Regulation Of Cdc42 Research Articles

Genetic and Pharmacologic Evidence Shows That Cdc42 GTPase Plays a Central Role in the Regulation of Both GPVI- and Non-GPVI-Dependent Activation of Platelets.

Abstract 2997Poster Board II-975Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton in platelets. Previously we have shown that Rac1 GTPase is involved in regulation of platelet secretion and aggregation by diverse signaling pathways (J Thromb Haemost 2007; 5: 1747-55). Others have reported that Rac1 is essential for GPVI-, but not G protein-dependent platelet aggregation (Pflugers Arch. 2009; 457:1173-85). Cdc42 was recently reported to be involved in collagen, but not collagen related peptide (CRP), a GPVI specific agonist, induced platelet aggregation (Platelets 2008; 19: 199-210). In this study we have investigated the role of Cdc42 in regulation of platelet function by using complementary approaches of (a) mouse gene targeting of Cdc42, and (b) specific inhibition of Cdc42 activity by a newly identified chemical inhibitor of Cdc42, CASIN (Cdc42 activity-specific inhibitor). Platelets from Cdc42−/− mice exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Threshold concentrations of collagen, CRP or thrombin failed to induce shape change or aggregation in platelets from Cdc42−/− mice compared with induction of shape change and maximal aggregation in platelets from Cdc42+/+ mice. Platelets from Cdc42−/− mice, as compared to Cdc42+/+ mice, exhibited a significant inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin from the dense- and alpha-granules respectively. Increasing concentrations of the agonists only partially corrected the defective aggregation and secretion responses in Cdc42−/−platelets. These data provide the genetic evidence that Cdc42 is required for collagen, CRP and thrombin mediated platelet signaling and activation. Treatment of platelets with CASIN, but not a pharmacologically inactive analog, blocked collagen induced activation of Cdc42 without detectably affecting the Rac1 activity. Human platelets pre-incubated with CASIN (10 micro-M) exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Further, treatment of platelets with CASIN (1-10 micro-M) inhibited: (a) aggregation induced by collagen, CRP, thrombin, ADP or U46619; (b) release of P-selectin and secretion of ATP induced by U46619; and (c) collagen induced phosphorylation of Akt. Addition of CASIN to platelets also blocked collagen or CRP induced aggregation in aspirinated platelets in the presence of apyrase. In other experiments, addition of CASIN to citrated platelet-rich plasma inhibited thrombin induced clot retraction. Significantly, removal of CASIN from the platelet samples by washing reversed inhibition of aggregation as well as clot retraction, reflecting a reversible suppression of Cdc42 activity by CASIN. Administration of CASIN into C57Bl/6 mice inhibited ex vivo platelet aggregation induced by collagen or ADP as well as significantly prolonged tail bleeding times. These data suggest that: (a) Cdc42 plays an essential, non-redundant role in platelet filopodia formation, spreading, secretion, aggregation and clot retraction; (b) Cdc42 is involved in GPVI, non-GPVI- and G protein-dependent signaling in platelets; (c) the pharmacologic inhibitor CASIN is capable of specifically and reversibly inhibiting Cdc42 activity in platelets, mimicking Cdc42 genetic knockout in mice. Altogether, our studies strongly implicate Cdc42 as a novel anti-platelet target, and present evidence that the Cdc42 specific small molecule inhibitor, CASIN, may have therapeutic potential. Disclosures:No relevant conflicts of interest to declare.

Pob1 Participates in the Cdc42 Regulation of Fission Yeast Actin Cytoskeleton

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1(+) as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745-2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1DeltaN) or the SAM domain (pob1DeltaSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1DeltaN, and pob1DeltaSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.

