and long-term imaging without excessive phototoxicity. By synchronizing cells in a way that increases resistance to photodamage, we discovered unexpected aspects of polarization including transient intermediates with more than one polarity cluster, oscillatory clustering of polarity factors, and mobile "wandering" polarity sites.

Authors
McClure, AW; Wu, C-F; Johnson, SA; Lew, DJ
MLA Citation
McClure, AW, Wu, C-F, Johnson, SA, and Lew, DJ. "Imaging Polarization in Budding Yeast." Methods in molecular biology (Clifton, N.J.) 1407 (January 2016): 13-23.
Website
http://hdl.handle.net/10161/13037
PMID
27271891
Source
epmc
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
1407
Publish Date
2016
Start Page
13
End Page
23
DOI
10.1007/978-1-4939-3480-5_2

Role of competition between polarity sites in establishing a unique front.

Polarity establishment in many cells is thought to occur via positive feedback that reinforces even tiny asymmetries in polarity protein distribution. Cdc42 and related GTPases are activated and accumulate in a patch of the cortex that defines the front of the cell. Positive feedback enables spontaneous polarization triggered by stochastic fluctuations, but as such fluctuations can occur at multiple locations, how do cells ensure that they make only one front? In polarizing cells of the model yeast Saccharomyces cerevisiae, positive feedback can trigger growth of several Cdc42 clusters at the same time, but this multi-cluster stage rapidly evolves to a single-cluster state, which then promotes bud emergence. By manipulating polarity protein dynamics, we show that resolution of multi-cluster intermediates occurs through a greedy competition between clusters to recruit and retain polarity proteins from a shared intracellular pool.

Authors
Wu, C-F; Chiou, J-G; Minakova, M; Woods, B; Tsygankov, D; Zyla, TR; Savage, NS; Elston, TC; Lew, DJ
MLA Citation
Wu, C-F, Chiou, J-G, Minakova, M, Woods, B, Tsygankov, D, Zyla, TR, Savage, NS, Elston, TC, and Lew, DJ. "Role of competition between polarity sites in establishing a unique front." eLife 4 (November 2, 2015).
PMID
26523396
Source
epmc
Published In
eLife
Volume
4
Publish Date
2015
DOI
10.7554/elife.11611

Role of competition between polarity sites in establishing a unique front

© Wu et al.Polarity establishment in many cells is thought to occur via positive feedback that reinforces even tiny asymmetries in polarity protein distribution. Cdc42 and related GTPases are activated and accumulate in a patch of the cortex that defines the front of the cell. Positive feedback enables spontaneous polarization triggered by stochastic fluctuations, but as such fluctuations can occur at multiple locations, how do cells ensure that they make only one front? In polarizing cells of the model yeast Saccharomyces cerevisiae, positive feedback can trigger growth of several Cdc42 clusters at the same time, but this multi-cluster stage rapidly evolves to a singlecluster state, which then promotes bud emergence. By manipulating polarity protein dynamics, we show that resolution of multi-cluster intermediates occurs through a greedy competition between clusters to recruit and retain polarity proteins from a shared intracellular pool.

Authors
Wu, CF; Chiou, JG; Minakova, M; Woods, B; Tsygankov, D; Zyla, TR; Savage, NS; Elston, TC; Lew, DJ
MLA Citation
Wu, CF, Chiou, JG, Minakova, M, Woods, B, Tsygankov, D, Zyla, TR, Savage, NS, Elston, TC, and Lew, DJ. "Role of competition between polarity sites in establishing a unique front." eLife 4.NOVEMBER2015 (November 2, 2015).
Source
scopus
Published In
eLife
Volume
4
Issue
NOVEMBER2015
Publish Date
2015
DOI
10.7554/eLife.11611.001

Dendritic spine geometry can localize GTPase signaling in neurons.

Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.

Authors
Ramirez, SA; Raghavachari, S; Lew, DJ
MLA Citation
Ramirez, SA, Raghavachari, S, and Lew, DJ. "Dendritic spine geometry can localize GTPase signaling in neurons." Molecular biology of the cell 26.22 (November 2015): 4171-4181.
PMID
26337387
Source
epmc
Published In
Molecular Biology of the Cell
Volume
26
Issue
22
Publish Date
2015
Start Page
4171
End Page
4181
DOI
10.1091/mbc.e15-06-0405

Role of Polarized G Protein Signaling in Tracking Pheromone Gradients.

Yeast cells track gradients of pheromones to locate mating partners. Intuition suggests that uniform distribution of pheromone receptors over the cell surface would yield optimal gradient sensing. However, yeast cells display polarized receptors. The benefit of such polarization was unknown. During gradient tracking, cell growth is directed by a patch of polarity regulators that wanders around the cortex. Patch movement is sensitive to pheromone dose, with wandering reduced on the up-gradient side of the cell, resulting in net growth in that direction. Mathematical modeling suggests that active receptors and associated G proteins lag behind the polarity patch and act as an effective drag on patch movement. In vivo, the polarity patch is trailed by a G protein-rich domain, and this polarized distribution of G proteins is required to constrain patch wandering. Our findings explain why G protein polarization is beneficial and illuminate a novel mechanism for gradient tracking.

Authors
McClure, AW; Minakova, M; Dyer, JM; Zyla, TR; Elston, TC; Lew, DJ
MLA Citation
McClure, AW, Minakova, M, Dyer, JM, Zyla, TR, Elston, TC, and Lew, DJ. "Role of Polarized G Protein Signaling in Tracking Pheromone Gradients." Developmental cell 35.4 (November 2015): 471-482.
PMID
26609960
Source
epmc
Published In
Developmental Cell
Volume
35
Issue
4
Publish Date
2015
Start Page
471
End Page
482
DOI
10.1016/j.devcel.2015.10.024

Polarity establishment requires localized activation of Cdc42.

Establishment of cell polarity in animal and fungal cells involves localization of the conserved Rho-family guanosine triphosphatase, Cdc42, to the cortical region destined to become the "front" of the cell. The high local concentration of active Cdc42 promotes cytoskeletal polarization through various effectors. Cdc42 accumulation at the front is thought to involve positive feedback, and studies in the budding yeast Saccharomyces cerevisiae have suggested distinct positive feedback mechanisms. One class of mechanisms involves localized activation of Cdc42 at the front, whereas another class involves localized delivery of Cdc42 to the front. Here we show that Cdc42 activation must be localized for successful polarity establishment, supporting local activation rather than local delivery as the dominant mechanism in this system.

Authors
Woods, B; Kuo, C-C; Wu, C-F; Zyla, TR; Lew, DJ
MLA Citation
Woods, B, Kuo, C-C, Wu, C-F, Zyla, TR, and Lew, DJ. "Polarity establishment requires localized activation of Cdc42." The Journal of cell biology 211.1 (October 2015): 19-26.
PMID
26459595
Source
epmc
Published In
The Journal of Cell Biology
Volume
211
Issue
1
Publish Date
2015
Start Page
19
End Page
26
DOI
10.1083/jcb.201506108

To avoid a mating mishap, yeast focus and communicate.

During mating, yeast cells must perforate their rigid cell walls at the right place to allow cell-cell fusion. In this issue, Dudin et al. (2015; J. Cell Biol. http://dx.doi.org/jcb.201411124) image mating fission yeast cells with unprecedented spatiotemporal resolution. The authors find that when mating cells come into contact, they form aster-like actin structures that direct cell wall remodeling precisely to the point of contact.

Authors
McClure, AW; Lew, DJ
MLA Citation
McClure, AW, and Lew, DJ. "To avoid a mating mishap, yeast focus and communicate." The Journal of cell biology 208.7 (March 2015): 867-868. (Review)
PMID
25825514
Source
epmc
Published In
The Journal of Cell Biology
Volume
208
Issue
7
Publish Date
2015
Start Page
867
End Page
868
DOI
10.1083/jcb.201502095

A GDI-independent mechanism to concentrate the Rho-GTPase Cdc42 during polarity establishment.

Authors
Woods, B; Wu, C; Zyla, T; Savage, N; Lew, DJ
MLA Citation
Woods, B, Wu, C, Zyla, T, Savage, N, and Lew, DJ. "A GDI-independent mechanism to concentrate the Rho-GTPase Cdc42 during polarity establishment." December 2014.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
25
Publish Date
2014

Regulation of pheromone-induced cell polarization by the polarisome proteins in yeast.

Authors
Lai, H; Zyla, T; Lew, DJ
MLA Citation
Lai, H, Zyla, T, and Lew, DJ. "Regulation of pheromone-induced cell polarization by the polarisome proteins in yeast." December 2014.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
25
Publish Date
2014

Cell polarity: netrin calms an excitable system.

In a tractable model for cell invasion, the Caenorhabditis elegans anchor cell migrates through basement membranes towards a polarity cue provided by netrin. A new study reveals that the anchor cell polarity network can break symmetry and oscillate in the absence of netrin, suggesting the presence of interlinked positive and negative feedback loops, which are common in polarity pathways.

Authors
McClure, AW; Lew, DJ
MLA Citation
McClure, AW, and Lew, DJ. "Cell polarity: netrin calms an excitable system." Current biology : CB 24.21 (November 3, 2014): R1050-R1052.
PMID
25517371
Source
epmc
Published In
Current Biology
Volume
24
Issue
21
Publish Date
2014
Start Page
R1050
End Page
R1052
DOI
10.1016/j.cub.2014.09.042

Inhibitory GEF phosphorylation provides negative feedback in the yeast polarity circuit.

Cell polarity is critical for the form and function of many cell types. During polarity establishment, cells define a cortical "front" that behaves differently from the rest of the cortex. The front accumulates high levels of the active form of a polarity-determining Rho-family GTPase (Cdc42, Rac, or Rop) that then orients cytoskeletal elements through various effectors to generate the polarized morphology appropriate to the particular cell type [1, 2]. GTPase accumulation is thought to involve positive feedback, such that active GTPase promotes further delivery and/or activation of more GTPase in its vicinity [3]. Recent studies suggest that once a front forms, the concentration of polarity factors at the front can increase and decrease periodically, first clustering the factors at the cortex and then dispersing them back to the cytoplasm [4-7]. Such oscillatory behavior implies the presence of negative feedback in the polarity circuit [8], but the mechanism of negative feedback was not known. Here we show that, in the budding yeast Saccharomyces cerevisiae, the catalytic activity of the Cdc42-directed GEF is inhibited by Cdc42-stimulated effector kinases, thus providing negative feedback. We further show that replacing the GEF with a phosphosite mutant GEF abolishes oscillations and leads to the accumulation of excess GTP-Cdc42 and other polarity factors at the front. These findings reveal a mechanism for negative feedback and suggest that the function of negative feedback via GEF inhibition is to buffer the level of Cdc42 at the polarity site.

Authors
Kuo, C-C; Savage, NS; Chen, H; Wu, C-F; Zyla, TR; Lew, DJ
MLA Citation
Kuo, C-C, Savage, NS, Chen, H, Wu, C-F, Zyla, TR, and Lew, DJ. "Inhibitory GEF phosphorylation provides negative feedback in the yeast polarity circuit." Current biology : CB 24.7 (March 13, 2014): 753-759.
PMID
24631237
Source
epmc
Published In
Current Biology
Volume
24
Issue
7
Publish Date
2014
Start Page
753
End Page
759
DOI
10.1016/j.cub.2014.02.024

Inhibitory GEF phosphorylation provides negative feedback in the yeast polarity circuit

Cell polarity is critical for the form and function of many cell types. During polarity establishment, cells define a cortical "front" that behaves differently from the rest of the cortex. The front accumulates high levels of the active form of a polarity-determining Rho-family GTPase (Cdc42, Rac, or Rop) that then orients cytoskeletal elements through various effectors to generate the polarized morphology appropriate to the particular cell type [1, 2]. GTPase accumulation is thought to involve positive feedback, such that active GTPase promotes further delivery and/or activation of more GTPase in its vicinity [3]. Recent studies suggest that once a front forms, the concentration of polarity factors at the front can increase and decrease periodically, first clustering the factors at the cortex and then dispersing them back to the cytoplasm [4-7]. Such oscillatory behavior implies the presence of negative feedback in the polarity circuit [8], but the mechanism of negative feedback was not known. Here we show that, in the budding yeast Saccharomyces cerevisiae, the catalytic activity of the Cdc42-directed GEF is inhibited by Cdc42-stimulated effector kinases, thus providing negative feedback. We further show that replacing the GEF with a phosphosite mutant GEF abolishes oscillations and leads to the accumulation of excess GTP-Cdc42 and other polarity factors at the front. These findings reveal a mechanism for negative feedback and suggest that the function of negative feedback via GEF inhibition is to buffer the level of Cdc42 at the polarity site. © 2014 Elsevier Ltd.

Authors
Kuo, C-C; Savage, NS; Chen, H; Wu, C-F; Zyla, TR; Lew, DJ
MLA Citation
Kuo, C-C, Savage, NS, Chen, H, Wu, C-F, Zyla, TR, and Lew, DJ. "Inhibitory GEF phosphorylation provides negative feedback in the yeast polarity circuit." Current Biology 24.7 (2014): 753-759.
Website
http://hdl.handle.net/10161/13038
Source
scival
Published In
Current Biology
Volume
24
Issue
7
Publish Date
2014
Start Page
753
End Page
759
DOI
10.1016/j.cub.2014.02.024

Beyond symmetry-breaking: competition and negative feedback in GTPase regulation.

Cortical domains are often specified by the local accumulation of active GTPases. Such domains can arise through spontaneous symmetry-breaking, suggesting that GTPase accumulation occurs via positive feedback. Here, we focus on recent advances in fungal and plant cell models - where new work suggests that polarity-controlling GTPases develop only one 'front' because GTPase clusters engage in a winner-takes-all competition. However, in some circumstances two or more GTPase domains can coexist, and the basis for the switch from competition to coexistence remains an open question. Polarity GTPases can undergo oscillatory clustering and dispersal, suggesting that these systems contain negative feedback. Negative feedback may prevent polarity clusters from spreading too far, regulate the balance between competition and coexistence, and provide directional flexibility for cells tracking gradients.

