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Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Here, using the Drosophila melanogaster border cells as a model, we report that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, we found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. We elucidate a molecular mechanism that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. We propose that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior. [ABSTRACT FROM AUTHOR]

Rho GTPases are central regulators of cell polarity and signaling. How Rho GTPases are directed to function in certain settings remains unclear. Here, we show the protein levels of the yeast Rho GTPase Cdc42p are regulated, which impacts a subset of its biological functions. Specifically, the active conformation of Cdc42p was ubiquitinated by the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones and turned over in the proteasome. A GTP-locked (Q61L) turnover-defective (TD) version, Cdc42pQ61L+TD, hyperactivated the MAPK pathway that regulates filamentous growth (fMAPK). Cdc42pQ61L+TD did not influence the activity of the mating pathway, which shares components with the fMAPK pathway. The fMAPK pathway adaptor, Bem4p, stabilized Cdc42p levels, which resulted in elevated fMAPK pathway signaling. Our results identify Cdc42p turnover regulation as being critical for the regulation of a MAPK pathway. The control of Rho GTPase levels by stabilization and turnover may be a general feature of signaling pathway regulation, which can result in the execution of a specific developmental program. [ABSTRACT FROM AUTHOR]

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture. [ABSTRACT FROM AUTHOR]

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbscompromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity. [ABSTRACT FROM AUTHOR]

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0–2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1. [ABSTRACT FROM AUTHOR]

Pulsed actomyosin contractility underlies diverse modes of tissue morphogenesis, but the underlying mechanisms remain poorly understood. Here, we combined quantitative imaging with genetic perturbations to identify a core mechanism for pulsed contractility in early Caenorhabditis elegans embryos. We show that pulsed accumulation of actomyosin is governed by local control of assembly and disassembly downstream of RhoA. Pulsed activation and inactivation of RhoA precede, respectively, the accumulation and disappearance of actomyosin and persist in the absence of Myosin II. We find that fast (likely indirect) autoactivation of RhoA drives pulse initiation, while delayed, F-actin-dependent accumulation of the RhoA GTPase-activating proteins RGA-3/4 provides negative feedback to terminate each pulse. A mathematical model, constrained by our data, suggests that this combination of feedbacks is tuned to generate locally excitable RhoA dynamics. We propose that excitable RhoA dynamics are a common driver for pulsed contractility that can be tuned or coupled differently to actomyosin dynamics to produce a diversity of morphogenetic outcomes. [ABSTRACT FROM AUTHOR]

The formation of a local zone of Cdc42 GTPase activity, which governs cell polarization in many cell types, requires not only local activation but also switch-off mechanisms. In this study, we identify Rga3, a paralog of Rga4, as a novel Cdc42 GTPaseactivating protein (GAP) in the fission yeast Schizosaccharomyces pombe. Contrary to Rga4, Rga3 localizes with Cdc42-GTP to sites of polarity. Rga3 is dispensable for cell polarization during mitotic growth, but it limits the lifetime of unstable Cdc42-GTP patches that underlie cell pairing during sexual reproduction, masking a partly compensatory patch-wandering motion. In consequence, cells lacking rga3 hyperpolarize and lose out in mating competition. Rga3 synergizes with the Cdc42 GAPs Rga4 and Rga6 to restrict Cdc42-GTP zone sizes during mitotic growth. Surprisingly, triple-mutant cells, which are almost fully round, retain pheromone-dependent dynamic polarization of Cdc42-GTP, extend a polarized projection, and mate. Thus, the requirement for Cdc42-GTP hydrolysis by GAPs is distinct during polarization by intrinsic or extrinsic cues. [ABSTRACT FROM AUTHOR]

Inflammation is pivotal to fight infection, clear debris, and orchestrate repair of injured tissues. Although Drosophila melanogaster have proven invaluable for studying extravascular recruitment of innate immune cells (hemocytes) to wounds, they have been somewhat neglected as viable models to investigate a key rate-limiting component of inflammation--that of immune cell extravasation across vessel walls--due to their open circulation. We have now identified a period during pupal development when wing hearts pulse hemolymph, including circulating hemocytes, through developing wing veins. Wounding near these vessels triggers local immune cell extravasation, enabling live imaging and correlative light-electron microscopy of these events in vivo. We show that RNAi knockdown of immune cell integrin blocks diapedesis, just as in vertebrates, and we uncover a novel role for Rho-like signaling through the GPCR Tre1, a gene previously implicated in the trans-epithelial migration of germ cells. We believe this new Drosophila model complements current murine models and provides new mechanistic insight into immune cell extravasation. [ABSTRACT FROM AUTHOR]

ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35. [ABSTRACT FROM AUTHOR]

Microtubule-dependent long-distance movement of peroxisomes occurs in mammalian cells. However, its molecular mechanisms remain undefined. In this study, we identified three distinct splicing variants of human mitochondrial Rho GTPase-1 (Miro1), each containing amino acid sequence insertions 1 (named Miro1-var2), 2 (Miro1-var3), and both 1 and 2 (Miro1-var4), respectively, at upstream of the transmembrane domain. Miro1-var4 and Miro1-var2 are localized to peroxisomes in a manner dependent on the insertion 1 that is recognized by the cytosolic receptor Pex19p. Exogenous expression of Miro1-var4 induces accumulation of peroxisomes at the cell periphery and augments long-range movement of peroxisomes along microtubules. Depletion of all Miro1 variants by knocking down MIRO1 suppresses the long-distance movement of peroxisomes. Such abrogated movement is restored by reexpression of peroxisomal Miro1 variants. Collectively, our findings identify for the first time peroxisome-localized Miro1 variants as adapter proteins that link peroxisomes to the microtubule-dependent transport complexes including TRAK2 in the intracellular translocation of peroxisomes in mammalian cells. [ABSTRACT FROM AUTHOR]

Rho GTPase-based signaling networks control cellular dynamics by coordinating protrusions and retractions in space and time. Here, we reveal a signaling network that generates pulses and propagating waves of cell contractions. These dynamic patterns emerge via self-organization from an activator-inhibitor network, in which the small GTPase Rho amplifies its activity by recruiting its activator, the guanine nucleotide exchange factor GEF-H1. Rho also inhibits itself by local recruitment of actomyosin and the associated RhoGAP Myo9b. This network structure enables spontaneous, self-limiting patterns of subcellular contractility that can explore mechanical cues in the extracellular environment. Indeed, actomyosin pulse frequency in cells is altered by matrix elasticity, showing that coupling of contractility pulses to environmental deformations modulates network dynamics. Thus, our study reveals a mechanism that integrates intracellular biochemical and extracellular mechanical signals into subcellular activity patterns to control cellular contractility dynamics. [ABSTRACT FROM AUTHOR]

Like tissues, single cells are subjected to continual stresses and damage. As such, cells have a robust wound repair mechanism comprised of dynamic membrane resealing and cortical cytoskeletal remodeling. One group of proteins, the Rho family of small guanosine triphosphatases (GTPases), is critical for this actin and myosin cytoskeletal response in which they form distinct dynamic spatial and temporal patterns/arrays surrounding the wound. A key mechanistic question, then, is how these GTPase arrays are formed. Here, we show that in the Drosophila melanogaster cell wound repair model Rho GTPase arrays form in response to prepatterning by Rho guanine nucleotide exchange factors (RhoGEFs), a family of proteins involved in the activation of small GTPases. Furthermore, we show that Annexin B9, a member of a class of proteins associated with the membrane resealing, is involved in an early, Rho family-independent, actin stabilization that is integral to the formation of one RhoGEF array. Thus, Annexin proteins may link membrane resealing to cytoskeletal remodeling processes in single cell wound repair. [ABSTRACT FROM AUTHOR]

Cell fusion is ubiquitous in eukaryotic fertilization and development. The highly conserved Rho-GTPase Cdc42p promotes yeast fusion through interaction with Fus2p, a pheromone-induced amphiphysin-like protein. We show that in prezygotes, Cdc42p forms a novel Fus2p-dependent focus at the center of the zone of cell fusion (ZCF) and remains associated with remnant cell walls after initial fusion. At the ZCF and during fusion, Cdc42p and Fus2p colocalized. In contrast, in shmoos, both proteins were near the cortex but spatially separate. Cdc42p focus formation depends on ZCF membrane curvature: mutant analysis showed that Cdc42p localization is negatively affected by shmoo-like positive ZCF curvature, consistent with the flattening of the ZCF during fusion. BAR-domain proteins such as the fusion proteins Fus2p and Rvs161p are known to recognize membrane curvature. We find that mutations that disrupt binding of the Fus2p/Rvs161p heterodimer to membranes affect Cdc42p ZCF localization. We propose that Fus2p localizes Cdc42p to the flat ZCF to promote cell wall degradation. [ABSTRACT FROM AUTHOR]