Alpha-synuclein regulates neurite outgrowth through the transcriptional regulation of Cdc42

Alpha-synuclein regulates neurite outgrowth through the transcriptional regulation of Cdc42

A Phosphatidylinositol-Transfer Protein and Phosphatidylinositol-4-phosphate 5-Kinase Control Cdc42 to Regulate the Actin Cytoskeleton and Secretory Pathway in Yeast

The actin cytoskeleton rapidly depolarizes in yeast secretory (sec) mutants at restrictive temperatures. Thus, an unknown signal conferred upon secretion is necessary for actin polarity and exocytosis. Here, we show that a phosphatidylinositol (PI) transfer protein, Sfh5, and a phosphatidylinositol-4-phosphate 5-kinase, Mss4, facilitate Cdc42 activation to concomitantly regulate both actin and protein trafficking. Defects in Mss4 function led to actin depolarization, an inhibition of secretion, reduced levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] in membranes, mislocalization of a pleckstrin homology domain fused to green fluorescent protein, and the mislocalization of Cdc42. Similar defects were observed in sec, myo2-66, and cdc42-6 mutants at elevated temperatures and were rescued by the overexpression of MSS4. Likewise, the overexpression of SFH5 or CDC42 could ameliorate these defects in many sec mutants, most notably in sec3Delta cells, indicating that Cdc42-mediated effects upon actin and secretion do not necessitate Sec3 function. Moreover, mutation of the residues involved in PI binding in Sfh5 led to the mislocalization and loss of function of both Sfh5 and Cdc42. Based upon these findings, we propose that the exocytic signal involves PI delivery to the PI kinases (i.e., Mss4) by Sfh5, generation of PI(4,5)P(2), and PI(4,5)P(2)-dependent regulation of Cdc42 and the actin cytoskeleton.

Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines

BackgroundHomer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1α and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.ResultsCupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191–283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191–230 (CPDΔ191–230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDΔ191–230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDΔ191–230 was distributed in dendritic shafts as well as spines. Overexpression of CPDΔ191–230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDΔ191–230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.ConclusionThese results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

Cdo promotes neuronal differentiationviaactivation of the p38 mitogen‐activated protein kinase pathway

Neural basic helix-loop-helix transcription factors (bHLHs) control many aspects of neurogenesis, such as proliferation, fate determination, and differentiation. We have previously shown that the promyogenic cell surface receptor Cdo modulates the Cdc42 and p38 mitogen-activated protein kinase (MAPK) pathways via a direct association with two scaffold-type proteins, JLP and Bnip-2, to regulate activities of myogenic bHLH factors and myogenic differentiation. We report here that Cdo uses similar regulatory mechanisms to promote neuronal differentiation. Expression of JLP, a scaffold protein for p38MAPK, and Bnip-2, a regulator of Cdc42, is increased during differentiation of C17.2 neural precursor cells and P19 embryonal carcinoma cells. These molecules regulate Cdc42 and p38MAPK activities, which increase in a Cdo-dependent manner during neuronal differentiation of C17.2 cells and retinoic acid-treated P19 cells. Furthermore, enhancement or reduction of Cdc42 and p38MAPK activities enhances or reduces, respectively, neuronal differentiation of these cell lines. Cdc42 and p38MAPK activities also promote heterodimerization of neurogenin1 and E47, suggesting that one way they promote neurogenesis is via regulation of neural bHLH factor activities. These results imply that a conserved intracellular signaling mechanism initiated by Cdo regulates the activities of tissue-specific bHLH factors and therefore functions as a key regulator of differentiation of several different cell lineages.

START-GAP2/DLC2 is localized in focal adhesions via its N-terminal region

START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a “FAT (focal adhesion targeting)” region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions.

A Cdo–Bnip-2–Cdc42 signaling pathway regulates p38α/β MAPK activity and myogenic differentiation

The p38α/β mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38α/β pathway scaffold protein JLP and, via JLP, p38α/β itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo–Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38α/β activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38α/β activation during cell differentiation.

