scholarly journals Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division

2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Hannah E. Opalko ◽  
Kristi E. Miller ◽  
Hyun-Soo Kim ◽  
Cesar Augusto Vargas-Garcia ◽  
Abhyudai Singh ◽  
...  
Keyword(s):  
Surface Area ◽  
Control Cell ◽  
Size Control ◽  
Bound State ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Node Localization ◽  
Nucleotide Exchange Factor ◽  
Proper Size

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

scholarly journals Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division

2021 ◽  
Author(s):  
Hannah E. Opalko ◽  
Kristi E. Miller ◽  
Hyun-Soo Kim ◽  
Cesar Augusto Vargas-Garcia ◽  
Abhyudai Singh ◽  
...  
Keyword(s):  
Surface Area ◽  
Control Cell ◽  
Size Control ◽  
Bound State ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Node Localization ◽  
Nucleotide Exchange Factor ◽  
Proper Size

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its GEF (guanine nucleotide exchange factor) Syt22 was required for both Arf6 node localization and proper size at division. In arf6∆ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6∆ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

scholarly journals A role for Gic1 and Gic2 in Cdc42 polarization

2018 ◽  
Author(s):  
Christine N. Daniels ◽  
Trevin R. Zyla ◽  
Daniel J. Lew
Keyword(s):  
Feedback Loop ◽  
Cell Types ◽  
Effector Proteins ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rho Family ◽  
Cytoskeletal Elements

AbstractThe conserved Rho-family GTPase Cdc42 is a master regulator of polarity establishment in many cell types. Cdc42 becomes activated and concentrated in a region of the cell cortex, and recruits a variety of effector proteins to that site. In turn, many effectors participate in regulation of cytoskeletal elements in order to remodel the cytoskeleton in a polarized manner. The budding yeast Saccharomyces cerevisiae has served as a tractable model system for studies of cell polarity. In yeast cells, Cdc42 polarization involves a positive feedback loop in which effectors called p21-activated kinases (PAKs) act to recruit a Cdc42-directed guanine nucleotide exchange factor (GEF), generating more GTP-Cdc42 in areas that already have GTP-Cdc42. The GTPase-interacting components (GICs) Gic1 and Gic2 are also Cdc42 effectors, and have been implicated in regulation of the actin and septin cytoskeleton. However, we report that cells lacking GICs are primarily defective in polarizing Cdc42 itself, suggesting that they act upstream as well as downstream of Cdc42 in yeast. Our findings suggest that feedback pathways involving GTPase effectors may be more prevalent than had been appreciated.

scholarly journals Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

2009 ◽  
Vol 29 (12) ◽  
pp. 3297-3306 ◽  
Author(s):  
Adam Mor ◽  
Joseph P. Wynne ◽  
Ian M. Ahearn ◽  
Michael L. Dustin ◽  
Guangwei Du ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Bound State ◽  
Small Gtpase ◽  
Resting Cells ◽  
Nucleotide Exchange ◽  
Lymphocyte Adhesion ◽  
Nucleotide Exchange Factor ◽  
Phospholipase D1

ABSTRACT Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.

scholarly journals Arf3p GTPase is a key regulator of Bud2p activation for invasive growth in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (15) ◽  
pp. 2328-2339 ◽  
Author(s):  
Jia-Wei Hsu ◽  
Fang-Jen S. Lee
Keyword(s):  
Bound State ◽  
Invasive Growth ◽  
Nucleotide Exchange ◽  
General Applicability ◽  
Glucose Depletion ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Adp Ribosylation Factor

The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. Bud2p, which functions as a GTPase-activating protein (GAP) for Bud1p/Rsr1p, is required for appropriate budding patterns and filamentous growth. The regulatory mechanisms leading to Bud2p activation, however, are poorly understood. In this study, we report that ADP-ribosylation factor 3p (Arf3p) acts as a regulator of Bud2p activation during invasive growth. Arf3p binds directly to the N-terminal region of Bud2p and promotes its GAP activity both in vitro and in vivo. Genetic analysis shows that deletion of BUD1 suppresses the defect of invasive growth in arf3Δ or bud2Δ cells. Lack of Arf3p, like that of Bud2p, causes the intracellular accumulation of Bud1p-GTP. The Arf3p–Bud2p interaction is important for invasive growth and facilitates the Bud2p–Bud1p association in vivo. Finally, we show that under glucose depletion–induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state, and the activation is independent of Arf3p guanine nucleotide-exchange factor Yel1p. Thus we demonstrate that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose depletion–induced invasive growth.

scholarly journals CGEF-1 and CHIN-1 Regulate CDC-42 Activity during Asymmetric Division in the Caenorhabditis elegans Embryo

2010 ◽  
Vol 21 (2) ◽  
pp. 266-277 ◽  
Author(s):  
Kraig T. Kumfer ◽  
Steven J. Cook ◽  
Jayne M. Squirrell ◽  
Kevin W. Eliceiri ◽  
Nina Peel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rho Gtpase ◽  
Maintenance Phase ◽  
Early Embryo ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Cell Stage ◽  
Nucleotide Exchange Factor ◽  
The One ◽  
Regulatory Loop

The anterior–posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.

scholarly journals The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo.

