Yeast oxysterol-binding proteins: sterol transporters or regulators of cell polarization?

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) are a conserved family of soluble cytoplasmic proteins that can bind sterols, translocate between membrane compartments, and affect sterol trafficking. These properties make ORPs attractive candidates for lipid transfer proteins (LTPs) that directly mediate nonvesicular sterol transfer to the plasma membrane. To test whether yeast ORPs (the Osh proteins) are sterol LTPs, we studied endoplasmic reticulum (ER)-to-plasma membrane (PM) sterol transport in OSH deletion mutants lacking one, several, or all Osh proteins. In conditional OSH mutants, ER-PM ergosterol transport slowed ~20-fold compared with cells expressing a full complement of Osh proteins. Although this initial finding suggested that Osh proteins act as sterol LTPs, the situation is far more complex. Osh proteins have established roles in Rho small GTPase signaling. Osh proteins reinforce cell polarization and they specifically affect the localization of proteins involved in polarized cell growth such as septins, and the GTPases Cdc42p, Rho1p, and Sec4p. In addition, Osh proteins are required for a specific pathway of polarized secretion to sites of membrane growth, suggesting that this is how Osh proteins affect Cdc42p- and Rho1p-dependent polarization. Our findings suggest that Osh proteins integrate sterol trafficking and sterol-dependent cell signaling with the control of cell polarization.     

Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Rho; actin; swelling     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

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     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Ras1 Acts through Duplicated Cdc42 and Rac Proteins to Regulate Morphogenesis and Pathogenesis in the Human Fungal Pathogen Cryptococcus neoformans

Proliferation and morphogenesis in eukaryotic cells depend on the concerted activity of Rho-type GTPases, including Ras, Cdc42, and Rac. The sexually dimorphic fungus Cryptococcus neoformans, which encodes paralogous, non-essential copies of all three, provides a unique model in which to examine the interactions of these conserved proteins. Previously, we demonstrated that RAS1 mediates C. neoformans virulence by acting as a central regulator of both thermotolerance and mating. We report here that ras1Δ mutants accumulate defects in polarized growth, cytokinesis, and cell cycle progression. We demonstrate that the ras1Δ defects in thermotolerance and mating can be largely explained by the compromised activity of four downstream Rho-GTPases: the Cdc42 paralogs, Cdc42 and Cdc420; and the Rac paralogs, Rac1 and Rac2. Further, we demonstrate that the separate GTPase classes play distinct Ras-dependent roles in C. neoformans morphogenesis and pathogenesis. Cdc42 paralogs primarily control septin localization and cytokinesis, while Rac paralogs play a primary role in polarized cell growth. Together, these duplicate, related signaling proteins provide a robust system to allow microbial proliferation in the presence of host-derived cell stresses.     

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia.     

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).     

Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK₁₀ and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations. Rac; Cdc42; actin; ezrin; adenosine 5'-triphosphate; guanosine 5'-triphosphate     

Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1. actin cytoskeleton; Cdc42; E-cadherin     

Cross-talk-dependent cortical patterning of Rho GTPases during cell repair

Rho GTPases such as Rho, Rac, and Cdc42 are important regulators of the cortical cytoskeleton in processes including cell division, locomotion, and repair. In these processes, Rho GTPases assume characteristic patterns wherein the active GTPases occupy mutually exclusive “zones” in the cell cortex. During cell wound repair, for example, a Rho zone encircles the wound edge and is in turn encircled by a Cdc42 zone. Here we evaluated the contributions of cross-talk between Rho and Cdc42 to the patterning of their respective zones in wounded Xenopus oocytes using experimental manipulations in combination with mathematical modeling. The results show that the position of the Cdc42 zone relative to the Rho zone and relative to the wound edge is controlled by the level of Rho activity. In contrast, the outer boundary of the Rho zone is limited by the level of Cdc42 activity. Models based on positive feedback within zones and negative feedback from Rho to the GEF-GAP Abr to Cdc42 capture some, but not all, of the observed behaviors. We conclude that GTPase zone positioning is controlled at the level of Rho activity and we speculate that the Cdc42 zone or something associated with it limits the spread of Rho activity.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane.     