Authors
Wu, C-F; Lew, DJ
MLA Citation
Wu, C-F, and Lew, DJ. "Beyond symmetry-breaking: competition and negative feedback in GTPase regulation." Trends Cell Biol 23.10 (October 2013): 476-483. (Review)
PMID
23731999
Source
pubmed
Published In
Trends in Cell Biology
Volume
23
Issue
10
Publish Date
2013
Start Page
476
End Page
483
DOI
10.1016/j.tcb.2013.05.003

Inhibition of Cdc42 during mitotic exit is required for cytokinesis

Authors
Atkins, BD; Yoshida, S; Saito, K; Wu, C-F; Lew, DJ; Pellman, D
MLA Citation
Atkins, BD, Yoshida, S, Saito, K, Wu, C-F, Lew, DJ, and Pellman, D. "Inhibition of Cdc42 during mitotic exit is required for cytokinesis." JOURNAL OF CELL BIOLOGY 202.2 (July 22, 2013): 231-240.
PMID
23878274
Source
wos-lite
Published In
The Journal of Cell Biology
Volume
202
Issue
2
Publish Date
2013
Start Page
231
End Page
240
DOI
10.1083/jcb.201301090

Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint.

Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.

Authors
King, K; Kang, H; Jin, M; Lew, DJ
MLA Citation
King, K, Kang, H, Jin, M, and Lew, DJ. "Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint." Mol Biol Cell 24.7 (April 2013): 914-922.
PMID
23389636
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
24
Issue
7
Publish Date
2013
Start Page
914
End Page
922
DOI
10.1091/mbc.E12-11-0812

Tracking shallow chemical gradients by actin-driven wandering of the polarization site.

BACKGROUND: Many cells are remarkably proficient at tracking very shallow chemical gradients, despite considerable noise from stochastic receptor-ligand interactions. Motile cells appear to undergo a biased random walk: spatial noise in receptor activity may determine the instantaneous direction, but because noise is spatially unbiased, it is filtered out by time averaging, resulting in net movement upgradient. How nonmotile cells might filter out noise is unknown. RESULTS: Using yeast chemotropic mating as a model, we demonstrate that a polarized patch of polarity regulators "wanders" along the cortex during gradient tracking. Computational and experimental findings suggest that actin-directed membrane traffic contributes to wandering by diluting local polarity factors. The pheromone gradient appears to bias wandering via interactions between receptor-activated Gβγ and polarity regulators. Artificially blocking patch wandering impairs gradient tracking. CONCLUSIONS: We suggest that the polarity patch undergoes an intracellular biased random walk that enables noise filtering by time averaging, allowing nonmotile cells to track shallow gradients.

Authors
Dyer, JM; Savage, NS; Jin, M; Zyla, TR; Elston, TC; Lew, DJ
MLA Citation
Dyer, JM, Savage, NS, Jin, M, Zyla, TR, Elston, TC, and Lew, DJ. "Tracking shallow chemical gradients by actin-driven wandering of the polarization site." Curr Biol 23.1 (January 7, 2013): 32-41.
PMID
23200992
Source
pubmed
Published In
Current Biology
Volume
23
Issue
1
Publish Date
2013
Start Page
32
End Page
41
DOI
10.1016/j.cub.2012.11.014

The yeast guanine nucleotide dissociation inhibitor (GDI) enforces singularity by enhancing competition between polarity sites

Authors
Wu, C-F; Savage, NS; Zyla, TR; Lew, DJ
MLA Citation
Wu, C-F, Savage, NS, Zyla, TR, and Lew, DJ. "The yeast guanine nucleotide dissociation inhibitor (GDI) enforces singularity by enhancing competition between polarity sites." MOLECULAR BIOLOGY OF THE CELL 24 (2013).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
24
Publish Date
2013

Tracking shallow chemical gradients by actin-driven wandering of the polarization site

Background: Many cells are remarkably proficient at tracking very shallow chemical gradients, despite considerable noise from stochastic receptor-ligand interactions. Motile cells appear to undergo a biased random walk: spatial noise in receptor activity may determine the instantaneous direction, but because noise is spatially unbiased, it is filtered out by time averaging, resulting in net movement upgradient. How nonmotile cells might filter out noise is unknown. Results: Using yeast chemotropic mating as a model, we demonstrate that a polarized patch of polarity regulators "wanders" along the cortex during gradient tracking. Computational and experimental findings suggest that actin-directed membrane traffic contributes to wandering by diluting local polarity factors. The pheromone gradient appears to bias wandering via interactions between receptor-activated Gβγ and polarity regulators. Artificially blocking patch wandering impairs gradient tracking. Conclusions: We suggest that the polarity patch undergoes an intracellular biased random walk that enables noise filtering by time averaging, allowing nonmotile cells to track shallow gradients. © 2013 Elsevier Ltd.

Authors
Dyer, JM; Savage, NS; Jin, M; Zyla, TR; Elston, TC; Lew, DJ
MLA Citation
Dyer, JM, Savage, NS, Jin, M, Zyla, TR, Elston, TC, and Lew, DJ. "Tracking shallow chemical gradients by actin-driven wandering of the polarization site." Current Biology 23.1 (2013): 32-41.
Source
scival
Published In
Current Biology
Volume
23
Issue
1
Publish Date
2013
Start Page
32
End Page
41
DOI
10.1016/j.cub.2012.11.014

Interaction between bud-site selection and polarity-establishment machineries in budding yeast.

Saccharomyces cerevisiae yeast cells polarize in order to form a single bud in each cell cycle. Distinct patterns of bud-site selection are observed in haploid and diploid cells. Genetic approaches have identified the molecular machinery responsible for positioning the bud site: during bud formation, specific locations are marked with immobile landmark proteins. In the next cell cycle, landmarks act through the Ras-family GTPase Rsr1 to promote local activation of the conserved Rho-family GTPase, Cdc42. Additional Cdc42 accumulates by positive feedback, creating a concentrated patch of GTP-Cdc42, which polarizes the cytoskeleton to promote bud emergence. Using time-lapse imaging and mathematical modelling, we examined the process of bud-site establishment. Imaging reveals unexpected effects of the bud-site-selection system on the dynamics of polarity establishment, raising new questions about how that system may operate. We found that polarity factors sometimes accumulate at more than one site among the landmark-specified locations, and we suggest that competition between clusters of polarity factors determines the final location of the Cdc42 cluster. Modelling indicated that temporally constant landmark-localized Rsr1 would weaken or block competition, yielding more than one polarity site. Instead, we suggest that polarity factors recruit Rsr1, effectively sequestering it from other locations and thereby terminating landmark activity.

Authors
Wu, C-F; Savage, NS; Lew, DJ
MLA Citation
Wu, C-F, Savage, NS, and Lew, DJ. "Interaction between bud-site selection and polarity-establishment machineries in budding yeast. (Published online)" Philos Trans R Soc Lond B Biol Sci 368.1629 (2013): 20130006-.
PMID
24062579
Source
pubmed
Published In
Philosophical Transactions B
Volume
368
Issue
1629
Publish Date
2013
Start Page
20130006
DOI
10.1098/rstb.2013.0006

Beyond symmetry-breaking: Competition and negative feedback in GTPase regulation

Cortical domains are often specified by the local accumulation of active GTPases. Such domains can arise through spontaneous symmetry-breaking, suggesting that GTPase accumulation occurs via positive feedback. Here, we focus on recent advances in fungal and plant cell models - where new work suggests that polarity-controlling GTPases develop only one 'front' because GTPase clusters engage in a winner-takes-all competition. However, in some circumstances two or more GTPase domains can coexist, and the basis for the switch from competition to coexistence remains an open question. Polarity GTPases can undergo oscillatory clustering and dispersal, suggesting that these systems contain negative feedback. Negative feedback may prevent polarity clusters from spreading too far, regulate the balance between competition and coexistence, and provide directional flexibility for cells tracking gradients. © 2013 Elsevier Ltd.

Authors
Wu, CF; Lew, DJ
MLA Citation
Wu, CF, and Lew, DJ. "Beyond symmetry-breaking: Competition and negative feedback in GTPase regulation." Trends in Cell Biology 23.10 (2013): 476-483.
Source
scival
Published In
Trends in Cell Biology
Volume
23
Issue
10
Publish Date
2013
Start Page
476
End Page
483
DOI
10.1016/j.tcb.2013.05.003

Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p.

Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase-formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p-Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via "polarisome" components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.

Authors
Chen, H; Kuo, C-C; Kang, H; Howell, AS; Zyla, TR; Jin, M; Lew, DJ
MLA Citation
Chen, H, Kuo, C-C, Kang, H, Howell, AS, Zyla, TR, Jin, M, and Lew, DJ. "Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p." Mol Biol Cell 23.19 (October 2012): 3814-3826.
PMID
22918946
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
23
Issue
19
Publish Date
2012
Start Page
3814
End Page
3826
DOI
10.1091/mbc.E12-05-0400

Mechanistic mathematical model of polarity in yeast.

The establishment of cell polarity involves positive-feedback mechanisms that concentrate polarity regulators, including the conserved GTPase Cdc42p, at the "front" of the polarized cell. Previous studies in yeast suggested the presence of two parallel positive-feedback loops, one operating as a diffusion-based system, and the other involving actin-directed trafficking of Cdc42p on vesicles. F-actin (and hence directed vesicle traffic) speeds fluorescence recovery of Cdc42p after photobleaching, suggesting that vesicle traffic of Cdc42p contributes to polarization. We present a mathematical modeling framework that combines previously developed mechanistic reaction-diffusion and vesicle-trafficking models. Surprisingly, the combined model recapitulated the observed effect of vesicle traffic on Cdc42p dynamics even when the vesicles did not carry significant amounts of Cdc42p. Vesicle traffic reduced the concentration of Cdc42p at the front, so that fluorescence recovery mediated by Cdc42p flux from the cytoplasm took less time to replenish the bleached pool. Simulations in which Cdc42p was concentrated into vesicles or depleted from vesicles yielded almost identical predictions, because Cdc42p flux from the cytoplasm was dominant. These findings indicate that vesicle-mediated delivery of Cdc42p is not required to explain the observed Cdc42p dynamics, and raise the question of whether such Cdc42p traffic actually contributes to polarity establishment.

Authors
Savage, NS; Layton, AT; Lew, DJ
MLA Citation
Savage, NS, Layton, AT, and Lew, DJ. "Mechanistic mathematical model of polarity in yeast." Mol Biol Cell 23.10 (May 2012): 1998-2013.
PMID
22438587
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
23
Issue
10
Publish Date
2012
Start Page
1998
End Page
2013
DOI
10.1091/mbc.E11-10-0837

Negative feedback enhances robustness in the yeast polarity establishment circuit.

Many cells undergo symmetry-breaking polarization toward a randomly oriented "front" in the absence of spatial cues. In budding yeast, such polarization involves a positive feedback loop that enables amplification of stochastically arising clusters of polarity factors. Previous mathematical modeling suggested that, if more than one cluster were amplified, the clusters would compete for limiting resources and the largest would "win," explaining why yeast cells always make one and only one bud. Here, using imaging with improved spatiotemporal resolution, we show the transient coexistence of multiple clusters during polarity establishment, as predicted by the model. Unexpectedly, we also find that initial polarity factor clustering is oscillatory, revealing the presence of a negative feedback loop that disperses the factors. Mathematical modeling predicts that negative feedback would confer robustness to the polarity circuit and make the kinetics of competition between polarity factor clusters relatively insensitive to polarity factor concentration. These predictions are confirmed experimentally.

Authors
Howell, AS; Jin, M; Wu, C-F; Zyla, TR; Elston, TC; Lew, DJ
MLA Citation
Howell, AS, Jin, M, Wu, C-F, Zyla, TR, Elston, TC, and Lew, DJ. "Negative feedback enhances robustness in the yeast polarity establishment circuit." Cell 149.2 (April 13, 2012): 322-333.
PMID
22500799
Source
pubmed
Published In
Cell
Volume
149
Issue
2
Publish Date
2012
Start Page
322
End Page
333
DOI
10.1016/j.cell.2012.03.012

Morphogenesis and the cell cycle.

Studies of the processes leading to the construction of a bud and its separation from the mother cell in Saccharomyces cerevisiae have provided foundational paradigms for the mechanisms of polarity establishment, cytoskeletal organization, and cytokinesis. Here we review our current understanding of how these morphogenetic events occur and how they are controlled by the cell-cycle-regulatory cyclin-CDK system. In addition, defects in morphogenesis provide signals that feed back on the cyclin-CDK system, and we review what is known regarding regulation of cell-cycle progression in response to such defects, primarily acting through the kinase Swe1p. The bidirectional communication between morphogenesis and the cell cycle is crucial for successful proliferation, and its study has illuminated many elegant and often unexpected regulatory mechanisms. Despite considerable progress, however, many of the most puzzling mysteries in this field remain to be resolved.

Authors
Howell, AS; Lew, DJ
MLA Citation
Howell, AS, and Lew, DJ. "Morphogenesis and the cell cycle." Genetics 190.1 (January 2012): 51-77. (Review)
PMID
22219508
Source
pubmed
Published In
Genetics
Volume
190
Issue
1
Publish Date
2012
Start Page
51
End Page
77
DOI
10.1534/genetics.111.128314

The yeast guanine nucleotide dissociation inhibitor (GDI) enforces singularity by enhancing competition between polarity sites.

