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Sample records for rho gtpases cdc42

  1. Rho GTPase protein Cdc42 is critical for postnatal cartilage development.

    PubMed

    Nagahama, Ryo; Yamada, Atsushi; Tanaka, Junichi; Aizawa, Ryo; Suzuki, Dai; Kassai, Hidetoshi; Yamamoto, Matsuo; Mishima, Kenji; Aiba, Atsu; Maki, Koutaro; Kamijo, Ryutaro

    2016-02-19

    Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 (fl/fl); Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 (fl/fl)) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages. PMID:26820532

  2. The Rho GTPase Cdc42 Is Essential for the Activation and Function of Mature B Cells

    PubMed Central

    Gerasimcik, Natalija; Dahlberg, Carin I. M.; Baptista, Marisa A. P.; Massaad, Michel J.; Geha, Raif S.; Westerberg, Lisa S.

    2015-01-01

    The Rho GTPase Cdc42 coordinates regulation of the actin and the microtubule cytoskeleton by binding and activating the Wiskott–Aldrich syndrome protein. We sought to define the role of intrinsic expression of Cdc42 by mature B cells in their activation and function. Mice with inducible deletion of Cdc42 in mature B cells formed smaller germinal centers and had a reduced Ab response, mostly of low affinity to T cell–dependent Ag, compared with wild-type (WT) controls. Spreading formation of long protrusions that contain F-actin, microtubules, and Cdc42-interacting protein 4, and assumption of a dendritic cell morphology in response to anti-CD40 plus IL-4 were impaired in Cdc42-deficient B cells compared with WT B cells. Cdc42-deficient B cells had an intact migratory response to chemokine in vitro, but their homing to the B cell follicles in the spleen in vivo was significantly impaired. Cdc42-deficient B cells induced a skewed cytokine response in CD4+ T cells, compared with WT B cells. Our results demonstrate a critical role for Cdc42 in the motility of mature B cells, their cognate interaction with T cells, and their differentiation into Ab-producing cells. PMID:25870239

  3. Regulation of hematopoietic stem cell aging by the small RhoGTPase Cdc42

    PubMed Central

    Geiger, Hartmut; Zheng, Yi

    2015-01-01

    Summary Aging of stem cells might be the underlying cause of tissue aging in tissue that in the adult heavily rely on stem cell activity, like the blood forming system. Hematopoiesis, the generation of blood forming cells, is sustained by hematopoietic stem cells. In this review article, we introduce the canonical set of phenotypes associated with aged HSCs, focus on the novel aging-associated phenotype apolarity caused by elevated activity of the small RhoGTPase in aged HSCs, disuccs the role of Cdc42 in hematopoiesis and describe that pharmacological inhibition of Cdc42 activity in aged HSCs results in functionally young and thus rejuvenated HSCs. PMID:25220425

  4. Roles of Aspergillus nidulans Cdc42/Rho GTPase regulators in hyphal morphogenesis and development.

    PubMed

    Si, Haoyu; Rittenour, William R; Harris, Steven D

    2016-01-01

    The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A nidulans. PMID:26932184

  5. The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea

    PubMed Central

    Kirjavainen, Anna; Laos, Maarja; Anttonen, Tommi; Pirvola, Ulla

    2015-01-01

    Hair cells of the organ of Corti (OC) of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubule cytoskeletons. The Rho GTPase Cdc42 regulates cytoskeletal dynamics and polarization of various cell types, and, thus, serves as a candidate regulator of hair cell polarity. We have here induced Cdc42 inactivation in the late-embryonic OC. We show the role of Cdc42 in the establishment of planar polarity of hair cells and in cellular patterning. Abnormal planar polarity was displayed as disturbances in hair bundle orientation and morphology and in kinocilium/basal body positioning. These defects were accompanied by a disorganized cell-surface microtubule network. Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion. Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network. The data also suggest that defects in apical polarization are influenced by disturbed cellular patterning in the OC. In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea. PMID:25770185

  6. Cdc42 GTPase dynamics control directional growth responses

    PubMed Central

    Brand, Alexandra C.; Morrison, Emma; Milne, Stephen; Gonia, Sara; Gale, Cheryl A.; Gow, Neil A. R.

