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Richard A Cerione - One of the best experts on this subject based on the ideXlab platform.
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the two splice variant forms of CDC42 exert distinct and essential functions in neurogenesis
Journal of Biological Chemistry, 2020Co-Authors: Makoto Endo, Joseph E Druso, Richard A CerioneAbstract:The small GTPase cell division cycle 42 (CDC42) plays essential roles in neurogenesis and brain development. Previously, using murine embryonic P19 cells as a model system, we showed that CDC42 stimulates mTOR complex 1 (mTORC1) activity and thereby up-regulates transcription factors required for the formation of neural progenitor cells. However, paradoxically, although endogenous CDC42 is required for both the initial transition of undifferentiated P19 cells to neural progenitors and their ultimate terminal differentiation into neurons, ectopic CDC42 overexpression promotes only the first stage of neurogenesis (i.e. the formation of neuroprogenitors) and not the second phase (differentiation into neurons). Here, using both P19 cells and mouse embryonic stem cells, we resolve this paradox, demonstrating that two splice variants of CDC42, differing only in nine amino acid residues in their very C-terminal regions, play distinct roles in neurogenesis. We found that a CDC42 splice variant that has a ubiquitous tissue distribution, termed here as CDC42u, specifically drives the formation of neuroprogenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neuroprogenitor cells to neurons. We further show that the specific roles of CDC42u and CDC42b in neurogenesis are due to their opposing effects on mTORC1 activity. Specifically, CDC42u stimulated mTORC1 activity and thereby induced neuroprogenitor formation, whereas CDC42b worked together with activated CDC42-associated kinase (ACK) in down-regulating mTOR expression and promoting neuronal differentiation. These findings highlight the remarkable functional specificities of two highly similar CDC42 splice variants in regulating distinct stages of neurogenesis.
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deletion of CDC42 enhances adam17 mediated vascular endothelial growth factor receptor 2 shedding and impairs vascular endothelial cell survival and vasculogenesis
Molecular and Cellular Biology, 2013Co-Authors: Yixin Jin, Qiong Lin, Yang Liu, Joseph E Druso, Marc A Antonyak, Junlin Guan, Cynthia J Meininger, Shenyuan L Zhang, David E Dostal, Richard A CerioneAbstract:CDC42 is a Ras-related GTPase that plays an important role in the regulation of a range of cellular functions, including cell migration, proliferation, and survival. Consistent with its critical functions in vitro, the inactivation of CDC42 in mice has been shown to result in embryonic lethality at embryonic day 6.5 (E6.5) before blood vessel formation. To determine the role of CDC42 in new blood vessel formation, we have generated vascular endothelial cell (EC)-specific CDC42 knockout mice by crossing CDC42flox/flox mice with Tie2-Cre mice. The deletion of CDC42 in ECs caused embryonic lethality with vasculogenesis and angiogenesis defects. We observed that CDC42 is critical for EC migration and survival but not for cell cycle progression. Moreover, we found that the inactivation of CDC42 in ECs decreased the level of vascular endothelial growth factor receptor 2 (VEGFR2) protein on the EC surface and promoted the production of a 75-kDa membrane-associated C-terminal VEGFR2 fragment. Using cultured primary mouse ECs and human umbilical vein ECs, we have demonstrated that the deletion of CDC42 increased ADAM17-mediated VEGFR2 shedding. Notably, inhibition of ADAM17 or overexpression of VEGFR2 can partially reverse CDC42 deletion-induced EC apoptosis. These data indicate that CDC42 is essential for VEGFR2-mediated signal transduction in blood vessel formation.