Authors
Wu, C-F; Lew, DJ
MLA Citation
Wu, C-F, and Lew, DJ. "The yeast guanine nucleotide dissociation inhibitor (GDI) enforces singularity by enhancing competition between polarity sites." MOLECULAR BIOLOGY OF THE CELL 23 (2012).
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
23
Publish Date
2012

Roles of hsl1p and Hsl7p in Swe1p degradation: Beyond septin tethering

The morphogenesis checkpoint in Saccharomyces cerevisiae couples bud formation to the cell division cycle by delaying nuclear division until cells have successfully constructed a bud. The cell cycle delay is due to the mitosis-inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. In unperturbed cells, Swe1p is degraded via a mechanism thought to involve its tethering to a cortical scaffold of septin proteins at the mother-bud neck. In cells that experience stresses that delay bud formation, Swe1p is stabilized, accumulates, and promotes a G2 delay. The tethering of Swe1p to the neck requires two regulators, called Hsl1p and Hsl7p. Hsl1p interacts with septins, and Hsl7p interacts with Swe1p; tethering occurs when Hsl1p interacts with Hsl7p. Here we created a version of Swe1p that is artificially tethered to the neck by fusion to a septin so that Swe1p no longer requires Hsl1p or Hsl7p for its localization to the neck. We show that the interaction between Hsl1p and Hsl7p, required for normal Swe1p degradation, is no longer needed for septin-Swe1p degradation, supporting the idea that the Hsl1p-Hsl7p interaction serves mainly to tether Swe1p to the neck. However, both Hsl1p and Hsl7p are still required for Swe1p degradation, implying that these proteins play additional roles beyond localizing Swe1p to the neck. © 2012, American Society for Microbiology. All Rights Reserved.

Authors
King, K; Jin, M; Lew, D
MLA Citation
King, K, Jin, M, and Lew, D. "Roles of hsl1p and Hsl7p in Swe1p degradation: Beyond septin tethering." Eukaryotic Cell 11.12 (2012): 1496-1502.
PMID
23042131
Source
scival
Published In
Eukaryotic cell
Volume
11
Issue
12
Publish Date
2012
Start Page
1496
End Page
1502
DOI
10.1128/EC.00196-12

Symmetry breaking and the establishment of cell polarity in budding yeast.

Cell polarity is typically oriented by external cues such as cell-cell contacts, chemoattractants, or morphogen gradients. In the absence of such cues, however, many cells can spontaneously polarize in a random direction, suggesting the existence of an internal polarity-generating mechanism whose direction can be spatially biased by external cues. Spontaneous 'symmetry-breaking' polarization is likely to involve an autocatalytic process set off by small random fluctuations. Here we review recent work on the nature of the autocatalytic process in budding yeast and on the question of why polarized cells only develop a single 'front'.

Authors
Johnson, JM; Jin, M; Lew, DJ
MLA Citation
Johnson, JM, Jin, M, and Lew, DJ. "Symmetry breaking and the establishment of cell polarity in budding yeast." Curr Opin Genet Dev 21.6 (December 2011): 740-746. (Review)
PMID
21955794
Source
pubmed
Published In
Current Opinion in Genetics and Development
Volume
21
Issue
6
Publish Date
2011
Start Page
740
End Page
746
DOI
10.1016/j.gde.2011.09.007

Dynamics of septin ring and collar formation in Saccharomyces cerevisiae.

Although the septin ring and collar in budding yeast were described over 20 years ago, there is still controversy regarding the organization of septin filaments within these structures and about the way in which the ring first forms and about how it converts into a collar at the mother-bud neck. Here we present quantitative analyses of the recruitment of fluorescently-tagged septins to the ring and collar through the cell cycle. Septin ring assembly began several minutes after polarity establishment and this interval was longer in daughter than in mother cells, suggesting asymmetric inheritance of septin regulators. Septins formed an initial faint and irregular ring, which became more regular as septins were recruited at a constant rate. This steady rate of septin recruitment continued for several minutes after the ring converted to a collar at bud emergence. We did not detect a stepwise change in septin fluorescence during the ring-to-collar transition. After collar formation, septins continued to accumulate at the bud neck, though at a reduced rate, until the onset of cytokinesis when the amount of neck-localized septins rapidly decreased. Implications for the mechanism of septin ring assembly are discussed.

Authors
Chen, H; Howell, AS; Robeson, A; Lew, DJ
MLA Citation
Chen, H, Howell, AS, Robeson, A, and Lew, DJ. "Dynamics of septin ring and collar formation in Saccharomyces cerevisiae." Biol Chem 392.8-9 (August 2011): 689-697.
PMID
21736496
Source
pubmed
Published In
Biological Chemistry
Volume
392
Issue
8-9
Publish Date
2011
Start Page
689
End Page
697
DOI
10.1515/BC.2011.075

Modeling vesicle traffic reveals unexpected consequences for Cdc42p-mediated polarity establishment.

BACKGROUND: Polarization in yeast has been proposed to involve a positive feedback loop whereby the polarity regulator Cdc42p orients actin cables, which deliver vesicles carrying Cdc42p to the polarization site. Previous mathematical models treating Cdc42p traffic as a membrane-free flux suggested that directed traffic would polarize Cdc42p, but it remained unclear whether Cdc42p would become polarized without the membrane-free simplifying assumption. RESULTS: We present mathematical models that explicitly consider stochastic vesicle traffic via exocytosis and endocytosis, providing several new insights. Our findings suggest that endocytic cargo influences the timing of vesicle internalization in yeast. Moreover, our models provide quantitative support for the view that integral membrane cargo proteins would become polarized by directed vesicle traffic given the experimentally determined rates of vesicle traffic and diffusion. However, such traffic cannot effectively polarize the more rapidly diffusing Cdc42p in the model without making additional assumptions that seem implausible and lack experimental support. CONCLUSIONS: Our findings suggest that actin-directed vesicle traffic would perturb, rather than reinforce, polarization in yeast.

Authors
Layton, AT; Savage, NS; Howell, AS; Carroll, SY; Drubin, DG; Lew, DJ
MLA Citation
Layton, AT, Savage, NS, Howell, AS, Carroll, SY, Drubin, DG, and Lew, DJ. "Modeling vesicle traffic reveals unexpected consequences for Cdc42p-mediated polarity establishment." Curr Biol 21.3 (February 8, 2011): 184-194.
PMID
21277209
Source
pubmed
Published In
Current Biology
Volume
21
Issue
3
Publish Date
2011
Start Page
184
End Page
194
DOI
10.1016/j.cub.2011.01.012

How does Cdc42p polarize actin cables?

Authors
Chen, H; Howell, AS; Kang, H; Lew, DJ
MLA Citation
Chen, H, Howell, AS, Kang, H, and Lew, DJ. "How does Cdc42p polarize actin cables?." 2011.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
22
Publish Date
2011

Singularity in polarization: rewiring yeast cells to make two buds.

For budding yeast to ensure formation of only one bud, cells must polarize toward one, and only one, site. Polarity establishment involves the Rho family GTPase Cdc42, which concentrates at polarization sites via a positive feedback loop. To assess whether singularity is linked to the specific Cdc42 feedback loop, we disabled the yeast cell's endogenous amplification mechanism and synthetically rewired the cells to employ a different positive feedback loop. Rewired cells violated singularity, occasionally making two buds. Even cells that made only one bud sometimes initiated two clusters of Cdc42, but then one cluster became dominant. Mathematical modeling indicated that, given sufficient time, competition between clusters would promote singularity. In rewired cells, competition occurred slowly and sometimes failed to develop a single "winning" cluster before budding. Slowing competition in normal cells also allowed occasional formation of two buds, suggesting that singularity is enforced by rapid competition between Cdc42 clusters.

Authors
Howell, AS; Savage, NS; Johnson, SA; Bose, I; Wagner, AW; Zyla, TR; Nijhout, HF; Reed, MC; Goryachev, AB; Lew, DJ
MLA Citation
Howell, AS, Savage, NS, Johnson, SA, Bose, I, Wagner, AW, Zyla, TR, Nijhout, HF, Reed, MC, Goryachev, AB, and Lew, DJ. "Singularity in polarization: rewiring yeast cells to make two buds." Cell 139.4 (November 13, 2009): 731-743.
PMID
19914166
Source
pubmed
Published In
Cell
Volume
139
Issue
4
Publish Date
2009
Start Page
731
End Page
743
DOI
10.1016/j.cell.2009.10.024

Molecular dissection of the checkpoint kinase Hsl1p.

Cell shape can influence cell behavior. In Saccharomyces cerevisiae, bud emergence can influence cell cycle progression via the morphogenesis checkpoint. This surveillance pathway ensures that mitosis always follows bud formation by linking degradation of the mitosis-inhibitory kinase Swe1p (Wee1) to successful bud emergence. A crucial component of this pathway is the checkpoint kinase Hsl1p, which is activated upon bud emergence and promotes Swe1p degradation. We have dissected the large nonkinase domain of Hsl1p by using evolutionary conservation as a guide, identifying regions important for Hsl1p localization, function, and regulation. An autoinhibitory motif restrains Hsl1p activity when it is not properly localized to the mother-bud neck. Hsl1p lacking this motif is active as a kinase regardless of the assembly state of cytoskeletal septin filaments. However, the active but delocalized Hsl1p cannot promote Swe1p down-regulation, indicating that localization is required for Hsl1p function as well as Hsl1p activation. We also show that the septin-mediated Hsl1p regulation via the novel motif operates in parallel to a previously identified Hsl1p activation pathway involving phosphorylation of the Hsl1p kinase domain. We suggest that Hsl1p responds to alterations in septin organization, which themselves occur in response to the local geometry of the cell cortex.

Authors
Crutchley, J; King, KM; Keaton, MA; Szkotnicki, L; Orlando, DA; Zyla, TR; Bardes, ESG; Lew, DJ
MLA Citation
Crutchley, J, King, KM, Keaton, MA, Szkotnicki, L, Orlando, DA, Zyla, TR, Bardes, ESG, and Lew, DJ. "Molecular dissection of the checkpoint kinase Hsl1p." Mol Biol Cell 20.7 (April 2009): 1926-1936.
PMID
19211841
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
20
Issue
7
Publish Date
2009
Start Page
1926
End Page
1936
DOI
10.1091/mbc.E08-08-0848

Response: GEF localization, not just activation, is needed for yeast polarity establishment

In this issue, Li and Wedlich-Soldner [1] offer an alternative interpretation of the previously reported synthetic lethality of the bem1Δ rsr1Δ double mutant and suggest an alternative conclusion from our recent experiments involving rescue of that lethality, published in Current Biology [2]. However, findings presented in our recent paper [2] as well as in our earlier work [3] argue against their interpretation. © 2009 Elsevier Ltd. All rights reserved.

Authors
Kozubowski, L; Saito, K; Johnson, JM; Howell, AS; Lew, DJ
MLA Citation
Kozubowski, L, Saito, K, Johnson, JM, Howell, AS, and Lew, DJ. "Response: GEF localization, not just activation, is needed for yeast polarity establishment." Current Biology 19.5 (2009): R195-.
Source
scival
Published In
Current Biology
Volume
19
Issue
5
Publish Date
2009
Start Page
R195
DOI
10.1016/j.cub.2009.01.022

Cell structure and dynamics

Authors
Lew, DJ; Rout, MP
MLA Citation
Lew, DJ, and Rout, MP. "Cell structure and dynamics." Current Opinion in Cell Biology 21.1 (2009): 1-3.
PMID
19185481
Source
scival
Published In
Current Opinion in Cell Biology
Volume
21
Issue
1
Publish Date
2009
Start Page
1
End Page
3
DOI
10.1016/j.ceb.2009.01.010

Symmetry-breaking polarization driven by a Cdc42p GEF-PAK complex.

BACKGROUND: In 1952, Alan Turing suggested that spatial patterns could arise from homogeneous starting conditions by feedback amplification of stochastic fluctuations. One example of such self-organization, called symmetry breaking, involves spontaneous cell polarization in the absence of spatial cues. The conserved GTPase Cdc42p is essential for both guided and spontaneous polarization, and in budding yeast cells Cdc42p concentrates at a single site (the presumptive bud site) at the cortex. Cdc42p concentrates at a random cortical site during symmetry breaking in a manner that requires the scaffold protein Bem1p. The mechanism whereby Bem1p promotes this polarization was unknown. RESULTS: Here we show that Bem1p promotes symmetry breaking by assembling a complex in which both a Cdc42p-directed guanine nucleotide exchange factor (GEF) and a Cdc42p effector p21-activated kinase (PAK) associate with Bem1p. Analysis of Bem1p mutants indicates that both GEF and PAK must bind to the same molecule of Bem1p, and a protein fusion linking the yeast GEF and PAK bypasses the need for Bem1p. Although mammalian cells lack a Bem1p ortholog, they contain more complex multidomain GEFs that in some cases can directly interact with PAKs, and we show that yeast containing an artificial GEF with similar architecture can break symmetry even without Bem1p. CONCLUSIONS: Yeast symmetry-breaking polarization involves a GEF-PAK complex that binds GTP-Cdc42p via the PAK and promotes local Cdc42p GTP-loading via the GEF. By generating fresh GTP-Cdc42p near pre-existing GTP-Cdc42p, the complex amplifies clusters of GTP-Cdc42p at the cortex. Our findings provide mechanistic insight into an evolutionarily conserved pattern-forming positive-feedback pathway.

Authors
Kozubowski, L; Saito, K; Johnson, JM; Howell, AS; Zyla, TR; Lew, DJ
MLA Citation
Kozubowski, L, Saito, K, Johnson, JM, Howell, AS, Zyla, TR, and Lew, DJ. "Symmetry-breaking polarization driven by a Cdc42p GEF-PAK complex." Curr Biol 18.22 (November 25, 2008): 1719-1726.
PMID
19013066
Source
pubmed
Published In
Current Biology
Volume
18
Issue
22
Publish Date
2008
Start Page
1719
End Page
1726
DOI
10.1016/j.cub.2008.09.060

The checkpoint kinase Hsl1p is activated by Elm1p-dependent phosphorylation.

Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.

Authors
Szkotnicki, L; Crutchley, JM; Zyla, TR; Bardes, ESG; Lew, DJ
MLA Citation
Szkotnicki, L, Crutchley, JM, Zyla, TR, Bardes, ESG, and Lew, DJ. "The checkpoint kinase Hsl1p is activated by Elm1p-dependent phosphorylation." Mol Biol Cell 19.11 (November 2008): 4675-4686.
PMID
18768748
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
19
Issue
11
Publish Date
2008
Start Page
4675
End Page
4686
DOI
10.1091/mbc.E08-06-0663

Nucleocytoplasmic trafficking of G2/M regulators in yeast.

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

Authors
Keaton, MA; Szkotnicki, L; Marquitz, AR; Harrison, J; Zyla, TR; Lew, DJ
MLA Citation
Keaton, MA, Szkotnicki, L, Marquitz, AR, Harrison, J, Zyla, TR, and Lew, DJ. "Nucleocytoplasmic trafficking of G2/M regulators in yeast." Mol Biol Cell 19.9 (September 2008): 4006-4018.
PMID
18562688
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
19
Issue
9
Publish Date
2008
Start Page
4006
End Page
4018
DOI
10.1091/mbc.E08-03-0286

The immortal strand hypothesis: how could it work?

Authors
Lew, DJ; Burke, DJ; Dutta, A
MLA Citation
Lew, DJ, Burke, DJ, and Dutta, A. "The immortal strand hypothesis: how could it work?." Cell 133.1 (April 4, 2008): 21-23. (Letter)
PMID
18394982
Source
pubmed
Published In
Cell
Volume
133
Issue
1
Publish Date
2008
Start Page
21
End Page
23
DOI
10.1016/j.cell.2008.03.016

IP7 guards the CDK gate.

Authors
York, JD; Lew, DJ
MLA Citation
York, JD, and Lew, DJ. "IP7 guards the CDK gate." Nat Chem Biol 4.1 (January 2008): 16-17.
PMID
18084274
Source
pubmed
Published In
Nature Chemical Biology
Volume
4
Issue
1
Publish Date
2008
Start Page
16
End Page
17
DOI
10.1038/nchembio0108-16

Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition.

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase-activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site.

Authors
Tong, Z; Gao, X-D; Howell, AS; Bose, I; Lew, DJ; Bi, E
MLA Citation
Tong, Z, Gao, X-D, Howell, AS, Bose, I, Lew, DJ, and Bi, E. "Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition." J Cell Biol 179.7 (December 31, 2007): 1375-1384.
PMID
18166650
Source
pubmed
Published In
The Journal of Cell Biology
Volume
179
Issue
7
Publish Date
2007
Start Page
1375
End Page
1384
DOI
10.1083/jcb.200705160

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation.

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.

Authors
Keaton, MA; Bardes, ESG; Marquitz, AR; Freel, CD; Zyla, TR; Rudolph, J; Lew, DJ
MLA Citation
Keaton, MA, Bardes, ESG, Marquitz, AR, Freel, CD, Zyla, TR, Rudolph, J, and Lew, DJ. "Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation." Curr Biol 17.14 (July 17, 2007): 1181-1189.
PMID
17614281
Source
pubmed
Published In
Current Biology
Volume
17
Issue
14
Publish Date
2007
Start Page
1181
End Page
1189
DOI
10.1016/j.cub.2007.05.075

Microtubule organization: cell shape is destiny.

A simple self-assembly pathway generates cytoplasmic microtubule bundles that can locate the cell center and guide spindle assembly in fission yeast. The cylindrical cell shape automatically corrects spindle orientation errors, rendering a checkpoint unnecessary.

Authors
Haase, SB; Lew, DJ
MLA Citation
Haase, SB, and Lew, DJ. "Microtubule organization: cell shape is destiny." Curr Biol 17.7 (April 3, 2007): R249-R251.
PMID
17407755
Source
pubmed
Published In
Current Biology
Volume
17
Issue
7
Publish Date
2007
Start Page
R249
End Page
R251
DOI
10.1016/j.cub.2007.02.003

Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint.

The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial.

Authors
Keaton, MA; Lew, DJ
MLA Citation
Keaton, MA, and Lew, DJ. "Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint." Curr Opin Microbiol 9.6 (December 2006): 540-546. (Review)
PMID
17055334
Source
pubmed
Published In
Current Opinion in Microbiology
Volume
9
Issue
6
Publish Date
2006
Start Page
540
End Page
546
DOI
10.1016/j.mib.2006.10.004

Swe1p responds to cytoskeletal perturbation, not bud size, in S. cerevisiae.

BACKGROUND: S. cerevisiae cells must grow to a critical size in G1 in order to pass start and enter the cell cycle. A recent study proposed that in addition to the mother size control in G1, the bud must grow to a critical bud size in G2 in order to enter mitosis. Insufficient bud size would cause G2 arrest enforced by the mitotic inhibitor Swe1p, explaining previous findings that some perturbations that block bud growth also trigger Swe1p-dependent cell-cycle arrest. RESULTS: We tested the critical-bud-size hypothesis. We found that halting bud growth by inactivation of the myosin Myo2p did not trigger Swe1p-dependent arrest in budded cells, even when the buds were very small. Moreover, Swe1p did not affect cell-cycle progression in unstressed cells, even when bud size was decreased by overriding G1 size control. Actin depolymerization did cause Swe1p-dependent arrest in small-budded but not large-budded cells, as previously reported. However, we found that the key determinant of cell-cycle arrest in those circumstances was not bud size, but rather the relative abundance of the Swe1p mitotic inhibitor and the mitosis-promoting cyclins. CONCLUSIONS: Swe1p does not respond to insufficient bud size. Instead, actin stress empowers Swe1p to promote arrest. The effectiveness of Swe1p in promoting that arrest declines as cells progress through the cell cycle.

Authors
McNulty, JJ; Lew, DJ
MLA Citation
McNulty, JJ, and Lew, DJ. "Swe1p responds to cytoskeletal perturbation, not bud size, in S. cerevisiae." Curr Biol 15.24 (December 20, 2005): 2190-2198.
PMID
16360682
Source
pubmed
Published In
Current Biology
Volume
15
Issue
24
Publish Date
2005
Start Page
2190
End Page
2198
DOI
10.1016/j.cub.2005.11.039

Yeast polarity: negative feedback shifts the focus.

A new study of Cdc42p polarization in yeast suggests that the actin cytoskeleton can destabilize the polarity axis, causing Cdc42p foci to wander aimlessly around the cell cortex.

Authors
Lew, DJ
MLA Citation
Lew, DJ. "Yeast polarity: negative feedback shifts the focus." Curr Biol 15.24 (December 20, 2005): R994-R996. (Review)
PMID
16360678
Source
pubmed
Published In
Current Biology
Volume
15
Issue
24
Publish Date
2005
Start Page
R994
End Page
R996
DOI
10.1016/j.cub.2005.11.048

Interplay between septin organization, cell cycle and cell shape in yeast.

Septins are conserved filament-forming proteins that assemble into cortical cytoskeletal structures in animal and fungal cells. Although rapid progress has been made into the functions of septins, the mechanisms governing their localization and organization remain mysterious. In Saccharomyces cerevisiae, Cdc42p organizes the septin cytoskeleton into a ring in preparation for bud formation, following which septins remain as a collar at the mother-bud neck. We have dissected the phenotype of cdc42(V36T,K94E) cells that display an aberrant cell shape correlated with the development of ectopic septin caps and rings within the bud. The results suggest that a well-assembled septin cortex plays a novel role in directing growth to shape the nascent bud, and that a disorganized septin cortex directs improper growth generating an aberrant neck. Conversely, we found that the elongated bud shape arising as a result of the morphogenesis checkpoint cell cycle delay that accompanies septin perturbation can feed back to exacerbate minor defects in septin organization, by maintaining a bud-tip-localized septin assembly activity that competes with the neck-localized septin cortex. Using this exacerbation as a tool, we uncovered septin organization defects in many mutants not previously known to display such defects, expanding the cast of characters involved in proper assembly of the septin cortex to include CLN1, CLN2, BNI1, BNI4, BUD3, BUD4 and BUD5.

Authors
Gladfelter, AS; Kozubowski, L; Zyla, TR; Lew, DJ
MLA Citation
Gladfelter, AS, Kozubowski, L, Zyla, TR, and Lew, DJ. "Interplay between septin organization, cell cycle and cell shape in yeast." J Cell Sci 118.Pt 8 (April 15, 2005): 1617-1628.
PMID
15784684
Source
pubmed
Published In
Journal of cell science
Volume
118
Issue
Pt 8
Publish Date
2005
Start Page
1617
End Page
1628
DOI
10.1242/jcs.02286

Opposing roles for actin in Cdc42p polarization.

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.

Authors
Irazoqui, JE; Howell, AS; Theesfeld, CL; Lew, DJ
MLA Citation
Irazoqui, JE, Howell, AS, Theesfeld, CL, and Lew, DJ. "Opposing roles for actin in Cdc42p polarization." Mol Biol Cell 16.3 (March 2005): 1296-1304.
PMID
15616194
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
16
Issue
3
Publish Date
2005
Start Page
1296
End Page
1304
DOI
10.1091/mbc.E04-05-0430

Opposing roles for actin in Cdc42p polarization

Authors
Howell, AS; Irazoqui, JE; Theesfeld, CL; Lew, DJ
MLA Citation
Howell, AS, Irazoqui, JE, Theesfeld, CL, and Lew, DJ. "Opposing roles for actin in Cdc42p polarization." November 2004.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
15
Publish Date
2004
Start Page
335A
End Page
335A

Bud size doesnt matter

Authors
McNulty, JJ; Lew, DJ
MLA Citation
McNulty, JJ, and Lew, DJ. "Bud size doesnt matter." November 2004.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
15
Publish Date
2004
Start Page
20A
End Page
20A

Exploring the basis for the asymmetry of septin-dependent proteins at the mother-bud neck in Saccharomyces cerevisiae

Authors
Kozubowski, L; Larson, JR; Lew, DJ; Tatchell, K
MLA Citation
Kozubowski, L, Larson, JR, Lew, DJ, and Tatchell, K. "Exploring the basis for the asymmetry of septin-dependent proteins at the mother-bud neck in Saccharomyces cerevisiae." November 2004.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
15
Publish Date
2004
Start Page
46A
End Page
46A

Genetic interactions among regulators of septin organization.

Septins form a cortical scaffold at the yeast mother-bud neck that restricts the diffusion of cortical proteins between the mother and bud and serves as a signaling center that is important for governing various cell functions. After cell cycle commitment in late G(1), septins are assembled into a narrow ring at the future bud site, which spreads to form a mature septin hourglass immediately after bud emergence. Although several septin regulators have been identified, it is unclear how they cooperate to assemble the septin scaffold. We have examined septin localization in isogenic strains containing single or multiple mutations in eight septin organization genes (CDC42, RGA1, RGA2, BEM3, CLA4, GIN4, NAP1, and ELM1). Our results suggest that these regulators act largely in parallel to promote either the initial assembly of the septin ring (CDC42, RGA1, RGA2, BEM3, and CLA4) or the conversion of the ring to a stable hourglass structure at the neck (GIN4, NAP1, and ELM1). Aberrant septin localization patterns in mutant strains could be divided into apparently discrete categories, but individual strains displayed heterogeneous defects, and there was no clear-cut correspondence between the specific mutations and specific categories of defect. These findings suggest that when they are deprived of their normal regulators, septin scaffolds collapse into a limited repertoire of aberrant states in which the nature of the mutant regulators influences the probability of a given aberrant state.

Authors
Gladfelter, AS; Zyla, TR; Lew, DJ
MLA Citation
Gladfelter, AS, Zyla, TR, and Lew, DJ. "Genetic interactions among regulators of septin organization." Eukaryot Cell 3.4 (August 2004): 847-854.
PMID
15302817
Source
pubmed
Published In
Eukaryotic cell
Volume
3
Issue
4
Publish Date
2004
Start Page
847
End Page
854
DOI
10.1128/EC.3.4.847-854.2004

Cdc42p, GTP hydrolysis, and the cell's sense of direction.

The GTPase Cdc42p is essential for polarity establishment in animals and fungi.(1) Human Cdc42p can functionally replace yeast Cdc42p,(2) indicating a high degree of evolutionary conservation. Current models of Cdc42p action generally follow the signaling paradigm established for Ras, in which receptors responding to an initiating stimulus cause guanine nucleotide exchange factors (GEFs) to trigger GTP-loading of Ras, leading to engagement of downstream effectors and ensuing cell proliferation. Key support for the Ras paradigm came from the finding that oncogenic forms of Ras, unable to hydrolyze GTP and therefore constitutively GTP-bound, mimicked the effect of constitutive signaling by the upstream receptors even in the absence of stimuli. Attempts to assess whether or not this paradigm is valid for Cdc42p-induced polarization of yeast cells have yielded conflicting results.(3-6) Here, we discuss the available information on this issue and conclude that unlike Ras signaling, Cdc42p directed polarity establishment additionally requires cycling between GTP- and GDP-bound forms. We suggest that such cycling is critical for a little-studied "function" of Cdc42p: its ability to designate a unique portion of the cell cortex to become the polarization site, and to become concentrated at that site.

Authors
Irazoqui, JE; Gladfelter, AS; Lew, DJ
MLA Citation
Irazoqui, JE, Gladfelter, AS, and Lew, DJ. "Cdc42p, GTP hydrolysis, and the cell's sense of direction." Cell Cycle 3.7 (July 2004): 861-864. (Review)
PMID
15190213
Source
pubmed
Published In
Cell Cycle
Volume
3
Issue
7
Publish Date
2004
Start Page
861
End Page
864

Polarity establishment in yeast.

