Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

Activation of Rac1 or Cdc42 during early morphogenesis of eye discs induces ectopic antennae in Drosophila

The Rho family small guanosine triphosphatases (GTPases) play important roles in many cellular processes, especially in regulation of cytoskeletal organization. In this study, I examined the functions of Rac1 and Cdc42 for disc morphogenesis in Drosophila. I expressed either a constitutively active form or a dominant negative form of each protein during early morphogenesis of eye discs. Inactivation of Rac1 or Cdc42 resulted in small eye phenotypes. On the other hand, I found that activation of either Rac1 or Cdc42 induces ectopic antennae. In some cases, an almost complete antenna was observed instead of an eye, which was possibly transformation from an eye to an antenna. As a molecular evidence for the ectopic antennae, I found that the Distal-less protein, which is essential for the distalization process, was ectopically induced in the eye discs. I also found that the Decapentaplegic and Wingless proteins, which are upstream morphogenetic signaling proteins of the distalization process, could be ectopically induced by activation of Rac1 or Cdc42. My observations suggest novel functions of Rac1 and Cdc42 for disc morphogenesis.     

A New Look at Rho GTPases in the Cell Cycle: Their Role in Kinetochore-Microtubule Attachment

Rho GTPases including Rho, Rac and Cdc42 are involved in cell morphogenesis by inducing specific types of actin cytoskeleton and alignment and stabilization of microtubules. Previous studies suggest that they also regulate cell cycle progression; Rho, Rac and Cdc42 regulate the G1-S progression and Rho controls cytokinesis. However, a role of Rho GTPases in nuclear division has not been definitely shown. We have recently found that Cdc42 and its downstream effector mDia3 are involved in bi-orientation and stabilization of spindle microtubules attachment to kinetochores and regulate chromosome alignment and segregation. Here, we discuss how this is coordinated with other events in mitosis, particularly, with the action of Rho in cytokinesis and how attachment of microtubules to kinetochores is achieved and stabilized. We also discuss redundancy of Cdc42 and Cdc42-related GTPase(s) and potential mechanisms of chromosome instability in cancer     

A Role for Cdc42 in Macrophage Chemotaxis

Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)–induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1–induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished.

We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.

     

The Ras and Rho GTPases genetically interact to co-ordinately regulate cell polarity during development in Penicillium marneffei

Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarization and development. Studies in Saccharomyces cerevisiae and mammalian cells have defined some of the central activities of these GTPases. However, these paradigms do not explain the role of these proteins in all eukaryotes. Unlike yeast, but like more complex eukaryotes, filamentous fungi have Rac-like proteins in addition to Ras and Cdc42. To investigate the unique functions of these proteins and determine how they interact to co-ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from the dimorphic pathogenic fungus, Penicillium marneffei. P. marneffei growth at 25 degrees C is as multinucleate, septate, branched hyphae which are capable of undergoing asexual development (conidiation), while at 37 degrees C, uninucleate pathogenic yeast cells which divide by fission are produced. Here we show that RasA (Ras) acts upstream of CflA (Cdc42) to regulate germination of spores and polarized growth of both hyphal and yeast cells, while also exhibiting CflA-independent activities. CflA (Cdc42) and CflB (Rac) co-ordinately control hyphal cell polarization despite also having unique roles in regulating conidial germination and polarized growth of yeast cells (CflA) and polarized growth of conidiophore cell types and hyphal branching (CflB).     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

The TRE17 oncogene encodes a component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling

The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.     

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.     

Regulation of hyphal morphogenesis by cdc42 and rac1 homologues in Aspergillus nidulans

The ability of filamentous fungi to form hyphae requires the establishment and maintenance of a stable polarity axis. Based on studies in yeasts and animals, the GTPases Cdc42 and Rac1 are presumed to play a central role in organizing the morphogenetic machinery to enable axis formation and stabilization. Here, we report that Cdc42 (ModA) and Rac1 (RacA) share an overlapping function required for polarity establishment in Aspergillus nidulans. Nevertheless, Cdc42 appears to have a more important role in hyphal morphogenesis in that it alone is required for the timely formation of lateral branches. In addition, we provide genetic evidence suggesting that the polarisome components SepA and SpaA function downstream of Cdc42 in a pathway that may regulate microfilament formation. Finally, we show that microtubules become essential for the establishment of hyphal polarity when the function of either Cdc42 or SepA is compromised. Our results are consistent with the action of parallel Cdc42 and microtubule-based pathways in regulating the formation of a stable axis of hyphal polarity in A. nidulans.     

Polarity in filamentous fungi: establishment,maintenance and new axes

Germ tube emergence in filamentous fungi appears to be similar to bud emergence in yeast. Several key proteins (e.g. Cdc42, septins, Bni1 formin, Rho1 and Rho3) play common roles in polarity establishment and early polarity maintenance in both processes. Although germ tube extension, which can be thought of as extreme polarity maintenance, uses some of the same genes, they are likely to be regulated differently. Mutations in polarity maintenance genes often lead to a split tip in filamentous fungi, a phenotype without an analogue in yeast. Cell cycle regulation differs between tip splitting and subapical branching, but in both processes filamentous fungi maintain several axes of polar growth simultaneously.     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.     

Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth

Rho G‐proteins are critical for polarized growth, yet little is known about the dynamics of their activation during fungal filamentous growth. We first investigated the roles of Rho1 and Rho2 during Candida albicans filamentous growth. Our results show that Rho1 is required for invasive filamentous growth and that Rho2 is not functionally redundant with Rho1. Using fluorescent reporters, we examined the dynamics of the active form of Rho1 and Cdc42 during initiation and maintenance of hyphal growth. Quantitative analyses indicated that the distribution, but not the level, of these active G‐proteins is altered during initial polarization upon germ tube emergence. A comparison of the dynamics of these active G‐proteins during budding and hyphal growth indicates that a higher concentration of active Cdc42 was recruited to the germ tube tip than to the bud tip. During hyphal elongation, active Cdc42 remained tightly restricted to the hyphal tip, whereas active Rho1 was broadly associated with the apex and subsequently recruited to the cell division site. Furthermore, our data suggest that phosphoinositide‐bis‐phosphates are critical to stabilize active Rho1 at the growth site. Together, our results point towards different regulation of Cdc42 and Rho1 activity during initiation and maintenance of filamentous growth.     

The Borgs, a New Family of Cdc42 and TC10 GTPase-Interacting Proteins

The Rho family of GTPases plays key roles in the regulation of cell motility and morphogenesis. They also regulate protein kinase cascades, gene expression, and cell cycle progression. This multiplicity of roles requires that the Rho GTPases interact with a wide variety of downstream effector proteins. An understanding of their functions at a molecular level therefore requires the identification of the entire set of such effectors. Towards this end, we performed a two-hybrid screen using the TC10 GTPase as bait and identified a family of putative effector proteins related to MSE55, a murine stromal and epithelial cell protein of 55 kDa. We have named this family the Borg (binder of Rho GTPases) proteins. Complete open reading frames have been obtained for Borg1 through Borg3. We renamed MSE55 as Borg5. Borg1, Borg2, Borg4, and Borg5 bind both TC10 and Cdc42 in a GTP-dependent manner. Surprisingly, Borg3 bound only to Cdc42. An intact CRIB (Cdc42, Rac interactive binding) domain was required for binding. No interaction of the Borgs with Rac1 or RhoA was detectable. Three-hemagglutinin epitope (HA(3))-tagged Borg3 protein was mostly cytosolic when expressed ectopically in NIH 3T3 cells, with some accumulation in membrane ruffles. The phenotype induced by Borg3 was reminiscent of that caused by an inhibition of Rho function and was reversed by overexpression of Rho. Surprisingly, it was independent of the ability to bind Cdc42. Borg3 also inhibited Jun kinase activity by a mechanism that was independent of Cdc42 binding. HA(3)-Borg3 expression caused substantial delays in the spreading of cells on fibronectin surfaces after replating, and the spread cells lacked stress fibers. We propose that the Borg proteins function as negative regulators of Rho GTPase signaling.     

Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells

Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.     

The Rho GTPases in Macrophage Motility and Chemotaxis

The GTP-binding proteins, Rho, Rac and Cdc42 are known to regulate actin organisation. Rho induces the assembly of contractile actin-based microfilaments such as stress fibres, Rac regulates the formation of membrane ruffles and lamellipodia, and Cdc42 activation is necessary for the formation of filopodia. In addition, all three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. The orchestration of these distinct cytoskeletal changes is thought to form the basis of the co-ordination of cell motility and we have investigated the roles of Rho family proteins in migration using a model system. We have found that in the macrophage cell line Bacl, the cytokine CSF-1 rapidly induces actin reorganisation: it stimulates the formation of filopodia, lamellipodia and membrane ruffles, as well as the appearance of fine actin cables within the cell. We have shown that Cdc42, Rac and Rho regulate the CSF-1 induced formation of these distinct actin filament-based structures. Using a cell tracking procedure we found that both Rho and Rac were required for CSF-1 stimulated cell translocation. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient of this chemotactic cytokine. This implies that Cdc42, and thus probably filopodia, are required for gradient sensing and cell polarisation in macrophages.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Rac1 and Cdc42 have different roles in Candida albicans development

We investigated the role of the highly conserved G protein Rac1 in the opportunistic pathogen Candida albicans. We identified and disrupted RAC1 and show here that, in contrast to CDC42, it is not necessary for viability or serum-induced hyphal growth but is essential for filamentous growth when cells are embedded in a matrix. Rac1 is localized to the plasma membrane, yet its distribution is more homogenous than that of Cdc42, with no enrichment at the tips of either buds or hyphae. In addition, fluorescence recovery after photobleaching results indicate that Rac1 and Cdc42 have different dynamics at the membrane. Furthermore, overexpression of Rac1 does not complement Cdc42 function, and conversely, overexpression of Cdc42 does not complement Rac1 function. Thus, Rac1 and Cdc42, although highly similar to one another, have different roles in C. albicans development.