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inactivation of CDC42 in embryonic brain results in hydrocephalus with ependymal cell defects in mice
Protein & Cell, 2013Co-Authors: Xu Peng, Qiong Lin, Yang Liu, Yixin Jin, Joseph E Druso, Marc A Antonyak, Junlin Guan, Richard A CerioneAbstract:The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the brain. CDC42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton architecture. Previous studies have shown that CDC42 plays an indispensable role in telencephalon development in earlier embryo developmental stage (before E12.5). However, the functions of CDC42 in other parts of brain in later embryo developmental stage or in adult brain remain unclear. Thus, in order to address the role of CDC42 in the whole brain in later embryo developmental stage or in adulthood, we used Cre/loxP technology to generate two lines of tissuespecific CDC42-knock-out mice. Inactivation of CDC42 was achieved in neuroepithelial cells by crossing CDC42/ flox mice with Nestin-Cre mice and resulted in hydrocephalus, causing death to occur within the postnatal stage. Histological analyses of the brains from these mice showed that ependymal cell differentiation was disrupted, resulting in aqueductal stenosis. Deletion of CDC42 in the cerebral cortex also induced obvious defects in interkinetic nuclear migration and hypoplasia. To further explore the role of CDC42 in adult mice brain, we examined the effects of knocking-out CDC42 in radial glial cells by crossing CDC42/flox mice with human glial fibrillary acidic protein (GFAP)-Cre mice. Inactivation of CDC42 in radial glial cells resulted in hydrocephalus and ependymal cell denudation. Taken together, these results highlight the importance of CDC42 for ependymal cell differentiation and maintaining, and suggest that these functions likely contribute to the essential roles played by CDC42 in the development of the brain.
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A CDC42 mutant specifically activated by intersectin.
Biochemistry, 2005Co-Authors: William J. Smith, Richard A Cerione, Brant Hamel, Marielle E. Yohe, John Sondek, Jason T. SnyderAbstract:The Rho family GTPase CDC42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate CDC42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of CDC42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of CDC42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of CDC42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of CDC42. Moreover, CDC42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other CDC42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of CDC42(Y32F), have been defined. CDC42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, CDC42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on CDC42-mediated signaling phenomena.
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RhoGDI is required for CDC42-mediated cellular transformation.
Current biology : CB, 2003Co-Authors: Qiong Lin, Wannian Yang, Reina N. Fuji, Richard A CerioneAbstract:Abstract Background: CDC42, a Rho-related small GTP binding protein, plays pivotal roles in actin cytoskeletal organization, Golgi vesicular trafficking, receptor endocytosis, and cell cycle progression. However, the target/effectors mediating these cellular activities and, in particular, those responsible for CDC42-mediated cell growth regulation and transformation are still being determined. In this study, we set out to examine how the regulatory protein RhoGDI influences the cellular responses elicited by activated CDC42. Results: X-ray crystallographic analysis of the CDC42-RhoGDI complex suggested that arginine 66 of CDC42 is essential for its interaction with RhoGDI. Here we show that mutation of either arginine 66 or arginine 68 within the Switch II domain of CDC42 completely abolished the binding of CDC42 to RhoGDI without affecting the binding of other known regulators or target/effectors of this GTP binding protein. Introduction of the RhoGDI binding-defective mutation R66A within a constitutively active CDC42(F28L) background was accompanied by changes in cell shape and an accumulation of CDC42 in the Golgi when these cells were compared to those expressing CDC42(F28L). However, the most striking change was that unlike CDC42(F28L), which was able to induce the transformation of NIH 3T3 fibroblasts as assayed by their growth in low serum or their ability to form colonies in soft-agar, the CDC42(F28L,R66A) mutant was transformation-defective. Likewise, the introduction of RhoGDI siRNA into CDC42(F28L)-transfected cells inhibited their transformation. Conclusions: Taken together, the results reported here indicate that despite being a negative regulator of CDC42 activation and GTP hydrolysis, RhoGDI plays an essential role in CDC42-mediated cellular transformation.
Wannian Yang - One of the best experts on this subject based on the ideXlab platform.
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Cellular signaling for activation of Rho GTPase CDC42.
Cellular signalling, 2008Co-Authors: Soniya Sinha, Wannian YangAbstract:The Rho family GTPase CDC42 regulates cytoskeletal organization and membrane trafficking in physiological processes such as cell proliferation, motility and polarity. Aberrant activation of CDC42 results in pathogenesis, such as tumorigenesis and tumor progression, cardiovascular diseases, diabetes, and neuronal degenerative diseases. The activation of CDC42 in response to upstream signals is mediated by guanine nucleotide exchange factors (GEFs), which converse GDP-bound inactive form to the GTP-bound active form of CDC42. The activated CDC42 transduces signals to downstream effectors and generates cellular effects. This review will discuss the molecular mechanism of activation of CDC42 and postulate that signaling specificity of CDC42 is conferred by the GEF/GTPase/Effector (GGE) complexes in response to external stimuli.