1992 ◽  
Vol 12 (5) ◽  
pp. 2091-2099 ◽  
Author(s):  
T Munder ◽  
P Fürst
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Cell Cycle Control ◽  
Bound State ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Nucleotide Exchange ◽  
Protein Protein Interaction ◽  
Upstream Regulator

Genetic data suggest that the yeast cell cycle control gene CDC25 is an upstream regulator of RAS2. We have been able to show for the first time that the guanine nucleotide exchange proteins Cdc25 and Sdc25 from Saccharomyces cerevisiae bind directly to their targets Ras1 and Ras2 in vivo. Using the characteristics of the yeast Ace1 transcriptional activator to probe for protein-protein interaction, we found that the CDC25 gene product binds specifically to wild-type Ras2 but not to the mutated Ras2Val-19 and Ras2 delta Val-19 proteins. The binding properties of Cdc25 to Ras2 were strongly diminished in yeast cells expressing an inactive Ira1 protein, which normally acts as a negative regulator of Ras activity. On the basis of these data, we propose that the ability of Cdc25 to interact with Ras2 proteins is strongly dependent on the activation state of Ras2. Cdc25 binds predominantly to the catalytically inactive GDP-bound form of Ras2, whereas a conformational change of Ras2 to its activated GTP-bound state results in its loss of binding affinity to Cdc25.

scholarly journals Integrins activate trimeric G proteins via the nonreceptor protein GIV/Girdin

2015 ◽  
Vol 210 (7) ◽  
pp. 1165-1184 ◽  
Author(s):  
Anthony Leyme ◽  
Arthur Marivin ◽  
Lorena Perez-Gutierrez ◽  
Lien T. Nguyen ◽  
Mikel Garcia-Marcos
Keyword(s):  
Tumor Cells ◽  
G Protein ◽  
Control Cell ◽  
Integrin Signaling ◽  
Nucleotide Exchange ◽  
Tumor Invasiveness ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Dependent Cell ◽  
G Protein Coupled

Signal transduction via integrins and G protein–coupled receptors is critical to control cell behavior. These two receptor classes have been traditionally believed to trigger distinct and independent signaling cascades in response to extracellular cues. Here, we report a novel mechanism of integrin signaling that requires activation of the trimeric G protein Gαi by the nonreceptor guanine nucleotide exchange factor (GEF) GIV (also known as Girdin), a metastasis-associated protein. We demonstrate that GIV enhances integrin-dependent cell responses upon extracellular matrix stimulation and makes tumor cells more invasive. These responses include remodeling of the actin cytoskeleton and PI3K-dependent signaling, resulting in enhanced haptotaxis and invasion. We show that both GIV and its substrate Gαi3 are recruited to active integrin complexes and that tumor cells engineered to express GEF-deficient GIV fail to transduce integrin signals into proinvasive responses via a Gβγ-PI3K axis. Our discoveries delineate a novel mechanism by which integrin signaling is rewired during metastasis to result in increased tumor invasiveness.

scholarly journals The TRAPP Complex Is a Nucleotide Exchanger for Ypt1 and Ypt31/32

2000 ◽  
Vol 11 (12) ◽  
pp. 4403-4411 ◽  
Author(s):  
Sara Jones ◽  
Christina Newman ◽  
Fengli Liu ◽  
Nava Segev
Keyword(s):  
Protein Complex ◽  
Protein Transport ◽  
Apparent Molecular Weight ◽  
Yeast Cells ◽  
Mutant Protein ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Exocytic Pathway

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.

scholarly journals Vav Regulates Activation of Rac but Not Cdc42 during FcγR-mediated Phagocytosis

2002 ◽  
Vol 13 (4) ◽  
pp. 1215-1226 ◽  
Author(s):  
Jayesh C. Patel ◽  
Alan Hall ◽  
Emmanuelle Caron
Keyword(s):  
Bound State ◽  
Small Gtpases ◽  
Membrane Receptors ◽  
Actin Dynamics ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Extracellular Signals ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Migration

Phagocytosis is the process whereby cells direct the spatially localized, receptor-driven engulfment of particulate materials. It proceeds via remodeling of the actin cytoskeleton and shares many of the core cytoskeletal components involved in adhesion and migration. Small GTPases of the Rho family have been widely implicated in coordinating actin dynamics in response to extracellular signals and during diverse cellular processes, including phagocytosis, yet the mechanisms controlling their recruitment and activation are not known. We show herein that in response to ligation of Fc receptors for IgG (FcγR), the guanine nucleotide exchange factor Vav translocates to nascent phagosomes and catalyzes GTP loading on Rac, but not Cdc42. The Vav-induced Rac activation proceeds independently of Cdc42 function, suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. We conclude that Rac is recruited to Fcγ membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac.

Girds ‘n’ cleeks o' cytokinesis: microtubule sticks and contractile hoops in cell division

2008 ◽  
Vol 36 (3) ◽  
pp. 400-404 ◽  
Author(s):  
David M. Glover ◽  
Luisa Capalbo ◽  
Pier Paolo D'Avino ◽  
Melanie K. Gatt ◽  
Matthew S. Savoian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Cortex ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Microtubule Stability ◽  
Spindle Poles ◽  
Nucleotide Exchange Factor

Microtubules maintain an intimate relationship with the rings of anillin, septins and actomyosin filaments throughout cytokinesis. In Drosophila, peripheral microtubules emanating from the spindle poles contact the equatorial cell cortex to deliver the signal that initiates formation of the cytokinetic furrow. Mutations that affect microtubule stability lead to ectopic furrowing because peripheral microtubules contact inappropriate cortical sites. The PAV-KLP (Pavarotti-kinesin-like protein)/RacGAP50C (where GAP is GTPase-activating protein) centralspindlin complex moves towards the plus ends of microtubules to reach the cell equator. When RacGAP50C is tethered to the cell membrane, furrowing initiates at multiple non-equatorial sites, indicating that mis-localization of this single molecule is sufficient to promote furrowing. Furrow formation and ingression requires RhoA activation by the RhoGEF (guanine-nucleotide-exchange factor) Pebble, which interacts with RacGAP50C. RacGAP50C also binds anillin, which associates with actin, myosin and septins. Thus RacGAP50C plays a pivotal role during furrow formation by activating RhoA and linking the peripheral microtubules with the nascent rings through its interaction with anillin.

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