Regulation of Apical Dominance inAspergillus nidulansHyphae by Reactive Oxygen Species

In fungal hyphae, apical dominance refers to the suppression of secondary polarity axes in the general vicinity of a growing hyphal tip. The mechanisms underlying apical dominance remain largely undefined, although calcium signaling may play a role. Here, we describe the localized accumulation of reactive oxygen species (ROS) in the apical region of Aspergillus nidulans hyphae. Our analysis of atmA (ATM) and prpA (PARP) mutants reveals a correlation between localized production of ROS and enforcement of apical dominance. We also provide evidence that NADPH oxidase (Nox) or related flavoproteins are responsible for the generation of ROS at hyphal tips and characterize the roles of the potential Nox regulators NoxR, Rac1, and Cdc42 in this process. Notably, our genetic analyses suggest that Rac1 activates Nox, whereas NoxR and Cdc42 may function together in a parallel pathway that regulates Nox localization. Moreover, the latter pathway may also include Bem1, which we propose represents a p40phox analog in fungi. Collectively, our results support a model whereby localized Nox activity generates a pool of ROS that defines a dominant polarity axis at hyphal tips.

P4-288: α-synuclein regulates neurite outgrowth through the transcriptional regulation of CDC42

P4-288: α-synuclein regulates neurite outgrowth through the transcriptional regulation of CDC42

Nudel Binds Cdc42GAP to Modulate Cdc42 Activity at the Leading Edge of Migrating Cells

Cdc42GAP promotes inactivation of Cdc42, a small GTPase whose activation at the leading edge by guanine nucleotide exchange factors is critical for cell migration. How Cdc42GAP is regulated to ensure proper levels of active Cdc42 is poorly understood. Here we show that Nudel, a cytoplasmic dynein regulator, competes with Cdc42 for binding Cdc42GAP. Consequently, Nudel can inhibit Cdc42GAP-mediated inactivation of Cdc42 in a dose-dependent manner. Both Nudel and Cdc42GAP exhibit leading-edge localization in migrating cells. The localization of Nudel requires its phosphorylation by Erk1/2. Depleting Nudel by RNAi or overexpression of a nonphosphorylatable mutant abolishes Cdc42 activation and cell migration. Our data thus uncover Nudel as a regulator of Cdc42 during cell migration. Nudel facilitates cell migration by sequestering Cdc42GAP at the leading edge to stabilize active Cdc42 in response to extracellular stimuli. Excess active Cdc42 may in turn control its own activity by recruiting Cdc42GAP from Nudel.

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.

Rho GTPase Cdc42 Maintains Neutrophil Polarity during Directed Migration by Regulating CD11b Integrin Signals.