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Identification of the region in CDC42 that confers the binding specificity to activated CDC42-associated kinase.
The Journal of biological chemistry, 2004Co-Authors: Qiong Lin, Chandra Childress, Wannian YangAbstract:The Rho family small G-protein CDC42 has been implicated in a diversity of biological functions. Multiple downstream effectors have been identified. How CDC42 discriminates the interaction with its multiple downstream effectors is not known. Activated CDC42-associated tyrosine kinase (ACK) is a very specific effector of CDC42. To delineate the CDC42 signaling pathway mediated by ACK, we set about to identify the specific ACK-binding region in CDC42. We utilized TC10, another member of the Rho family of G-proteins that is 66.7% identical to CDC42, to construct TC10/CDC42 chimeras for screening the specific ACK-binding region in CDC42. A region between switch I and switch II has been identified as the specific ACK-binding (AB) region. The replacement of the AB region with the corresponding region in TC10 resulted in the complete loss of ACK-binding ability but did not affect the binding to WASP, suggesting that the AB region confers the binding specificity to ACK. On the other hand, replacement of the corresponding region of TC10 with the AB region enabled TC10 to acquire ACK-binding ability. Eight residues are different between the AB region and the corresponding region of TC10. The mutational analysis indicated that all eight residues contribute to the binding to ACK2. The assays for the CDC42-mediated activation of ACK2 indicated that the AB region is essential for CDC42 to activate ACK2 in cells. Thus, our studies have defined a specific ACK-binding region in CDC42 and have provided a molecular basis for generating ACK binding-defective mutants of CDC42 to delineate ACK-mediated signaling pathway.
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RhoGDI is required for CDC42-mediated cellular transformation.
Current biology : CB, 2003Co-Authors: Qiong Lin, Wannian Yang, Reina N. Fuji, Richard A CerioneAbstract:Abstract Background: CDC42, a Rho-related small GTP binding protein, plays pivotal roles in actin cytoskeletal organization, Golgi vesicular trafficking, receptor endocytosis, and cell cycle progression. However, the target/effectors mediating these cellular activities and, in particular, those responsible for CDC42-mediated cell growth regulation and transformation are still being determined. In this study, we set out to examine how the regulatory protein RhoGDI influences the cellular responses elicited by activated CDC42. Results: X-ray crystallographic analysis of the CDC42-RhoGDI complex suggested that arginine 66 of CDC42 is essential for its interaction with RhoGDI. Here we show that mutation of either arginine 66 or arginine 68 within the Switch II domain of CDC42 completely abolished the binding of CDC42 to RhoGDI without affecting the binding of other known regulators or target/effectors of this GTP binding protein. Introduction of the RhoGDI binding-defective mutation R66A within a constitutively active CDC42(F28L) background was accompanied by changes in cell shape and an accumulation of CDC42 in the Golgi when these cells were compared to those expressing CDC42(F28L). However, the most striking change was that unlike CDC42(F28L), which was able to induce the transformation of NIH 3T3 fibroblasts as assayed by their growth in low serum or their ability to form colonies in soft-agar, the CDC42(F28L,R66A) mutant was transformation-defective. Likewise, the introduction of RhoGDI siRNA into CDC42(F28L)-transfected cells inhibited their transformation. Conclusions: Taken together, the results reported here indicate that despite being a negative regulator of CDC42 activation and GTP hydrolysis, RhoGDI plays an essential role in CDC42-mediated cellular transformation.
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Antiapoptotic CDC42 mutants are potent activators of cellular transformation.