Efficient collective migration depends on a balance between contractility and cytoskeletal rearrangements, adhesion, and mechanical cell-cell communication, all controlled by GTPases of the RHO family. By comprehensive screening of guanine nucleotide exchange factors (GEFs) in human bronchial epithelial cell monolayers, we identified GEFs that are required for collective migration at large, such as SOS1 and ß-PIX, and RHOA GEFs that are implicated in intercellular communication. Down-regulation of the latter GEFs differentially enhanced front-to-back propagation of guidance cues through the monolayer and was mirrored by down-regulation of RHOA expression and myosin II activity. Phenotype- based clustering of knockdown behaviors identified RHOA-ARH GEF18 and ARH GEF3-ARH GEF28-ARH GEF11 clusters, indicating that the latter may signal through other RHO-family GTPases. Indeed, knockdown of RHOC produced an intermediate between the two phenotypes. We conclude that for effective collective migration, the RHOA-GEFs → RHOA/C → actomyosin pathways must be optimally tuned to compromise between generation of motility forces and restriction of intercellular communication. [ABSTRACT FROM AUTHOR]

Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1-mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2-protein phosphatase 2ACdc55 (PP2ACdc55) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2-PP2ACdc55 promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1-Mpk1 activity inhibits cortical PP2ACdc55, ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2ACdc55 specifies Rho1 signaling output and that reciprocal antagonism between Rho1-PP2ACdc55 and Rho1-Pkc1 explains how only one signaling pathway is robustly activated at a time. [ABSTRACT FROM AUTHOR]

Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth. [ABSTRACT FROM AUTHOR]

The article focuses on a study which aims to visualize and characterize pulses and waves of Rho activation in adherent cells and to recommend excitable Rho signaling networks underlying cell contractility.

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front-back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1 -driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front-back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines. [ABSTRACT FROM AUTHOR]

Cell-cell contact formation is a dynamic process requiring the coordination of cadherin-based cell-cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell-cell adhesion identified an Elmo-Dock complex. This was unexpected as Elmo-Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dockl to initial cell-cell contacts in Madin-Darby canine kidney cells. At cell-cell contacts, both Elmo2 and Dockl are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell-cell adhesion. Upon completion of cell-cell adhesion, Elmo2 and Dockl no longer localize to cell-cell contacts and are not required subsequently for the maintenance of cell-cell adhesion. These studies show that Elmo-Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell-cell adhesion. [ABSTRACT FROM AUTHOR]

DLC1 is a tumor suppressor protein whose full activity depends on its presence at focal adhesions, its Rho-GTPase activating protein (Rho-GAP) function, and its ability to bind several ligands, including tensin and talin. However, the mechanisms that regulate and coordinate these activities remain poorly understood. Here we identify CDK5, a predominantly cytoplasmic serine/threonine kinase, as an important regulator of DLC1 functions. The CDK5 kinase phosphorylates four serines in DLC1 located N-terminal to the Rho-GAP domain. When not phosphorylated, this N-terminal region functions as an autoinhibitory domain that places DLC1 in a closed, inactive conformation by efficiently binding to the Rho-GAP domain. CDK5 phosphorylation reduces this binding and orchestrates the coordinate activation DLC1, including its localization to focal adhesions, its Rho-GAP activity, and its ability to bind tensin and talin. In cancer, these anti-oncogenic effects of CDK5 can provide selective pressure for the down-regulation of DLC1, which occurs frequently in tumors, and can contribute to the pro-oncogenic activity of CDK5 in lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Although much is known about signaling factors downstream of Rho GTPases that contribute to epidermal differentiation, little is known about which upstream regulatory proteins (guanine nucleotide exchange factors [GEFs] or GTPase-activating proteins [GAPs]) are involved in coordinating Rho signaling in keratinocytes. Here we identify the GEF breakpoint cluster region (Bcr) as a major upstream regulator of RhoA activity, stress fibers, and focal adhesion formation in keratinocytes. Loss of Bcr reduced expression of multiple markers of differentiation (such as desmoglein-1 [Dsg1], keratin-1, and loricrin) and abrogated MAL/SRF signaling in differentiating keratinocytes. We further demonstrated that loss of Bcr or MAL reduced levels of Dsg1 mRNA in keratinocytes, and ectopic expression of Dsg1 rescued defects in differentiation seen upon loss of Bcr or MAL signaling. Taken together, these data identify the GEF Bcr as a regulator of RhoA/MAL signaling in keratinocytes, which in turn promotes differentiation through the desmosomal cadherin Dsg1. [ABSTRACT FROM AUTHOR]