Cell migration and signaling specificity is determined by the phosphatidylserine recognition motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.     

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.     

Arabidopsis RopGAPs are a novel family of rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for rop-specific GTPase stimulation

The plant-specific Rop subfamily of Rho GTPases, most closely related to the mammalian Cdc42 and Rac GTPases, plays an important role in the regulation of calcium-dependent pollen tube growth, H(2)O(2)-mediated cell death, and many other processes in plants. In a search for Rop interactors using the two-hybrid method, we identified a family of Rho GTPase-activating proteins (GAP) from Arabidopsis, termed RopGAPs. In addition to a GAP catalytic domain, RopGAPs contain a Cdc42/Rac-interactive binding (CRIB) motif known to allow Cdc42/Rac effector proteins to bind activated Cdc42/Rac. This novel combination of a GAP domain with a CRIB motif is widespread in higher plants and is unique to the regulation of the Rop GTPase. A critical role for CRIB in the regulation of in vitro RopGAP activity was demonstrated using point and deletion mutations. Both types of mutants have drastically reduced capacities to stimulate the intrinsic Rop GTPase activity and to bind Rop. Furthermore, RopGAPs preferentially stimulate the GTPase activity of Rop, but not Cdc42 in a CRIB-dependent manner. In vitro binding assays show that the RopGAP CRIB domain interacts with GTP- and GDP-bound forms of Rop, as well as the transitional state of Rop mimicked by aluminum fluoride. The CRIB domain also promotes the association of the GAP domain with the GDP-bound Rop, as does aluminum fluoride. These results reveal a novel CRIB-dependent mechanism for the regulation of the plant-specific family of Rho GAPs. We propose that the CRIB domain facilitates the formation of or enhanced GAP-mediated stabilization of the transitional state of the Rop GTPase.     

Rho GTPase Expression in Human Myeloid Cells

Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.     

Involvement of Rho GTPases and Their Effectors in the Secretory Process of PC12 Cells

We investigated the involvement of Rho GTPases in the secretory process of PC12 cells. Overexpression of wild-type RhoA, Rac1, or Cdc42 did affect exocytosis. In contrast, secretion elicited by depolarizing K⁺ concentrations was enhanced by the dominant negative mutants RhoA^N19, Rac1^N17, and Cdc42^N17 and was diminished by the constitutively active mutants RhoA^V14, Rac1^V12, and Cdc42^V12. The inhibition observed in the presence of RhoA^V14 was likely a result of the activation of ROKα, since the catalytic domain of this kinase was able to mimic both the reorganization of the actin cytoskeleton and the decrease in exocytosis induced by the RhoA mutant. Part of the effect of Rac1^V12 may be due to POR1 activation. Thus, overexpression of full-length POR1 diminished K⁺-stimulated exocytosis, and a point mutation in the effector domain of Rac1^V12 that prevents the interaction with POR1 abolished the inhibitory effect of the GTPase. We also searched for the Cdc42^V12 target but overexpression of the Cdc42 effector WASP did not mimic the inhibition of exocytosis observed in cells transfected with the activated GTPase. Our findings indicate that different signaling cascades resulting in the activation of RhoA, Rac1, or Cdc42 can modulate the exocytotic process of neuroendocrine cells.     

Burkholderia cenocepacia infection: Disruption of phagocyte immune functions through Rho GTPase inactivation

Phagocytosis is an important component of innate immunity that contributes to the eradication of infectious microorganisms; however, successful bacterial pathogens often evade different aspects of host immune responses. A common bacterial evasion strategy entails the production of toxins and/or effectors that disrupt normal host cell processes and because of their importance Rho-family GTPases are often targeted. Burkholderia cenocepacia, an opportunistic pathogen that has a propensity to infect cystic fibrosis patients, is an example of a pathogenic bacterium that has only recently been shown to disrupt Rho GTPase function in professional phagocytes. More specifically, B. cenocepacia disrupts Rac and Cdc42 seemingly through perturbation of guanine nucleotide exchange factor function. Inactivation of Rac, Cdc42 and conceivably other Rho GTPases seriously compromises phagocyte function.