Biochemistry, 2002Co-Authors: Wannian Yang, Klaus M. Hahn, Peri Nolbant, Richard A CerioneAbstract:CDC42 is a small GTP-binding protein which has been implicated in a number of cellular activities, including cell morphology, motility, cell-cycle progression, and malignant transformation. While GTPase-defective forms of CDC42 inhibit cell growth, a mutation [CDC42(F28L)] that allows the constitutive exchange of GDP for GTP and is GTPase-competent induces cellular transformation. These results suggest that CDC42 must cycle between its GTP- and GDP-bound states to stimulate cell growth. In attempting to design CDC42 molecules with more potent transforming activity, we set out to generate other types of CDC42 mutants capable of constitutive GDP−GTP exchange. Here, we describe one such mutant, generated by changing a conserved aspartic acid residue at position 118 to an asparagine. The CDC42(D118N) protein exchanges GDP for GTP more rapidly than wild-type CDC42, but significantly more slowly than the CDC42(F28L) mutant. Despite its slower rate of activation, the CDC42(D118N) mutant is more potent at inducin...
Yi Zheng - One of the best experts on this subject based on the ideXlab platform.
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The Rho GTPase CDC42 is essential for B lyphocyte development and Activation
Blood, 2008Co-Authors: Fukun Guo, Yi ZhengAbstract:Abstract CDC42 of the Rho GTPase family has been implicated in a wide range of fundamental cell functions including actin cytoskeleton dynamics, proliferation, and migration, but its cell-type specific functions have just begun to be appreciated. Previous studies of CDC42 in B cell regulation by using dominant mutant forms of CDC42 suggest that CDC42 is involved in B cell cytoskeleton organization and antigen-receptor signaling, however, its contribution to the B-lineage development and B cell physiology remains unknown. In this study we have achieved B cell- and hematopoietic stem cell-deletion of CDC42 by conditional gene targeting with the CD19 and Mx promoter-driven Cre expression, respectively, in the CDC42loxP/loxP mice. Deletion of CDC42 after the pre-proB cell stage significantly inhibited late B cell development, resulting in reduced mature B cell populations in spleen, bone marrow, peripheral blood, lymph node, and peritoneal cavity. Accordingly, antigen-specific IgM, IgG1 and IgG3, was reduced in the CDC42-deificient mice. The reduction of CDC42 knockout B cells are associated with impaired proliferation and survival. CDC42 deficiency caused a BCR signaling defect with increased Erk and decreased Akt activation, and a defect in BCR-mediated BAFF receptor upregulation and subsequent BAFF receptor signaling in the mature resting B cells. Interestingly, CDC42 is dispensable for SDF-1α- or BLC-induced B cell migration. Further, deletion of CDC42 from hematopoietic stem cells resulted in an unaltered common lymphoid progenitor production but severely reduced pre-proB/proB/preB and immature B cell populations, indicating that CDC42 is also critically involved in the B cell precursor differentiation. Together our results reveal multi-facet roles of CDC42 in B cell development and activation.
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Gene targeting of CDC42 and CDC42GAP affirms the critical involvement of CDC42 in filopodia induction, directed migration, and proliferation in primary mouse embryonic fibroblasts.
Molecular biology of the cell, 2006Co-Authors: Linda Yang, Lei Wang, Yi ZhengAbstract:Recent studies in CDC42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian CDC42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian CDC42, we have characterized the CDC42−/− and CDC42GAP−/− primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the CDC42 loss- or gain-of-activity cell model. The CDC42−/− cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the CDC42GAP−/− cells displayed spontaneous filopodia. The CDC42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3β, myosin light chain, and FAK phosphorylation. Furthermore, CDC42−/− cells were defective in G1/S-phase transition and survival, correlating with deficient NF-κB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of CDC42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that CDC42 plays cell-type–specific signaling roles.
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Localization of the PAK1-, WASP-, and IQGAP1-specifying Regions of CDC42
The Journal of biological chemistry, 1999Co-Authors: Balazs Debreceni, Baoqing Jia, Yuan Gao, Gabor Tigyi, Yi ZhengAbstract:Abstract The Rho family small GTPase CDC42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of CDC42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of CDC42-effector specificity, we utilized recombinant CDC42 mutants and chimeras made between CDC42 and RhoA to map the regions of CDC42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of CDC42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by CDC42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of CDC42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157–191 and 84–120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique “insert region,” residues 122–134, of CDC42 to achieve high affinity binding. Microinjection of the constitutively active CDC42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of CDC42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by CDC42 was insufficient for PAM-induction. Thus, CDC42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.