Authors
Irazoqui, JE; Lew, DJ
MLA Citation
Irazoqui, JE, and Lew, DJ. "Polarity establishment in yeast." J Cell Sci 117.Pt 11 (May 1, 2004): 2169-2171. (Review)
PMID
15126618
Source
pubmed
Published In
Journal of cell science
Volume
117
Issue
Pt 11
Publish Date
2004
Start Page
2169
End Page
2171
DOI
10.1242/jcs.00953

Stress-specific activation mechanisms for the "cell integrity" MAPK pathway.

Many environmental stresses trigger cellular responses by activating mitogen-activated protein kinase (MAPK) pathways. Once activated, these highly conserved protein kinase cascades can elicit cellular responses such as transcriptional activation of response genes, cytoskeletal rearrangement, and cell cycle arrest. The mechanism of pathway activation by environmental stresses is in most cases unknown. We have analyzed the activation of the budding yeast "cell integrity" MAPK pathway by heat shock, hypoosmotic shock, and actin perturbation, and we report that different stresses regulate this pathway at different steps. In no case can MAPK activation be explained by the prevailing view that stresses simply induce GTP loading of the Rho1p GTPase at the "top" of the pathway. Instead, our findings suggest that the stresses can modulate at least three distinct kinases acting between Rho1p and the MAPK. These findings suggest that stresses provide "lateral" inputs into this regulatory pathway, rather than operating in a linear "top-down" manner.

Authors
Harrison, JC; Zyla, TR; Bardes, ESG; Lew, DJ
MLA Citation
Harrison, JC, Zyla, TR, Bardes, ESG, and Lew, DJ. "Stress-specific activation mechanisms for the "cell integrity" MAPK pathway." J Biol Chem 279.4 (January 23, 2004): 2616-2622.
PMID
14610085
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
4
Publish Date
2004
Start Page
2616
End Page
2622
DOI
10.1074/jbc.M306110200

Scaffold-mediated symmetry breaking by Cdc42p.

Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking. In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally. The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization. We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments. Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour. Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch.

Authors
Irazoqui, JE; Gladfelter, AS; Lew, DJ
MLA Citation
Irazoqui, JE, Gladfelter, AS, and Lew, DJ. "Scaffold-mediated symmetry breaking by Cdc42p." Nat Cell Biol 5.12 (December 2003): 1062-1070.
PMID
14625559
Source
pubmed
Published In
Nature Cell Biology
Volume
5
Issue
12
Publish Date
2003
Start Page
1062
End Page
1070
DOI
10.1038/ncb1068

The morphogenesis checkpoint: how yeast cells watch their figures.

The morphogenesis checkpoint maintains coordination between the process of bud formation and the nuclear events of the cell cycle in yeast. This checkpoint regulates the Wee1 homolog, Swe1p, to induce cell-cycle delay or arrest when aspects of bud formation are defective. A variety of studies have suggested that this checkpoint can monitor actin organization, septin organization, the presence of a bud and even the size of a bud. The evidence for these proposals is reviewed, highlighting recent findings indicating that Swe1p degradation is controlled by the cell shape change that accompanies bud emergence.

Authors
Lew, DJ
MLA Citation
Lew, DJ. "The morphogenesis checkpoint: how yeast cells watch their figures." Curr Opin Cell Biol 15.6 (December 2003): 648-653. (Review)
PMID
14644188
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
15
Issue
6
Publish Date
2003
Start Page
648
End Page
653

A monitor for bud emergence in the yeast morphogenesis checkpoint.

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.

Authors
Theesfeld, CL; Zyla, TR; Bardes, EGS; Lew, DJ
MLA Citation
Theesfeld, CL, Zyla, TR, Bardes, EGS, and Lew, DJ. "A monitor for bud emergence in the yeast morphogenesis checkpoint." Mol Biol Cell 14.8 (August 2003): 3280-3291.
PMID
12925763
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
14
Issue
8
Publish Date
2003
Start Page
3280
End Page
3291
DOI
10.1091/mbc.E03-03-0154

The role of F-actin in Cdc42p polarization.

Authors
Irazoqui, JE; Lew, DJ
MLA Citation
Irazoqui, JE, and Lew, DJ. "The role of F-actin in Cdc42p polarization." July 2003.
Source
wos-lite
Published In
Yeast
Volume
20
Publish Date
2003
Start Page
S67
End Page
S67

The spindle assembly and spindle position checkpoints.

The mitotic spindle segregates chromosomes to opposite ends of the cell in preparation for cell division. Chromosome attachment to the spindle is monitored by the spindle assembly checkpoint, and at least in yeast cells, penetration of one spindle pole into the bud is monitored by the spindle position checkpoint. We review the historical origins of these checkpoints and recent progress in understanding their surveillance pathways. We also highlight fascinating but as yet unresolved questions, and examine crosstalk between the checkpoints.

Authors
Lew, DJ; Burke, DJ
MLA Citation
Lew, DJ, and Burke, DJ. "The spindle assembly and spindle position checkpoints." Annu Rev Genet 37 (2003): 251-282. (Review)
PMID
14616062
Source
pubmed
Published In
Annual Review of Genetics
Volume
37
Publish Date
2003
Start Page
251
End Page
282
DOI
10.1146/annurev.genet.37.042203.120656

Stress-specific activation mechanisms for the "cell integrity" MAPK pathway in Saccharomyces cerevisiae

Authors
Harrison, JC; Bardes, EG; Zyla, TR; Lew, DJ
MLA Citation
Harrison, JC, Bardes, EG, Zyla, TR, and Lew, DJ. "Stress-specific activation mechanisms for the "cell integrity" MAPK pathway in Saccharomyces cerevisiae." November 2002.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
13
Publish Date
2002
Start Page
294A
End Page
294A

Determinants of Swe1p degradation in Saccharomyces cerevisiae.

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.

Authors
McMillan, JN; Theesfeld, CL; Harrison, JC; Bardes, ESG; Lew, DJ
MLA Citation
McMillan, JN, Theesfeld, CL, Harrison, JC, Bardes, ESG, and Lew, DJ. "Determinants of Swe1p degradation in Saccharomyces cerevisiae." Mol Biol Cell 13.10 (October 2002): 3560-3575.
PMID
12388757
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
13
Issue
10
Publish Date
2002
Start Page
3560
End Page
3575
DOI
10.1091/mbc.E02-05-0283

The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint.

The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p, and we now show that it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p required GAP activity, the checkpoint role of Bem2p did not. Instead, this function required an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurred normally in bem2 cells, but they were nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint.

Authors
Marquitz, AR; Harrison, JC; Bose, I; Zyla, TR; McMillan, JN; Lew, DJ
MLA Citation
Marquitz, AR, Harrison, JC, Bose, I, Zyla, TR, McMillan, JN, and Lew, DJ. "The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint." EMBO J 21.15 (August 1, 2002): 4012-4025.
PMID
12145202
Source
pubmed
Published In
EMBO Journal
Volume
21
Issue
15
Publish Date
2002
Start Page
4012
End Page
4025
DOI
10.1093/emboj/cdf416

Formin' actin filament bundles.

Authors
Lew, DJ
MLA Citation
Lew, DJ. "Formin' actin filament bundles." Nat Cell Biol 4.2 (February 2002): E29-E30.
PMID
11835051
Source
pubmed
Published In
Nature Cell Biology
Volume
4
Issue
2
Publish Date
2002
Start Page
E29
End Page
E30
DOI
10.1038/ncb0202-e29

Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p.

At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

Authors
Gladfelter, AS; Bose, I; Zyla, TR; Bardes, ESG; Lew, DJ
MLA Citation
Gladfelter, AS, Bose, I, Zyla, TR, Bardes, ESG, and Lew, DJ. "Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p." J Cell Biol 156.2 (January 21, 2002): 315-326.
PMID
11807094
Source
pubmed
Published In
The Journal of Cell Biology
Volume
156
Issue
2
Publish Date
2002
Start Page
315
End Page
326
DOI
10.1083/jcb.200109062

The septin cortex at the yeast mother-bud neck.

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.

Authors
Gladfelter, AS; Pringle, JR; Lew, DJ
MLA Citation
Gladfelter, AS, Pringle, JR, and Lew, DJ. "The septin cortex at the yeast mother-bud neck." Curr Opin Microbiol 4.6 (December 2001): 681-689. (Review)
PMID
11731320
Source
pubmed
Published In
Current Opinion in Microbiology
Volume
4
Issue
6
Publish Date
2001
Start Page
681
End Page
689

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Authors
Adamo, JE; Moskow, JJ; Gladfelter, AS; Viterbo, D; Lew, DJ; Brennwald, PJ
MLA Citation
Adamo, JE, Moskow, JJ, Gladfelter, AS, Viterbo, D, Lew, DJ, and Brennwald, PJ. "Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud." J Cell Biol 155.4 (November 12, 2001): 581-592.
PMID
11706050
Source
pubmed
Published In
The Journal of Cell Biology
Volume
155
Issue
4
Publish Date
2001
Start Page
581
End Page
592
DOI
10.1083/jcb.200106065

The Rho GAP Bem2p plays a role in the morphogenesis checkpoint of Saccharomyces cerevisiae

Authors
Marquitz, AR; Harrison, JC; Theesfeld, CL; Bardes, ESG; Lew, DJ
MLA Citation
Marquitz, AR, Harrison, JC, Theesfeld, CL, Bardes, ESG, and Lew, DJ. "The Rho GAP Bem2p plays a role in the morphogenesis checkpoint of Saccharomyces cerevisiae." MOLECULAR BIOLOGY OF THE CELL 12 (November 2001): 408A-408A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
12
Publish Date
2001
Start Page
408A
End Page
408A

Septin-dependent monitoring of local cell shape in the yeast morphogenesis checkpoint

Authors
Theesfeld, CL; Lew, DJ
MLA Citation
Theesfeld, CL, and Lew, DJ. "Septin-dependent monitoring of local cell shape in the yeast morphogenesis checkpoint." MOLECULAR BIOLOGY OF THE CELL 12 (November 2001): 142A-143A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
12
Publish Date
2001
Start Page
142A
End Page
143A

Hsl1 as a sensor of cell shape.

Authors
Theesfeld, CL; Bardes, E; Lew, DJ
MLA Citation
Theesfeld, CL, Bardes, E, and Lew, DJ. "Hsl1 as a sensor of cell shape." YEAST 18 (August 2001): S142-S142.
Source
wos-lite
Published In
Yeast
Volume
18
Publish Date
2001
Start Page
S142
End Page
S142

Isolation and characterization of effector-loop mutants of CDC42 in yeast.

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

Authors
Gladfelter, AS; Moskow, JJ; Zyla, TR; Lew, DJ
MLA Citation
Gladfelter, AS, Moskow, JJ, Zyla, TR, and Lew, DJ. "Isolation and characterization of effector-loop mutants of CDC42 in yeast." Mol Biol Cell 12.5 (May 2001): 1239-1255.
PMID
11359919
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
12
Issue
5
Publish Date
2001
Start Page
1239
End Page
1255

A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint.

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.

Authors
Harrison, JC; Bardes, ES; Ohya, Y; Lew, DJ
MLA Citation
Harrison, JC, Bardes, ES, Ohya, Y, and Lew, DJ. "A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint." Nat Cell Biol 3.4 (April 2001): 417-420.
PMID
11283616
Source
pubmed
Published In
Nature Cell Biology
Volume
3
Issue
4
Publish Date
2001
Start Page
417
End Page
420
DOI
10.1038/35070104

Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p.

In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.

Authors
Bose, I; Irazoqui, JE; Moskow, JJ; Bardes, ES; Zyla, TR; Lew, DJ
MLA Citation
Bose, I, Irazoqui, JE, Moskow, JJ, Bardes, ES, Zyla, TR, and Lew, DJ. "Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p." J Biol Chem 276.10 (March 9, 2001): 7176-7186.
PMID
11113154
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
10
Publish Date
2001
Start Page
7176
End Page
7186
DOI
10.1074/jbc.M010546200

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Authors
Adamo, JE; Moskow, JJ; Gladfelter, AS; Viterbo, D; Lew, DJ; Brennwald, PJ
MLA Citation
Adamo, JE, Moskow, JJ, Gladfelter, AS, Viterbo, D, Lew, DJ, and Brennwald, PJ. "Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud." Journal of Cell Biology 155.3 (2001): 581-592.
Source
scival
Published In
The Journal of Cell Biology
Volume
155
Issue
3
Publish Date
2001
Start Page
581
End Page
592
DOI
10.1083/jcb.200106065

Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants.

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.

Authors
Yeh, E; Yang, C; Chin, E; Maddox, P; Salmon, ED; Lew, DJ; Bloom, K
MLA Citation
Yeh, E, Yang, C, Chin, E, Maddox, P, Salmon, ED, Lew, DJ, and Bloom, K. "Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants." Mol Biol Cell 11.11 (November 2000): 3949-3961.
PMID
11071919
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
11
Issue
11
Publish Date
2000
Start Page
3949
End Page
3961

Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae.

CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.

Authors
Moskow, JJ; Gladfelter, AS; Lamson, RE; Pryciak, PM; Lew, DJ
MLA Citation
Moskow, JJ, Gladfelter, AS, Lamson, RE, Pryciak, PM, and Lew, DJ. "Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae." Mol Cell Biol 20.20 (October 2000): 7559-7571.
PMID
11003652
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
20
Publish Date
2000
Start Page
7559
End Page
7571

Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae.

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).