Neutrophil (PMN) migration to sites of infection is the first line of cellular defense. Among others, a key event of cell migration is the maintenance of a polarized morphology characterized by a single protrusive leading edge of F-actin and a contractile uropod devoid of F-actin protrusions. Using mice genetically deficient in the Cdc42 negative regulator Cdc42 GTPase Activating Protein, we previously demonstrated that Cdc42 activity suppresses membrane protrusions at the uropod of the cells to maintain stable polarity during directed migration (Szczur et al, Blood 2006). However, the underlying molecular mechanism of Cdc42-mediated neutrophil polarity remains to be understood. Here, using mice with a conditional Cdc42 (flox) allele, we showed by video microscopy that Cdc42−/− PMNs exhibited multiple membrane extensions in various directions and failed to maintain cell polarity and directionality towards formyl-methionyl-leucyl-phenylalanin (fMLP) gradient compared to wild type (WT) cells. Consistent with this observation, Cdc42−/− PMNs exhibited increased lamellipodia protrusions of F-actin all around the cells compared to WT PMNs, in response to fMLP stimulation and fibrinogen ligation, confirming that Cdc42 maintains stable polarity by preventing abnormal membrane protrusions outside the leading edge. To understand how Cdc42 orchestrates neutrophil polarity at a mechanistic level, we explored the possibility of a role for integrins in this process since Cdc42 appears to regulate neutrophil polarity in a manner, at least in part, dependent on integrin ligation (Szczur et al, Blood 2006). Expression of the neutrophil integrin, CD11b/CD18, on resting or fMLP-stimulated PMNs was similar between the genotypes. Stimulation of WT PMNs with fMLP and ligation to fibrinogen induced a polarized distribution of CD11b into clusters mostly concentrated at the uropod of the cells. Remarkably, the numbers of CD11b clusters of Cdc42−/− PMNs were significantly decreased compared to WT cells. Furthermore, inhibition of CD11b clustering in WT PMNs, using anti-CD11b blocking antibody, significantly increased membrane protrusions associated with loss of stable polarity during directed migration, similarly to Cdc42-deficiency. Enforcing CD11b clustering by CD11b cross-linking in Cdc42−/− PMNs partially rescued cell polarity and F-actin distribution concentrated only at the leading edge of the cells to WT levels. These results strongly suggest that CD11b clustering is regulated by Cdc42 activity and contributes to suppress F-actin protrusions at the uropod of neutrophils during migration. The uropod distribution of CD11b suggests that CD11b may recruit contractile proteins, such as the myosin regulator myosin light chain (MLC), to antagonize membrane protrusions. To test this hypothesis, we analyzed the distribution of phosphorylated MLC (p-MLC). Upon stimulation, p-MLC strongly translocated to the uropod of WT cells. In contrast, p-MLC remained diffuse and non polarized in the cytoplasm of Cdc42−/− cells. Blocking CD11b function in WT cells abrogated the polarized distribution of p-MLC mimicking Cdc42−/− PMNs. Enforcing CD11b clustering in Cdc42−/− PMNs rescued p-MLC signals concentrated at the uropod of the cells. Altogether, this study suggests that Cdc42 activity maintains neutrophil polarity during directed migration by regulating CD11b clustering/distribution and subsequent outside/in signals to suppress lateral membrane protrusions. This study uncovers a critical role for CD11b in maintaining neutrophil polarity during migration.

Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

Cyclin-dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase-activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1-dependent hyperphosphorylation. Using the analog-sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Delta mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28-dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery.

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.

Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle

ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis in budding yeast. Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth. Second, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; because of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize the actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. Previous results indicate a connection between Rot1 and the cyclin Clb2. In fact, overexpression of CLB2 is toxic when ROT1 is partially inactivated, and reciprocally, deletion of CLB2 suppresses the lethality of the rot1 mutant, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and we show that the Clb2 cyclin is not properly degraded in rot1 cells.

Cdc42 GTPase-activating protein deficiency promotes genomic instability and premature aging-like phenotypes

Cdc42 is a member of the Rho GTPase family known to regulate cell actin cytoskeleton organization, polarity, and growth, but its function in mammalian organismal physiology remains unclear. We found that natural aging of WT mice is marked with increased Cdc42 activity in various tissues. Among the negative regulators of Cdc42, gene targeting of Cdc42 GTPase-activating protein (Cdc42GAP) results in constitutively elevated Cdc42-GTP level in diverse tissues of adult mice; significantly shortened life span of the animals; and multiple premature aging-like phenotypes, including a reduction in body mass, a loss of subdermal adipose tissue, severe lordokyphosis, muscle atrophy, osteoporosis, and reduction of reepithelialization ability in wound-healing. Cdc42GAP-/- mouse embryonic fibroblasts and/or tissues display reduced population doubling, significantly dampened DNA damage repair activity after DNA-damaging agent treatment, accumulated genomic abnormalities, and induction of p53, p16Ink4a, p21Cip1, and senescence-associated beta-galactosidase expressions. Furthermore, Cdc42 activation is sufficient to promote a premature cellular senescence phenotype that depends on p53. These results suggest a role of Cdc42 activity in regulating mammalian genomic stability and aging-related physiology.