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells. [ABSTRACT FROM AUTHOR]

The activity of Raf-1 and Rok-α kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-α, but the underlying mechanism is unknown. In this study, we show that Rok-α inhibition by Raf-1 relies on an intermolecular interaction between the Rok-α kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1 reg), which is similar to Rok-α's own autoinhibitory region. Thus, Raf-1 mediates Rok-α inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1 reg is sufficient to rescue all Rok-α-dependent defects of Raf-1-deficient cells. Downstream of Ras and Rho, the Raf-1-Rok-α interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility. [ABSTRACT FROM AUTHOR]

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6-atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical-basal polarity in forming epithelia, the Cdc42Par6-aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter's binding to RhoGDI1, which in turn enhances RhoGDI1's affinity for RhoG. S4 clustering activates PKCoα, which phosphorylates RhoGDI1 at Ser96] This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4-synectin-RhoGDI1 protein complex and activation via PKCα. [ABSTRACT FROM AUTHOR]

Maintenance and remodeling of adherens junctions (AJs) and cell shape in epithelia are necessary for the development of functional epithelia and are commonly altered during cancer progression/metastasis. Although formation of nascent AJs has received` much attention, whether shared mechanisms are responsible for the maintenance and remodeling of AJs in dynamic epithelia, particularly in vivo, is not clear. Using clonal analysis in the postmitotic Drosophila melanogaster pupal eye epithelium, we demonstrate that Rho1 is required to maintain AJ integrity independent of its role in sustaining apical cell tension. Rho1 depletion in a remodeling postmitotic epithelium disrupts AJs but only when depleted in adjacent cells. Surprisingly, neither of the Rho effectors, Rok or Dia, is necessary downstream of Rho1 to maintain AJs; instead, Rho1 maintains AJs by inhibiting Drosophila epithelial cadherin endocytosis in a Cdc42/Par6-dependent manner. In contrast, depletion of Rho1 in single cells decreases apical tension, and Rok and myosin are necessary, while Dia function also contributes, downstream of Rho1 to sustain apical cell tension. [ABSTRACT FROM AUTHOR]

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin α subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans. [ABSTRACT FROM AUTHOR]

p120 catenin regulates the activity of the Rho family guanosine triphosphatases (including RhoA aid Rac1) in an adhesion-dependent manner. Through this action, p120 promotes a sessile cellular phenotype when associated with epithelial cadherin (E-cadherin) or a motile phenotype when associated with mesenchymal cadherins. In this study, we show that p120 also exerts significant and diametrically opposing effects on tumor cell growth depending on E-cadherin expression. Endogenous p120 acts to stabilize E-cadherin complexes and to actively promote the tumor-suppressive function of E-cadherin, potently inhibiting Ras activation. Upon E-cadherin loss during tumor progression, the negative regulation of Ras is relieved; under these conditions, endogenous p120 promotes transformed cell growth both in vitro and in vivo by activating a Rac1-mitogen-activated protein kinase signaling pathway normally activated by the adhesion of cells to the extracellular matrix. These data indicate that both E-cadherin and p120 are important regulators of tumor cell growth and imply roles for both proteins in chemoresistance and targeted therapeutics. [ABSTRACT FROM AUTHOR]

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the, ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility. [ABSTRACT FROM AUTHOR]

Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. We show that activation of the G protein--coupled receptor (GPCR) trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gβ as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila melanogaster germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. We show that uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. Our findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion. [ABSTRACT FROM AUTHOR]

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family. [ABSTRACT FROM AUTHOR]

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK[sup -/-] or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK-p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility. [ABSTRACT FROM AUTHOR]

Development, angiogenesis, wound healing, and metastasis all involve the movement of cells in response to changes in the extracellular environment. To determine whether caveolin-1 plays a role in cell migration, we have used fibroblasts from knockout mice. Caveolin-1-deficient cells lose normal cell polarity, exhibit impaired wound healing, and have decreased Rho and increased Rac and Cdc42 GTPase activities. Directional persistency of migration is lost, and the cells show an impaired response to external directional stimuli. Both Src inactivation and p190RhoGAP knockdown restore the wild-type phenotype to caveolin-1-deficient cells, suggesting that caveolin-1 stimulates normal Rho GTP loading through inactivation of the Src-p190RhoGAP pathway. These findings highlight the importance of caveolin-1 in the establishment of cell polarity during directional migration through coordination of the signaling of Src kinase and Rho GTPases. [ABSTRACT FROM AUTHOR]