Authors
Longtine, MS; Theesfeld, CL; McMillan, JN; Weaver, E; Pringle, JR; Lew, DJ
MLA Citation
Longtine, MS, Theesfeld, CL, McMillan, JN, Weaver, E, Pringle, JR, and Lew, DJ. "Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae." Mol Cell Biol 20.11 (June 2000): 4049-4061.
PMID
10805747
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
11
Publish Date
2000
Start Page
4049
End Page
4061

Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae.

Cytoskeletal organization is crucial for several aspects of cell-cycle progression but cytoskeletal elements are quite sensitive to environmental perturbations. Two novel checkpoint controls monitor the function of the actin and microtubule systems in budding yeast and operate to delay cell-cycle progression in response to cytoskeletal perturbations. In cells whose actin cytoskeleton has been perturbed, bud formation is frequently delayed and the morphogenesis checkpoint introduces a compensatory delay of nuclear division until a bud has been formed. In cells whose microtubule cytoskeleton has been perturbed, anaphase spindle elongation often occurs entirely within the mother cell, and the post-anaphase nuclear migration checkpoint introduces a compensatory delay of cytokinesis until one pole of the anaphase nucleus enters the bud. Recent studies indicate that regulators of entry into mitosis are localized to the daughter side of the mother-bud neck whereas regulators of exit from mitosis are localized to the spindle pole bodies. Thus, specific cell-cycle regulators are well-placed to monitor whether a cell has formed a bud and whether a daughter nucleus has been delivered accurately to the bud following mitosis.

Authors
Lew, DJ
MLA Citation
Lew, DJ. "Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae." Curr Opin Genet Dev 10.1 (February 2000): 47-53. (Review)
PMID
10679396
Source
pubmed
Published In
Current Opinion in Genetics & Development
Volume
10
Issue
1
Publish Date
2000
Start Page
47
End Page
53

Seroprevalence of HTLV-I in Cheju Island, a Korean island adjacent to the endemic area of Japan.

Authors
Kim, JM; Chang, KH; Choi, YH; Song, YG; Kang, SM; Yoon, TY; Choi, JM; Park, SY; Lew, DJ
MLA Citation
Kim, JM, Chang, KH, Choi, YH, Song, YG, Kang, SM, Yoon, TY, Choi, JM, Park, SY, and Lew, DJ. "Seroprevalence of HTLV-I in Cheju Island, a Korean island adjacent to the endemic area of Japan." J Acquir Immune Defic Syndr 22.4 (December 1, 1999): 409-412. (Letter)
PMID
10634207
Source
pubmed
Published In
Journal of Acquired Immune Deficiency Syndromes
Volume
22
Issue
4
Publish Date
1999
Start Page
409
End Page
412

Analysis of cdc42effector loop mutants in budding yeast

Authors
Gladfelter, AS; Moskow, JJ; Lew, DJ
MLA Citation
Gladfelter, AS, Moskow, JJ, and Lew, DJ. "Analysis of cdc42effector loop mutants in budding yeast." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 54A-54A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
54A
End Page
54A

Cell cycle and checkpoint control of Swe1p localization and degradation.

Authors
Lew, DJ
MLA Citation
Lew, DJ. "Cell cycle and checkpoint control of Swe1p localization and degradation." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 3A-3A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
3A
End Page
3A

The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p.

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.

Authors
McMillan, JN; Longtine, MS; Sia, RA; Theesfeld, CL; Bardes, ES; Pringle, JR; Lew, DJ
MLA Citation
McMillan, JN, Longtine, MS, Sia, RA, Theesfeld, CL, Bardes, ES, Pringle, JR, and Lew, DJ. "The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p." Mol Cell Biol 19.10 (October 1999): 6929-6939.
PMID
10490630
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
10
Publish Date
1999
Start Page
6929
End Page
6939

The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

Authors
Theesfeld, CL; Irazoqui, JE; Bloom, K; Lew, DJ
MLA Citation
Theesfeld, CL, Irazoqui, JE, Bloom, K, and Lew, DJ. "The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle." J Cell Biol 146.5 (September 6, 1999): 1019-1032.
PMID
10477756
Source
pubmed
Published In
The Journal of Cell Biology
Volume
146
Issue
5
Publish Date
1999
Start Page
1019
End Page
1032

Phosphorylation-independent inhibition of Cdc28p by the tyrosine kinase Swe1p in the morphogenesis checkpoint.

The morphogenesis checkpoint in budding yeast delays cell cycle progression in G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G(2) arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28(Y19F) allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28(E12K) allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28(Y19F) allele suggested that Swe1p is still able to inhibit CDC28(Y19F) in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms.

Authors
McMillan, JN; Sia, RA; Bardes, ES; Lew, DJ
MLA Citation
McMillan, JN, Sia, RA, Bardes, ES, and Lew, DJ. "Phosphorylation-independent inhibition of Cdc28p by the tyrosine kinase Swe1p in the morphogenesis checkpoint." Mol Cell Biol 19.9 (September 1999): 5981-5990.
PMID
10454545
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
9
Publish Date
1999
Start Page
5981
End Page
5990

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.

Authors
Sia, RA; Bardes, ES; Lew, DJ
MLA Citation
Sia, RA, Bardes, ES, and Lew, DJ. "Control of Swe1p degradation by the morphogenesis checkpoint." EMBO J 17.22 (November 16, 1998): 6678-6688.
PMID
9822611
Source
pubmed
Published In
EMBO Journal
Volume
17
Issue
22
Publish Date
1998
Start Page
6678
End Page
6688
DOI
10.1093/emboj/17.22.6678

A morphogenesis checkpoint monitors the actin cytoskeleton in yeast.

A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I. Reed. 1995. J. Cell Biol. 129:739- 749). The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Mol. Biol. Cell. 7:1657- 1666). In this report, we have investigated the nature of the defect(s) that trigger this checkpoint. A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact. Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation. In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle. However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.

Authors
McMillan, JN; Sia, RA; Lew, DJ
MLA Citation
McMillan, JN, Sia, RA, and Lew, DJ. "A morphogenesis checkpoint monitors the actin cytoskeleton in yeast." J Cell Biol 142.6 (September 21, 1998): 1487-1499.
PMID
9744879
Source
pubmed
Published In
The Journal of Cell Biology
Volume
142
Issue
6
Publish Date
1998
Start Page
1487
End Page
1499

Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis.

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother-bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother-bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Delta strains divide nearly as efficiently as wild type; other myo1Delta strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell-wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

Authors
Bi, E; Maddox, P; Lew, DJ; Salmon, ED; McMillan, JN; Yeh, E; Pringle, JR
MLA Citation
Bi, E, Maddox, P, Lew, DJ, Salmon, ED, McMillan, JN, Yeh, E, and Pringle, JR. "Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis." J Cell Biol 142.5 (September 7, 1998): 1301-1312.
PMID
9732290
Source
pubmed
Published In
The Journal of Cell Biology
Volume
142
Issue
5
Publish Date
1998
Start Page
1301
End Page
1312

Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1.

Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein. MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30 complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity.

Authors
Kaiser, P; Sia, RA; Bardes, EG; Lew, DJ; Reed, SI
MLA Citation
Kaiser, P, Sia, RA, Bardes, EG, Lew, DJ, and Reed, SI. "Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1." Genes Dev 12.16 (August 15, 1998): 2587-2597.
PMID
9716410
Source
pubmed
Published In
Genes & development
Volume
12
Issue
16
Publish Date
1998
Start Page
2587
End Page
2597

Flow cytometric analysis of DNA content in budding yeast.

Authors
Haase, SB; Lew, DJ
MLA Citation
Haase, SB, and Lew, DJ. "Flow cytometric analysis of DNA content in budding yeast." Methods Enzymol 283 (1997): 322-332.
PMID
9251030
Source
pubmed
Published In
Methods in Enzymology
Volume
283
Publish Date
1997
Start Page
322
End Page
332

Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control.

Cyclins and cyclin-dependent kinases (Cdks) are universal regulators of cell cycle progression in eukaryotic cells. Cdk activity is controlled by phosphorylation at three conserved sites, and many of the enzymes that act on these sites have now been identified. Although the biochemistry of Cdk phosphorylation is relatively well understood, the regulatory roles of such phosphorylation are, in many cases, obscure. Recent studies have uncovered new and unexpected potential roles, and prompted re-examination of previously assumed roles, of Cdk phosphorylation.

Authors
Lew, DJ; Kornbluth, S
MLA Citation
Lew, DJ, and Kornbluth, S. "Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control." Curr Opin Cell Biol 8.6 (December 1996): 795-804. (Review)
PMID
8939679
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
8
Issue
6
Publish Date
1996
Start Page
795
End Page
804

Genetic screen to uncover proteins involved in the morphogenesis cell cycle checkpoint in budding yeast.

Authors
McMillan, JN; Lew, DJ
MLA Citation
McMillan, JN, and Lew, DJ. "Genetic screen to uncover proteins involved in the morphogenesis cell cycle checkpoint in budding yeast." MOLECULAR BIOLOGY OF THE CELL 7 (December 1996): 2077-2077.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
7
Publish Date
1996
Start Page
2077
End Page
2077

Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast.

A morphogenesis checkpoint in budding yeast delays nuclear division (and subsequent cell cycle progression) in cells that have failed to make a bud. We show that the ability of this checkpoint to delay nuclear division requires the SWE1 gene, encoding a protein kinase that inhibits the master cell cycle regulatory kinase Cdc28. The timing of nuclear division in cells that cannot make a bud is exquisitely sensitive to the dosage of SWE1 and MIH1 genes, which control phosphorylation of Cdc28 at tyrosine 19. In contrast, the timing of nuclear division in budded cells does not rely on Cdc28 phosphorylation, suggesting that the morphogenesis checkpoint somehow turns on this regulatory pathway. We show that SWE1 mRNA levels fluctuate during the cell cycle and are elevated in cells that cannot make a bud. However, regulation of SWE1 mRNA levels by the checkpoint is indirect, acting through a feedback loop requiring Swe1 activity. Further, the checkpoint is capable of delaying nuclear division even when SWE1 transcription is deregulated. We propose that the checkpoint delays nuclear division through post-translational regulation of Swe1 and that transcriptional feedback loops enhance the efficacy of the checkpoint.

Authors
Sia, RA; Herald, HA; Lew, DJ
MLA Citation
Sia, RA, Herald, HA, and Lew, DJ. "Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast." Mol Biol Cell 7.11 (November 1996): 1657-1666.
PMID
8930890
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
7
Issue
11
Publish Date
1996
Start Page
1657
End Page
1666

A cell cycle checkpoint monitors cell morphogenesis in budding yeast.

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.

Authors
Lew, DJ; Reed, SI
MLA Citation
Lew, DJ, and Reed, SI. "A cell cycle checkpoint monitors cell morphogenesis in budding yeast." J Cell Biol 129.3 (May 1995): 739-749.
PMID
7730408
Source
pubmed
Published In
The Journal of Cell Biology
Volume
129
Issue
3
Publish Date
1995
Start Page
739
End Page
749

Phase-sensitive measurements of vortex dynamics in the terahertz domain.

Authors
Parks, B; Spielman, S; Orenstein, J; Nemeth, DT; Ludwig, F; Clarke, J; Merchant, P; Lew, DJ
MLA Citation
Parks, B, Spielman, S, Orenstein, J, Nemeth, DT, Ludwig, F, Clarke, J, Merchant, P, and Lew, DJ. "Phase-sensitive measurements of vortex dynamics in the terahertz domain." Phys Rev Lett 74.16 (April 17, 1995): 3265-3268.
PMID
10058153
Source
pubmed
Published In
Physical Review Letters
Volume
74
Issue
16
Publish Date
1995
Start Page
3265
End Page
3268
DOI
10.1103/PhysRevLett.74.3265

Cell cycle control of morphogenesis in budding yeast.

A detailed description of the cytoskeletal rearrangements that orchestrate bud formation is beginning to emerge from studies on yeast morphogenesis. In this review, we focus on recent advances in our understanding of how the timing of these rearrangements is controlled. Dramatic changes in cell polarity that occur in G1 (polarization to the bud site), G2 (depolarization within the bud), and mitosis (repolarization to the mother/bud neck) are triggered by changes in the kinase activity of Cdc28, the universal regulator of cell cycle progression. The hunt for Cdc28 morphogenesis substrates is on.

Authors
Lew, DJ; Reed, SI
MLA Citation
Lew, DJ, and Reed, SI. "Cell cycle control of morphogenesis in budding yeast." Curr Opin Genet Dev 5.1 (February 1995): 17-23. (Review)
PMID
7749320
Source
pubmed
Published In
Current Opinion in Genetics & Development
Volume
5
Issue
1
Publish Date
1995
Start Page
17
End Page
23

Observation of the quasiparticle Hall effect in superconducting YBa2Cu3O7- delta.

Authors
Spielman, S; Parks, B; Orenstein, J; Nemeth, DT; Ludwig, F; Clarke, J; Merchant, P; Lew, DJ
MLA Citation
Spielman, S, Parks, B, Orenstein, J, Nemeth, DT, Ludwig, F, Clarke, J, Merchant, P, and Lew, DJ. "Observation of the quasiparticle Hall effect in superconducting YBa2Cu3O7- delta." Phys Rev Lett 73.11 (September 12, 1994): 1537-1540.
PMID
10056818
Source
pubmed
Published In
Physical Review Letters
Volume
73
Issue
11
Publish Date
1994
Start Page
1537
End Page
1540
DOI
10.1103/PhysRevLett.73.1537

Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation.

Authors
Stueland, CS; Lew, DJ; Cismowski, MJ; Reed, SI
MLA Citation
Stueland, CS, Lew, DJ, Cismowski, MJ, and Reed, SI. "Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae." Mol Cell Biol 13.6 (June 1993): 3744-3755.
PMID
8388545
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
13
Issue
6
Publish Date
1993
Start Page
3744
End Page
3755

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins.

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.