Characterization of IQGAP1-Containing Complexes in NK-Like Cells: Evidence for Rac 2 and RACK1 Association during Homotypic Adhesion

IQGAP1 is a scaffolding protein that binds to a diverse array of signaling and structural molecules that are often associated with cell polarization and adhesion. Through interaction with its target proteins, IQGAP1 participates in multiple cellular functions, including Ca2+-calmodulin signaling, definition of cytoskeletal architecture, regulation of Cdc42 and Rac1 dependent cytoskeletal changes, and control of E-cadherin mediated intercellular adhesion. These analysis have been largely restricted to cells of epithelial and fibroblast origin. The present studies were initiated to examine the role of IQGAP1 in cellular interactions involving the lymphoid cells. A mass spectrometric based analysis of IQGAP1 containing complexes isolated from the human NK-like cell line, YTS, identified several known and new potential IQGAP1 interaction partners including receptor of activated C kinase 1 (RACK1) and the small GTPase, Rac2. Immunofluorescence analysis of YTS cells indicated that a minor component of IQGAP1 was localized at the cell membrane with the remainder diffusely distributed through out the cytoplasm. However, at sites of cellular contact, there was a marked accumulation of IQGAP1. Staining for RACK1 and Rac2 revealed that both of these proteins accumulated these contact sites. Antibody-based studies suggested that a subset of RACK1 was associated in an IQGAP1-containing complex, which prevented recognition of RACK1 by monoclonal antibody. These results suggest that RACK1, Rac2, and IQGAP1 are components of complexes involved in NK cell homotypic adhesion.

Rho GTPase CDC42 regulates directionality and random movement via distinct MAPK pathways in neutrophils

AbstractNeutrophil transmigration into tissue is a multiple-step process that results from a coordinated rearrangement of the cytoskeleton and adhesion complexes. Assembly and disassembly of actin and adhesion structures dictate motility behavior, while polarity and gradient sensing provide directionality to the cell movement. Here, using mice deficient in the CDC42 regulator CDC42 GTPase-activating protein (CDC42GAP), we demonstrate that CDC42 activity separately regulates neutrophil motility and directionality. CDC42GAP–/– neutrophils showed increased motility, while directed migration was defective. Podosome-like structures present at the leading edge in wild-type neutrophils were significantly reduced in CDC42GAP–/– cells. CDC42GAP–/– neutrophils also showed increased lateral and tail filopodia-like formation, and excess membrane protrusions. We further suggest that CDC42GAP-mediated extracellular signal–regulated kinase (ERK) activity regulates motility associated with podosome-like structures at the cell leading edge, while CDC42GAP-induced p38MAPK phosphorylation regulates directed migration by antagonizing filopodia assembly. Overall, this study reveals that CDC42 activity regulates both motility and directionality in neutrophils, but via distinct mitogen-activated protein kinase (MAPK) pathways.

Regulation of Cdc42 GTPase Activity in the Formation of Hyphae inCandida albicans

The human fungal pathogen Candida albicans can switch between yeast, pseudohyphal, and hyphal morphologies. To investigate whether the distinctive characteristics of hyphae are due to increased activity of the Cdc42 GTPase, strains lacking negative regulators of Cdc42 were constructed. Unexpectedly, the deletion of the Cdc42 Rho guanine dissociation inhibitor RDI1 resulted in reduced rather than enhanced polarized growth. However, when cells lacking both Cdc42 GTPase-activating proteins, encoded by RGA2 and BEM3, were grown under pseudohyphal-promoting conditions the bud was highly elongated and lacked a constriction at its base, so that its shape resembled a hyphal germ tube. Moreover, a Spitzenkörper was present at the bud tip, a band of disorganized septin was present at bud base, true septin rings formed within the bud, and nuclei migrated out of the mother cell before the first mitosis. These are all characteristic features of a hyphal germ tube. Intriguingly, we observed hyphal-specific phosphorylation of Rga2, suggesting a possible mechanism for Cdc42 activation during normal hyphal development. In contrast, expression of Cdc42(G12V), which is constitutively GTP bound because it lacks GTPase activity, resulted in swollen cells with prominent and stable septin bars. These results suggest the development of hyphal-specific characteristics is promoted by Cdc42-GTP in a process that also requires the intrinsic GTPase activity of Cdc42.