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Characterization of the Interactions between the Small GTPase CDC42 and Its GTPase-activating Proteins and Putative Effectors COMPARISON OF KINETIC PROPERTIES OF CDC42 BINDING TO THE CDC42-INTERACTIVE DOMAINS
The Journal of biological chemistry, 1997Co-Authors: Baolin Zhang, Zhi-xin Wang, Yi ZhengAbstract:Abstract The small GTPase CDC42 interacts with multiple factors to transduce diverse intracellular signals. The factors that preferentially recognize the GTP-bound, active state of CDC42 include a panel of GTPase-activating proteins (GAPs), the CDC42/Rac interactive binding (CRIB) motif-containing molecules, and the RasGAP domain containing IQGAP1 and IQGAP2. In the present study, we have determined the kinetic parameters underlying the functional interactions between the CDC42-binding domains of some of these factors and CDC42 by monitoring the continuous release of γPi and have compared the ability of the domains to bind to CDC42. The catalytic efficiencies (K cat/K m) of the GAP domains of Bcr, 3BP-1, and p190 on CDC42 are found to be 60-, 160-, and over 500-fold less than that of CDC42GAP, respectively, and the differences are due, to a large part, to differences inK m. The K m values of the GAP domains compare well to the binding affinity to the guanylyl imidodiphosphate-bound CDC42, suggesting a rapid equilibrium reaction mechanism. The affinity of the CDC42-binding domains of the CRIB motif of Wiskott-Aldrich Syndrome protein and p21CDC42/rac-activated kinase 1, and the RasGAP-related domain of IQGAP1, which all inhibit the intrinsic rate of GTP hydrolysis of CDC42, are found to be 4, 0.7, and 0.08 μm, respectively. These quantitative analysis provide insight that CDC42GAP functions as an effective negative regulator of CDC42 by fast, relatively tight binding to the GTP-bound CDC42, whereas IQGAP1 interacts with CDC42 as a putative effector with over 10-fold higher affinity than the CRIB domains and GAPs, and suggest that various GAPs and effectors employ distinct mechanism to play roles in CDC42-mediated signaling pathways.
Derek Mccusker - One of the best experts on this subject based on the ideXlab platform.
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Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions.
The EMBO Journal, 2019Co-Authors: Julien Meca, Aurélie Massoni‐laporte, Denis Martinez, Elodie Sartorel, Antoine Loquet, Birgit Habenstein, Derek MccuskerAbstract:While Rho GTPases are indispensible regulators of cellular polarity, the mechanisms underlying their anisotropic activation at membranes have been elusive. Using the budding yeast CDC42 GTPase module, which includes a guanine nucleotide exchange factor (GEF) Cdc24 and the scaffold Bem1, we find that avidity generated via multivalent anionic lipid interactions is a critical mechanistic constituent of polarity establishment. We identify basic cluster (BC) motifs in Bem1 that drive the interaction of the scaffold–GEF complex with anionic lipids at the cell pole. This interaction appears to influence lipid acyl chain ordering, thus regulating membrane rigidity and feedback between CDC42 and the membrane environment. Sequential mutation of the Bem1 BC motifs, PX domain, and the PH domain of Cdc24 lead to a progressive loss of cellular polarity stemming from defective CDC42 nanoclustering on the plasma membrane and perturbed signaling. Our work demonstrates the importance of avidity via multivalent anionic lipid interactions in the spatial control of GTPase activation.
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Phosphatidylserine and GTPase activation control CDC42 nanoclustering to counter dissipative diffusion
Molecular Biology of the Cell, 2018Co-Authors: Elodie Sartorel, Caner Unlu, Julien Meca, Mini Jose, Aurélie Massoni-laporte, Jean-baptiste Sibarita, Derek MccuskerAbstract:The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatiotemporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor CDC42 in budding yeast, a system in which CDC42 exhibits anisotropic organization. CDC42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The CDC42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing CDC42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by CDC42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating CDC42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing CDC42 on the plasma membrane and sustaining cell polarity.