Authors
Lew, DJ; Reed, SI
MLA Citation
Lew, DJ, and Reed, SI. "Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins." J Cell Biol 120.6 (March 1993): 1305-1320.
PMID
8449978
Source
pubmed
Published In
The Journal of Cell Biology
Volume
120
Issue
6
Publish Date
1993
Start Page
1305
End Page
1320

A suppressor of cln3 for size control

Authors
Lew, DJ; Marini, NJ; Reed, SI
MLA Citation
Lew, DJ, Marini, NJ, and Reed, SI. "A suppressor of cln3 for size control." Cell 72.4 (1993): 488-489.
PMID
8440017
Source
scival
Published In
Cell
Volume
72
Issue
4
Publish Date
1993
Start Page
488
End Page
489
DOI
10.1016/0092-8674(93)90068-2

Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2.

We have cloned four cyclin-B homologs from Saccharomyces cerevisiae, CLB1-CLB4, using the polymerase chain reaction and low stringency hybridization approaches. These genes form two classes based on sequence relatedness: CLB1 and CLB2 show highest homology to the Schizosaccharomyces pombe cyclin-B homolog cdc13 involved in the initiation of mitosis, whereas CLB3 and CLB4 are more highly related to the S. pombe cyclin-B homolog cig1, which appears to play a role in G1 or S phase. CLB1 and CLB2 mRNA levels peak late in the cell cycle, whereas CLB3 and CLB4 are expressed earlier in the cell cycle but peak later than the G1-specific cyclin, CLN1. Analysis of null mutations suggested that the CLB genes exhibit some degree of redundancy, but clb1,2 and clb2,3 cells were inviable. Using clb1,2,3,4 cells rescued by conditional overproduction of CLB1, we showed that the CLB genes perform an essential role at the G2/M-phase transition, and also a role in S phase. CLB genes also appear to share a role in the assembly and maintenance of the mitotic spindle. Taken together, these analyses suggest that CLB1 and CLB2 are crucial for mitotic induction, whereas CLB3 and CLB4 might participate additionally in DNA replication and spindle assembly.

Authors
Richardson, H; Lew, DJ; Henze, M; Sugimoto, K; Reed, SI
MLA Citation
Richardson, H, Lew, DJ, Henze, M, Sugimoto, K, and Reed, SI. "Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2." Genes Dev 6.11 (November 1992): 2021-2034.
PMID
1427070
Source
pubmed
Published In
Genes & development
Volume
6
Issue
11
Publish Date
1992
Start Page
2021
End Page
2034

A proliferation of cyclins.

Cyclins are regulatory subunits of the serine/threonine protein kinases that play key roles in cell cycle control. The roster of known cyclins has expanded significantly in the past year, revealing a large and very diverse family of proteins. Although cyclins were originally characterized by their periodic accumulation during interphase and destruction in mitosis (these were the 'mitotic' cyclins that control entry into mitosis), the newly identified cyclins do not conform to this pattern. Here we review what is known about the functions of the nonmitotic cyclins in yeast and in mammalian cells.

Authors
Lew, DJ; I Reed, S
MLA Citation
Lew, DJ, and I Reed, S. "A proliferation of cyclins." Trends Cell Biol 2.3 (March 1992): 77-81.
PMID
14731948
Source
pubmed
Published In
Trends in Cell Biology
Volume
2
Issue
3
Publish Date
1992
Start Page
77
End Page
81

G1 control in yeast and animal cells.

In budding yeast, Saccharomyces cerevisiae, the cell cycle is controlled at the G1/S phase transition by regulating the activity of the CDC28 protein kinase. This is the budding yeast homologue of the cdc2 protein kinase associated in most organisms with control of mitosis. In budding yeast CDC28 controls both the G1/S phase transition and the G2/M phase transition by being differentially activated by two distinct classes of positive regulatory subunits known as G1 cyclins or CLNs and B-type cyclins or CLBs, respectively. To establish whether a similar dual role for Cdc2-related kinases exists in animal cells, we and others have sought human homologues of yeast G1 cyclins. Of several candidates, cyclin E is the most promising in that it accumulates prior to S phase and is associated with a pre-S phase protein kinase activity. The kinetics of accumulation of cyclin E-associated protein kinase activity is consistent with a role at the mammalian cell cycle restriction point.

Authors
Reed, SI; Dulic, V; Lew, DJ; Richardson, HE; Wittenberg, C
MLA Citation
Reed, SI, Dulic, V, Lew, DJ, Richardson, HE, and Wittenberg, C. "G1 control in yeast and animal cells." Ciba Found Symp 170 (1992): 7-15. (Review)
PMID
1483351
Source
pubmed
Published In
Ciba Foundation symposium
Volume
170
Publish Date
1992
Start Page
7
End Page
15

Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration

Cycling and cyclin-associated cdc kinases are key regulators of oocyte maturation (Mailer, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr ∼32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.

Authors
Lu, XP; Koch, KS; Lew, DJ; Dulic, V; Pines, J; Reed, SI; Hunter, T; Leffert, HL
MLA Citation
Lu, XP, Koch, KS, Lew, DJ, Dulic, V, Pines, J, Reed, SI, Hunter, T, and Leffert, HL. "Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration." Journal of Biological Chemistry 267.5 (1992): 2841-2844.
PMID
1310673
Source
scival
Published In
The Journal of biological chemistry
Volume
267
Issue
5
Publish Date
1992
Start Page
2841
End Page
2844

Different G1 cyclins control the timing of cell cycle commitment in mother and daughter cells of the budding yeast S. cerevisiae

Growth of S. cerevisiae cells by budding gives rise to asymmetric progeny cells: a larger "mother" cell and a smaller "daughter" cell. The mother cell transits a brief G1 phase before forming a new bud and beginning DNA replication. The daughter cell stays in G1 for a longer period, growing in size before initiating a new cell cycle. We show that the timing of cell cycle initiation in mother and daughter cells is governed by different G1 cyclins. In daughter cells, transcription of CLN1 and CLN2 is induced in a size-dependent manner, and these cyclins are necessary for the normal timing of cell cycle initiation. CLN3 is not required in daughter cells, but is crucial for mother cells, in which the G1 phase is much longer in the absence of this cyclin. Copyright © 1992 by Cell Press.

Authors
Lew, DJ; Marini, NJ; Reed, SI
MLA Citation
Lew, DJ, Marini, NJ, and Reed, SI. "Different G1 cyclins control the timing of cell cycle commitment in mother and daughter cells of the budding yeast S. cerevisiae." Cell 69.2 (1992): 317-327.
PMID
1533176
Source
scival
Published In
Cell
Volume
69
Issue
2
Publish Date
1992
Start Page
317
End Page
327
DOI
10.1016/0092-8674(92)90412-6

Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene.

The promoter of the gene encoding a cytoplasmic guanylate-binding protein (GBP) contains two overlapping elements: the interferon stimulation response element (ISRE), which mediates alpha interferon (IFN-alpha)-dependent transcription, and the IFN-gamma activation site (GAS), which is required for IFN-gamma-mediated stimulation. The ISRE binds a factor called ISGF-3 that is activated by IFN-alpha but not by IFN-gamma. The GAS binds a protein that is activated by IFN-gamma, which we have termed GAF (IFN-gamma activation factor; T. Decker, D. J. Lew, J. Mirkovitch, and J. E. Darnell, Jr., EMBO J., in press; D. J. Lew, T. Decker, I. Strehlow, and J. E. Darnell, Jr., Mol. Cell. Biol. 11:182-191, 1991). We now find that the GAS is also an IFN-alpha-responsive element in vivo and that IFN-alpha (in addition to activating ISGF-3) rapidly activates a GAS-binding factor, the IFN-alpha activation factor (AAF). The AAF has characteristics very similar to those of the previously described GAF. Through the use of inhibitors of protein synthesis and inhibitors of protein kinases, the activation conditions of AAF, GAF, and ISGF-3 could be distinguished. Therefore, not only do IFN-alpha and IFN-gamma stimulate transcription of GBP through different receptors linked to different signaling molecules, but occupation of the IFN-alpha receptor apparently leads to the rapid activation of two different DNA-binding proteins through the use of different intracellular pathways.

Authors
Decker, T; Lew, DJ; Darnell, JE
MLA Citation
Decker, T, Lew, DJ, and Darnell, JE. "Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene." Mol Cell Biol 11.10 (October 1991): 5147-5153.
PMID
1833631
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
11
Issue
10
Publish Date
1991
Start Page
5147
End Page
5153

Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast.

We have isolated a number of cDNAs derived from human mRNAs that are able to substitute for G1 cyclin genes in S. cerevisiae. Several of these encode human cyclins A, B1, and B2. Three novel genes have been identified, which we call cyclins C, D, and E. The novel proteins are sufficiently distantly related to the other members of the cyclin family and to each other as to constitute three new classes of cyclins. Cyclin C and E mRNAs accumulate periodically through the cell cycle, peaking at different times in G1.

Authors
Lew, DJ; Dulić, V; Reed, SI
MLA Citation
Lew, DJ, Dulić, V, and Reed, SI. "Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast." Cell 66.6 (September 20, 1991): 1197-1206.
PMID
1833066
Source
pubmed
Published In
Cell
Volume
66
Issue
6
Publish Date
1991
Start Page
1197
End Page
1206

Characterization of constitutive exocytosis in the yeast Saccharomyces cerevisiae.

Constitutive exocytosis was investigated in the yeast Saccharomyces cerevisiae using temperature-sensitive mutant (sec) strains which do not allow vesicle fusion to the plasma membrane at the restrictive temperature. Secretory vesicles were accumulated in the cell at the restrictive temperature and then protein synthesis was blocked with cycloheximide. Upon returning the cells to the permissive temperature the contents of the accumulated vesicles were secreted. This allowed the study of constitutive exocytosis independent of the processes responsible for vesicular biosynthesis. Neither the kinetics nor magnitude of exocytosis were affected by removal of external Ca2+ or perturbations of cytosolic Ca2+. This suggests that in those systems where calcium is required for exocytosis it is a regulatory molecule and not part of the mechanism of membrane fusion. Release occurred over a very broad range of pH and in media with different ionic compositions, suggesting that ionic and potential gradients across the plasma membrane play no role in exocytosis in yeast. High osmolarity inhibited the rate, but not the extent, of release. A novel inhibitory effect of azide was detected which occurred only at low pH. Vanadate also inhibited release in a pH-independent manner. Secretion occurred at the same rate in cells with or without accumulated vesicles. This infers a rate-limiting step following vesicle accumulation, perhaps a limiting number of release sites on the plasma membrane.

Authors
Lew, DJ; Simon, SM
MLA Citation
Lew, DJ, and Simon, SM. "Characterization of constitutive exocytosis in the yeast Saccharomyces cerevisiae." J Membr Biol 123.3 (September 1991): 261-268.
PMID
1744905
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
123
Issue
3
Publish Date
1991
Start Page
261
End Page
268

A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

Authors
Ghiara, JB; Richardson, HE; Sugimoto, K; Henze, M; Lew, DJ; Wittenberg, C; Reed, SI
MLA Citation
Ghiara, JB, Richardson, HE, Sugimoto, K, Henze, M, Lew, DJ, Wittenberg, C, and Reed, SI. "A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis." Cell 65.1 (April 5, 1991): 163-174.
PMID
1849458
Source
pubmed
Published In
Cell
Volume
65
Issue
1
Publish Date
1991
Start Page
163
End Page
174

Cytoplasmic activation of GAF, an IFN-gamma-regulated DNA-binding factor.

We have investigated events following treatment of cells with interferon-gamma (IFN-gamma) that lead to the immediate transcriptional activation of an inducible gene. A gamma-interferon activation factor (GAF) was activated in the cytoplasm of human fibroblasts immediately after IFN-gamma treatment and bound to a newly identified target DNA sequence, the gamma-interferon activation site (GAS). The time course of activation of GAF was different in fibroblasts and HeLa cells and correlated well with IFN-gamma-induced transcriptional activation in both cell types. IFN-gamma-dependent activation of GAF also occurred in enucleated cells (cytoplasts), showing that an inactive cytoplasmic precursor is converted to the active factor. These findings support the concept that ligand-specific signals originating at the cell surface are transmitted through latent cytoplasmic proteins which are activated to bind specific DNA sites and then move to the nucleus to activate the transcription of specific sets of genes.

Authors
Decker, T; Lew, DJ; Mirkovitch, J; Darnell, JE
MLA Citation
Decker, T, Lew, DJ, Mirkovitch, J, and Darnell, JE. "Cytoplasmic activation of GAF, an IFN-gamma-regulated DNA-binding factor." EMBO J 10.4 (April 1991): 927-932.
PMID
1901265
Source
pubmed
Published In
EMBO Journal
Volume
10
Issue
4
Publish Date
1991
Start Page
927
End Page
932

Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons.

The gene encoding a 67-kDa cytoplasmic guanylate-binding protein (GBP) is transcriptionally induced in cells exposed to interferon of either type I (alpha interferon [IFN-alpha] or type II (IFN-gamma). The promoter of the GBP gene was cloned and found to contain an IFN-alpha-stimulated response element, which mediated the response of the GBP gene to IFN-alpha. On the basis of transfection experiments with recombinant plasmids, two different elements were delineated. Both were required to obtain the maximal response of the GBP gene to IFN-gamma: the IFN-alpha-stimulated response element and an overlapping element termed the IFN-gamma activation site. Different proteins that act on each element were investigated, and their possible involvement in IFN-gamma-induced transcriptional regulation is discussed.

Authors
Lew, DJ; Decker, T; Strehlow, I; Darnell, JE
MLA Citation
Lew, DJ, Decker, T, Strehlow, I, and Darnell, JE. "Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons." Mol Cell Biol 11.1 (January 1991): 182-191.
PMID
1898761
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
11
Issue
1
Publish Date
1991
Start Page
182
End Page
191

G1 control in yeast and animal cells.