Salmonella enterica, the cause of food poisoning and typhoid fever, has evolved sophisticated mechanisms to modulate Rho family guanosine triphosphatases (GTPases) to mediate specific cellular responses such as actin remodeling, macropinocytosis, and nuclear responses. These responses are largely the result of the activity of a set of bacterial proteins (SopE, SopE2, and SopB) that, upon delivery into host cells via a type III secretion system, activate specific Rho family GTPases either directly (SopE and SopE2) or indirectly (SopB) through the stimulation of an endogenous exchange factor. We show that different Rho family GTPases play a distinct role in Salmonella-induced cellular responses. In addition, we report that SopB stimulates cellular responses by activating SH3-containing guanine nucleotide exchange factor (SGEF), an exchange factor for RhoG, which we found plays a central role in the actin cytoskeleton remodeling stimulated by Salmonella. These results reveal a remarkable level of complexity in the manipulation of Rho family GTPases by a bacterial pathogen. [ABSTRACT FROM AUTHOR]

αMβ2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. αMβ2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during αMβ2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to αMβ2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of β2, but not of αM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the β2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for β2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling. [ABSTRACT FROM AUTHOR]

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix. [ABSTRACT FROM AUTHOR]

Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rhol- Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis. [ABSTRACT FROM AUTHOR]

The Rho GTPases ploy a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Racl/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidyl- inositol-4,5-bisphosphate (Pl(4,5)P2) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of Pl(4,5)P2 hydrolysis or increased Pl(4,5)P2 generation by overexpression of phosphatidylinositol phosphate kinase l prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of Pl(4,5)P2 dictates the remodeling of actin necessary for completion of phagocytosis. [ABSTRACT FROM AUTHOR]

Tip growth in neuronal cells, plant cells, and fungal hyphae is known to require tip-localized Rho GTPase, calcium, and filamentous actin (F-actin), but how they interact with each other is unclear. The pollen tube is an exciting model to study spatiotemporal regulation of tip growth and F-actin dynamics. An Arobidopsis thaliana Rho family GTPase, ROP1, controls pollen tube growth by regulating apical F-actin dynamics. This paper shows that ROP1 activates two counteracting pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4. RIC4 promotes F-actin assembly, whereas RIC3 activates Ca2+ signaling that leads to F-actin disassembly. Overproduction or depletion of either RIC4 or RIC3 causes tip growth defects that are rescued by overproduction or depletion of RIC3 or RIC4, respectively. Thus, ROP1 controls actin dynamics and tip growth through a check and balance between the two pathways. The dual and antagonistic roles of this GTPase may provide a unifying mechanism by which Rho modulates various processes dependent on actin dynamics in eukaryotic cells. [ABSTRACT FROM AUTHOR]

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vavl/3ko), we show that Vav proteins are required for multiple β2 integrin- dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial β2 activation asVav2/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vavl/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. 132-induced activation of Cdc42, Rac1, and RhoA is defective in Vavl/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. in contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling β2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis. [ABSTRACT FROM AUTHOR]

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference-mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis. [ABSTRACT FROM AUTHOR]

Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross- rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context. [ABSTRACT FROM AUTHOR]

The article discusses a study that examined the role of inhibition of mammalian Rac in releasing cell adhesion at the division plane and constriction of the contractile ring. The researchers first tested a recombinant human CYK4 GAP domain against a panel of small GTPases. Results indicate that the GTPase-activating protein, CYK4, suppresses equatorial cell substrate adhesion by inhibiting Rac and its effectors ARFGEF7 and PAK1/2.

The article discusses the study of Emmanuel Martin and colleagues on behavior of individual epidermal cells during embryonic elongation, in which they found that Rho and Rac GTPases are responsible for regulating myosin contractility during tissue morphogenesis and mentions RhoA and Rac1 pathways.

An introduction is presented in which the editor discusses various reports within the issue on topics including the complexity of the signaling network that controls neurite growth, the extracellular matrix (ECM) properties that control epithelial development, and the capability of the GTPase Rho1 to spur a yeast cell to grow or to repair damage and resist stress.

An introduction to the journal is presented in which the author discusses a report within the issue regarding myosin light chain kinase (MLCK) that controls the number of protrusion extended by crawling cells.