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scaffold mediated gating of CDC42 signalling flux
eLife, 2017Co-Authors: Peter Rapali, Romain Mitteau, Craig R Braun, Aurelie Massonilaporte, Caner Unlu, Laure Bataille, Floriane Saint Arramon, Steven P Gygi, Derek MccuskerAbstract:Scaffold proteins modulate signalling pathway activity spatially and temporally. In budding yeast, the scaffold Bem1 contributes to polarity axis establishment by regulating the GTPase CDC42. Although different models have been proposed for Bem1 function, there is little direct evidence for an underlying mechanism. Here, we find that Bem1 directly augments the guanine exchange factor (GEF) activity of Cdc24. Bem1 also increases GEF phosphorylation by the p21-activated kinase (PAK), Cla4. Phosphorylation abrogates the scaffold-dependent stimulation of GEF activity, rendering Cdc24 insensitive to additional Bem1. Thus, Bem1 stimulates GEF activity in a reversible fashion, contributing to signalling flux through CDC42. The contribution of Bem1 to GTPase dynamics was borne-out by in vivo imaging: active CDC42 was enriched at the cell pole in hypophosphorylated cdc24 mutants, while hyperphosphorylated cdc24 mutants that were resistant to scaffold stimulation displayed a deficit in active CDC42 at the pole. These findings illustrate the self-regulatory properties that scaffold proteins confer on signalling pathways.
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Cdk1 coordinates cell-surface growth with the cell cycle
Nature Cell Biology, 2007Co-Authors: Derek Mccusker, Carilee Denison, Scott Anderson, Thea Egelhofer, John Yates, Steven Gygi, Douglas KelloggAbstract:The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the CDC42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin-Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the CDC42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.
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CDC42p GDP/GTP Cycling Is Necessary for Efficient Cell Fusion during Yeast Mating
Molecular Biology of the Cell, 2006Co-Authors: Sophie Barale, Derek Mccusker, Robert ArkowitzAbstract:The highly conserved small Rho G-protein, CDC42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, CDC42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of CDC42p in the mating process by screening for specific mating defective CDC42 alleles. We have identified and characterized novel mating defective CDC42 alleles that are unaffected in vegetative cell polarity. Replacement of the CDC42p Val36 residue with Met resulted in a specific cell fusion defect. This CDC42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling CDC42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the CDC42[V36M] fusion defect. Taken together, our results indicate that CDC42p GDP-GTP cycling is critical for efficient cell fusion.
Suncheol Choi - One of the best experts on this subject based on the ideXlab platform.
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xenopus CDC42 regulates convergent extension movements during gastrulation through wnt ca2 signaling pathway
Developmental Biology, 2002Co-Authors: Suncheol ChoiAbstract:Abstract Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. We have isolated the Xenopus homolog of Rho GTPase CDC42 and examined its potential role during gastrulation movements in early Xenopus embryos. XCDC42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCDC42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCDC42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-α, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCDC42, implying that XCDC42 acts downstream of the Wnt/Ca2+ signaling pathway involving PKC activation. XCDC42 also lies downstream of XWnt-5a in the regulation of Ca2+-dependent cell adhesion. Taken together, our results suggest that XCDC42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca2+ pathway.
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Xenopus CDC42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway.
Developmental Biology, 2002Co-Authors: Suncheol ChoiAbstract:Abstract Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. We have isolated the Xenopus homolog of Rho GTPase CDC42 and examined its potential role during gastrulation movements in early Xenopus embryos. XCDC42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCDC42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCDC42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-α, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCDC42, implying that XCDC42 acts downstream of the Wnt/Ca2+ signaling pathway involving PKC activation. XCDC42 also lies downstream of XWnt-5a in the regulation of Ca2+-dependent cell adhesion. Taken together, our results suggest that XCDC42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca2+ pathway.