In yeast G1, cyclins control the Cdc28 protein kinase in order to regulate the primary cell cycle gating event known as START. Environmental and internal signals that control the cell cycle do so, apparently, by controlling the synthesis and/or stability of G1 cyclins, hence controlling the activity of the Cdc28 kinase. The substrates of the Cdc28 kinase that are critical for passage through START are not known. One simple hypothesis is that the G1 kinase phosphorylates and thus activates a transcription factor required for the initiation of S phase. The synthesis of an origin of replication-binding factor might be regulated in this fashion. Recent evidence suggests that Cdc28 protein kinase activity directly regulates the transcription of a family of genes whose products are required for DNA replication (N. Marini and S. Reed, in prep.). However, it is not yet known whether this transcriptional activation constitutes the execution of START. The situation in animal cells is more complex. A number of new cyclins and p34s have been identified. It is not clear yet which of these, if any, have functions in G1 and if they do, what functions these might be. If G1 cyclins and p34 kinases do have critical G1 roles, by analogy with yeast, they may couple signals mediated by both positive and negative growth factors to cell cycle progression. Candidates for the critical G1 substrates of these putative G1 protein kinases are the tumor suppressors such as the RB (retinoblastoma) gene product (p105RB).(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Reed, SI; Wittenberg, C; Lew, DJ; Dulic, V; Henze, M
MLA Citation
Reed, SI, Wittenberg, C, Lew, DJ, Dulic, V, and Henze, M. "G1 control in yeast and animal cells." Cold Spring Harb Symp Quant Biol 56 (1991): 61-67.
PMID
1840266
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
56
Publish Date
1991
Start Page
61
End Page
67

G1 control in yeast and animal cells

In yeast G1, cyclins control the Cdc28 protein kinase in order to regulate the primary cell cycle gating event known as START. Environmental and internal signals that control the cell cycle do so, apparently, by controlling the synthesis and/or stability of G1 cyclins, hence controlling the activity of the Cdc28 kinase. The substrates of the Cdc28 kinase that are critical for passage through START are not known. One simple hypothesis is that the G1 kinase phosphorylates and thus activates a transcription factor required for the initiation of S phase. The synthesis of an origin of replication-binding factor might be regulated in this fashion. Recent evidence suggests that Cdc28 protein kinase activity directly regulates the transcription of a family of genes whose products are required for DNA replication (N. Marini and S. Reed, in prep.). However, it is not yet known whether this transcriptional activation constitutes the execution of START. The situation in animal cells is more complex. A number of new cyclins and p34s have been identified. It is not clear yet which of these, if any, have functions in G1 and if they do, what functions these might be. If G1 cyclins and p34 kinases do have critical G1 roles, by analogy with yeast, they may couple signals mediated by both positive and negative growth factors to cell cycle progression. Candidates for the critical G1 substrates of these putative G1 protein kinases are the tumor suppressors such as the RB (retinoblastoma) gene product (p105(RB)). It has been proposed that phosphorylation of p105(RB) is associated with loss of its inhibitory effects on cell cycle progression (Buchkovich et al. 1989; Chen et al. 1989; Ludlow et al. 1990). Furthermore, it has been demonstrated that cdc2 in its mitotic form phosphorylates p105(RB). Yet the critical phosphorylation events relevant to cell cycle progression must occur before the mitotic kinase is assembled. Hence, G1 kinases likely to be associated with cyclins C and E may be responsible for regulating p105(RB). The discovery of these novel cyclins clearly opens up new avenues of investigation into G1 control in animal cells.

Authors
Reed, SI; Wittenberg, C; Lew, DJ; Dulic, V; Henze, M
MLA Citation
Reed, SI, Wittenberg, C, Lew, DJ, Dulic, V, and Henze, M. "G1 control in yeast and animal cells." Cold Spring Harbor Symposia on Quantitative Biology 56 (1991): 61-68.
Source
scival
Published In
Cold Spring Harbor Symposia on Quantitative Biology
Volume
56
Publish Date
1991
Start Page
61
End Page
68

Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3.

Interferon-alpha (IFN alpha) and interferon-gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell-surface receptors to induce expression of largely non-overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10-fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis-acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10-fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Levy, DE; Lew, DJ; Decker, T; Kessler, DS; Darnell, JE
MLA Citation
Levy, DE, Lew, DJ, Decker, T, Kessler, DS, and Darnell, JE. "Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3." EMBO J 9.4 (April 1990): 1105-1111.
PMID
2108862
Source
pubmed
Published In
EMBO Journal
Volume
9
Issue
4
Publish Date
1990
Start Page
1105
End Page
1111

Alpha interferon and gamma interferon stimulate transcription of a single gene through different signal transduction pathways.

Interferons (IFNs) play a key role in the defense against virus infection and the regulation of cell growth and differentiation, in part through changes in specific gene transcription in target cells. We describe several differences between the signal transduction events that result in transcriptional activation of the human gene coding for a guanylate-binding protein (GBP) by alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma). Activation by IFN-alpha was rapid, transient, and cycloheximide resistant. Activation by IFN-gamma was slower, sustained, and delayed by cycloheximide. IFN-gamma led to the formation of a stable intracellular signal which led to continued GBP transcription even if the ligand was withdrawn, whereas IFN-alpha-induced GBP transcription decayed rapidly if IFN-alpha was withdrawn. Perturbations of signaling pathways involving classical second messengers (cyclic AMP, Ca2+, protein kinase C) did not induce GBP transcription. However, various kinase inhibitors blocked the transcriptional response to IFN-gamma but not IFN-alpha, suggesting that a specific and possibly novel kinase is involved in gene activation by IFN-gamma.

Authors
Lew, DJ; Decker, T; Darnell, JE
MLA Citation
Lew, DJ, Decker, T, and Darnell, JE. "Alpha interferon and gamma interferon stimulate transcription of a single gene through different signal transduction pathways." Mol Cell Biol 9.12 (December 1989): 5404-5411.
PMID
2555698
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
9
Issue
12
Publish Date
1989
Start Page
5404
End Page
5411

Interactions of alpha- and gamma-interferon in the transcriptional regulation of the gene encoding a guanylate-binding protein.

Transcriptional regulation of the gene encoding a guanylate-binding protein (GBP) by the two interferon (IFN) types was studied. GBP gene transcription was regulated by alpha IFN in a manner identical to that of previously described IFN-stimulated genes (ISGs): rapid induction, without a need for protein synthesis, followed by a protein synthesis-dependent suppression of transcription to basal levels within 6 h. Transcriptional induction by gamma IFN was equally rapid and independent of ongoing protein synthesis but remained at elevated levels for greater than 24 h. Experiments employing combined treatments with IFNs of both types revealed that induction of the GBP gene by gamma IFN overrides the alpha IFN-induced active repression and reverses the alpha IFN-induced repressed state. Moreover, the alpha IFN-mediated repression of ISG54, a gene normally responsive to only alpha IFN, is also reversed by gamma IFN. Induction of GBP by gamma IFN is presumably mediated by a factor different from the recently described activator Interferon Stimulated Gene Factor 3 (ISGF3) because induction of this factor was not observed upon treatment of cells with gamma IFN. Finally, a complex set of reinforcing or synergistic effects were observed when induction of the GBP gene was evoked by a combined treatment with the two IFN types.

Authors
Decker, T; Lew, DJ; Cheng, YS; Levy, DE; Darnell, JE
MLA Citation
Decker, T, Lew, DJ, Cheng, YS, Levy, DE, and Darnell, JE. "Interactions of alpha- and gamma-interferon in the transcriptional regulation of the gene encoding a guanylate-binding protein." EMBO J 8.7 (July 1989): 2009-2014.
PMID
2507314
Source
pubmed
Published In
EMBO Journal
Volume
8
Issue
7
Publish Date
1989
Start Page
2009
End Page
2014

Dehydration and delayed proton equilibria of red blood cells suspended in isosmotic phosphate buffers. Implications for studies of sickled cells.

PO4 buffers isosmotic with plasma or phosphate-buffered saline solution with a substantial proportion of PO4 are often used to wash and suspend red blood cells in studies of respiratory or sickling behavior. Measurements of sequential changes in mean cell hemoglobin concentration, pH, and ion content of red blood cells suspended in 295 mOsm Na-phosphate, pH 7.4, at 23 degrees or 37 degrees C, showed (1) rapid, persistent cell dehydration (mean cell hemoglobin concentration greater than 40 gm/dl) caused initially by Cl- efflux and later by replacement of monovalent Cl- by divalent HPO=4; and (2) temporary reversal of membrane pH gradients with normalization time (30 to 120 minutes) dependent on factors controlling the rate of phosphate-chloride exchange. Sequential equilibration of red blood cells in isosmotic citrate (impermeable) followed by PO4 demonstrated the two stages of the observed shifts in PO4 alone, and red blood cells suspended in 0.15 mol/L 32PO4 at 37 degrees C showed PO4 influx consistent with pH equilibrium kinetics. Sickle trait red blood cells deoxygenated at 37 degrees C, pH 7.4, in plasma or 10 mmol/L HEPES-buffered saline solution showed only 6% to 20% sickling. In isosmotic PO4, mean cell hemoglobin concentration was 40 to 41 gm/dl with approximately 80% sickling. In phosphate-buffered saline solution containing 70 mmol/L PO4, red blood cells showed smaller, similar changes (mean cell hemoglobin concentration approximately 38 gm/dl) with a longer equilibration period and deoxygenated sickle trait cells showed 40% sickling. The altered properties of red blood cells suspended in PO4 or phosphate-buffered saline solution were neither intended nor appropriate for many studies using these media, particularly with hemoglobin S-containing red blood cells, and interpretations of reported results must be reassessed in light of these findings.

Authors
Bookchin, RM; Lew, DJ; Balazs, T; Ueda, Y; Lew, VL
MLA Citation
Bookchin, RM, Lew, DJ, Balazs, T, Ueda, Y, and Lew, VL. "Dehydration and delayed proton equilibria of red blood cells suspended in isosmotic phosphate buffers. Implications for studies of sickled cells." J Lab Clin Med 104.6 (December 1984): 855-866.
PMID
6094692
Source
pubmed
Published In
Journal of Laboratory and Clinical Medicine
Volume
104
Issue
6
Publish Date
1984
Start Page
855
End Page
866
Show More

Research Areas:

  • Actin Cytoskeleton
  • Actins
  • Actomyosin
  • Adaptor Proteins, Signal Transducing
  • Alleles
  • Amino Acid Sequence
  • Binding Sites
  • CDC28 Protein Kinase, S cerevisiae
  • Cell Cycle
  • Cell Division
  • Cell Nucleus
  • Cell Polarity
  • Cell Shape
  • Cell Wall
  • Chemotaxis
  • Chromosome Segregation
  • Computer Simulation
  • Cullin Proteins
  • Cyclin B
  • Cyclin-Dependent Kinase Inhibitor Proteins
  • Cyclin-Dependent Kinases
  • Cyclins
  • Cycloheximide
  • Cytochalasin B
  • Cytokinesis
  • Cytoplasm
  • Cytoskeleton
  • DNA Mutational Analysis
  • Drug Synergism
  • Enhancer Elements, Genetic
  • Exodeoxyribonucleases
  • F-Box Proteins
  • Feedback, Physiological
  • Flow Cytometry
  • Fluorescence Recovery After Photobleaching
  • G1 Phase
  • G2 Phase
  • GTP-Binding Proteins
  • GTPase-Activating Proteins
  • Gene Deletion
  • Gene Expression Regulation, Fungal
  • Genes
  • Genes, Fungal
  • Genes, cdc
  • Glutathione Transferase
  • Guanine Nucleotide Exchange Factors
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • Hot Temperature
  • Humans
  • Hydrolases
  • Image Processing, Computer-Assisted
  • Interferon Type I
  • Interferon-gamma
  • Kinetochores
  • MAP Kinase Signaling System
  • Maturation-Promoting Factor
  • Metaphase
  • Microfilament Proteins
  • Microscopy, Confocal
  • Microtubules
  • Mitogen-Activated Protein Kinases
  • Mitosis
  • Molecular Sequence Data
  • Morphogenesis
  • Multigene Family
  • Myosin Heavy Chains
  • Myosins
  • Nucleosome Assembly Protein 1
  • Organelles
  • Pheromones
  • Phosphoprotein Phosphatases
  • Phosphoric Monoester Hydrolases
  • Phosphothreonine
  • Phosphotyrosine
  • Polarity
  • Polymerization
  • Precipitin Tests
  • Profilins
  • Promoter Regions, Genetic
  • Protein Kinase C
  • Protein Kinases
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Protein Transport
  • Protein Tyrosine Phosphatases
  • Protein-Arginine N-Methyltransferases
  • Protein-Serine-Threonine Kinases
  • Protein-Tyrosine Kinases
  • Proteolysis
  • Proto-Oncogene Proteins
  • Quorum Sensing
  • RNA, Fungal
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Regulatory Sequences, Nucleic Acid
  • Repressor Proteins
  • Reproduction, Asexual
  • S Phase
  • S-Phase Kinase-Associated Proteins
  • SNARE Proteins
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Saccharomycetales
  • Schizosaccharomyces pombe Proteins
  • Septins
  • Sequence Deletion
  • Signal Transduction
  • Stress, Mechanical
  • Suppression, Genetic
  • Systems Biology
  • Thiazolidines
  • Time-Lapse Imaging
  • Transcription, Genetic
  • Transport Vesicles
  • Tyrosine
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligase Complexes
  • Ubiquitin-Protein Ligases
  • Yeast
  • Yeasts
  • cdc42 GTP-Binding Protein
  • cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
  • p21-Activated Kinases
  • ras-GRF1
  • rho GTP-Binding Proteins