    2014-01-01

    Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca2+ influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca2+ influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca2+. Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca2+. Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca2+. The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca2+ influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca2+-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance. PMID:24385582

  7. Cloning, sequencing and phylogenetic analysis of the small GTPase gene cdc-42 from Ancylostoma caninum.

    PubMed

    Yang, Yurong; Zheng, Jing; Chen, Jiaxin

    2012-12-01

    CDC-42 is a member of the Rho GTPase subfamily that is involved in many signaling pathways, including mitosis, cell polarity, cell migration and cytoskeleton remodeling. Here, we present the first characterization of a full-length cDNA encoding the small GTPase cdc-42, designated as Accdc-42, isolated from the parasitic nematode Ancylostoma caninum. The encoded protein contains 191 amino acid residues with a predicted molecular weight of 21 kDa and displays a high level of identity with the Rho-family GTPase protein CDC-42. Phylogenetic analysis revealed that Accdc-42 was most closely related to Caenorhabditis briggsae cdc-42. Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Danio rerio, Mus musculus and human genomes showed that Accdc-42 is highly conserved. AcCDC-42 demonstrates the highest identity to CDC-42 from C. briggsae (94.2%), and it also exhibits 91.6% identity to CDC-42 from C. elegans and 91.1% from Brugia malayi. Additionally, the transcript of Accdc-42 was analyzed during the different developmental stages of the worm. Accdc-42 was expressed in the L1/L2 larvae, L3 larvae and female and male adults of A. caninum. PMID:23000556

  8. Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

    SciTech Connect

    Logan, Michael R.; Jones, Lynden; Eitzen, Gary

    2010-03-26

    Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P{sub 2} specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.

  9. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    SciTech Connect

    Sato, Mai; Kitaguchi, Tetsuya; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  10. Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans

    PubMed Central

    Neukomm, L J; Zeng, S; Frei, A P; Huegli, P A; Hengartner, M O

    2014-01-01

    The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans. PMID:24632947

  11. Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases.

    PubMed

    Oprea, Tudor I; Sklar, Larry A; Agola, Jacob O; Guo, Yuna; Silberberg, Melina; Roxby, Joshua; Vestling, Anna; Romero, Elsa; Surviladze, Zurab; Murray-Krezan, Cristina; Waller, Anna; Ursu, Oleg; Hudson, Laurie G; Wandinger-Ness, Angela

    2015-01-01

    Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses-using the rotationally constrained carboxylate in R-naproxen-led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and

  12. Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases

    PubMed Central

    Oprea, Tudor I.; Sklar, Larry A.; Agola, Jacob O.; Guo, Yuna; Silberberg, Melina; Roxby, Joshua; Vestling, Anna; Romero, Elsa; Surviladze, Zurab; Murray-Krezan, Cristina; Waller, Anna; Ursu, Oleg; Hudson, Laurie G.; Wandinger-Ness, Angela

    2015-01-01

    Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses—using the rotationally constrained carboxylate in R-naproxen—led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and

  13. Rho GTPases and their effector proteins.

    PubMed Central

    Bishop, A L; Hall, A

    2000-01-01

    Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field. PMID:10816416

  14. Mechanism and rate constants of the Cdc42 GTPase binding with intrinsically disordered effectors.

    PubMed

    Pang, Xiaodong; Zhou, Huan-Xiang

    2016-05-01

    Intrinsically disordered proteins (IDPs) are often involved in signaling and regulatory functions, through binding to cellular targets. Many IDPs undergo disorder-to-order transitions upon binding. Both the binding mechanisms and the magnitudes of the binding rate constants can have functional importance. Previously we have found that the coupled binding and folding of any IDP generally follows a sequential mechanism that we term dock-and-coalesce, whereby one segment of the IDP first docks to its subsite on the target surface and the remaining segments subsequently coalesce around their respective subsites. Here we applied our TransComp method within the framework of the dock-and-coalesce mechanism to dissect the binding kinetics of two Rho-family GTPases, Cdc42 and TC10, with two intrinsically disordered effectors, WASP and Pak1. TransComp calculations identified the basic regions preceding the GTPase binding domains (GBDs) of the effectors as the docking segment. For Cdc42 binding with both WASP and Pak1, the calculated docking rate constants are close to the observed overall binding rate constants, suggesting that basic-region docking is the rate-limiting step and subsequent conformational coalescence of the GBDs on the Cdc42 surface is fast. The possibility that conformational coalescence of the WASP GBD on the TC10 surface is slow warrants further experimental investigation. The account for the differences in binding rate constants among the three GTPase-effector systems and mutational effects therein yields deep physical and mechanistic insight into the binding processes. Our approach may guide the selection of mutations that lead to redesigned binding pathways. Proteins 2016; 84:674-685. © 2016 Wiley Periodicals, Inc. PMID:26879470

  15. Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast

    PubMed Central

    Lee, Mid Eum; Lo, Wing-Cheong; Miller, Kristi E.; Chou, Ching-Shan; Park, Hay-Oak

    2015-01-01

    ABSTRACT Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42–GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on Rsr1 and its GTPase-activating protein (GAP) Bud2. Interestingly, Rga1, a Cdc42 GAP, exhibits transient localization to a site near the bud neck and to the division site during cytokinesis and G1, and this temporal change of Rga1 distribution is necessary for determination of a proper growth site. Mathematical modeling suggests that a proper axis of Cdc42 polarization in haploid cells might be established through a biphasic mechanism involving sequential positive feedback and transient negative feedback. PMID:25908844

  16. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    PubMed Central

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2013-01-01

    Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts. PMID:23524265

  17. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    SciTech Connect

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2013-04-19

    Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm{sup 2}) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

  18. Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

    PubMed Central

    Revilla-Guarinos, M. T.; Martín-García, Rebeca; Villar-Tajadura, M. Antonia; Estravís, Miguel; Coll, Pedro M.; Pérez, Pilar

    2016-01-01

    Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions. PMID:26960792

  19. Pattern formation of Rho GTPases in single cell wound healing

    PubMed Central

    Simon, Cory M.; Vaughan, Emily M.; Bement, William M.; Edelstein-Keshet, Leah

    2013-01-01

    The Rho GTPases—Rho, Rac, and Cdc42—control an enormous variety of processes, many of which reflect activation of these GTPases in spatially confined and mutually exclusive zones. By using mathematical models and experimental results to establish model parameters, we analyze the formation and segregation of Rho and Cdc42 zones during Xenopus oocyte wound repair and the role played by Abr, a dual guanine nucleotide exchange factor–GTPase-activating protein, in this process. The Rho and Cdc42 zones are found to be best represented as manifestations of spatially modulated bistability, and local positive feedback between Abr and Rho can account for the maintenance and dynamic properties of the Rho zone. In contrast, the invocation of an Abr-independent positive feedback loop is required to account for Cdc42 spatial bistability. In addition, the model replicates the results of previous in vivo experiments in which Abr activity is manipulated. Further, simulating the model with two closely spaced wounds made nonintuitive predictions about the Rho and Cdc42 patterns; these predictions were confirmed by experiment. We conclude that the model is a useful tool for analysis of Rho GTPase signaling and that the Rho GTPases can be fruitfully considered as components of intracellular pattern formation systems. PMID:23264464

  20. RhoGAPs and Rho GTPases in platelets.

    PubMed

    Elvers, Margitta

    2016-08-01

    Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets responsible for the formation of filopodia and lamellipodia to strongly increase the platelet surface upon activation. They are involved in platelet activation and aggregate formation including platelet secretion, integrin activation and arterial thrombus formation. The activity of Rho GTPases is tightly controlled by different proteins such as GTPase-activating proteins (GAPs). GAPs stimulate GTP hydrolysis to terminate Rho signaling. The role and impact of GAPs in platelets is not well-defined and many of the RhoGAPs identified are not known to be present in platelets or to have any function in platelets. The recently identified RhoGAPs Oligophrenin1 (OPHN1) and Nadrin regulate the activity of RhoA, Rac1 and Cdc42 and subsequent platelet cytoskeletal reorganization, platelet activation and thrombus formation. In the last years, the analysis of genetically modified mice helped to gain the understanding of Rho GTPases and their regulators in cytoskeletal rearrangements and other Rho mediated cellular processes in platelets. PMID:25639730

  1. Shear stress–induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

    PubMed Central

    Wojciak-Stothard, Beata; Ridley, Anne J.

    2003-01-01

    Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress–induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow. PMID:12719476

  2. Rho GTPases at the crossroad of signaling networks in mammals

    PubMed Central

    Wojnacki, José; Quassollo, Gonzalo; Marzolo, María-Paz; Cáceres, Alfredo

    2014-01-01

    Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization. PMID:24691223

  3. Rho GTPase signalling in cell migration

    PubMed Central

    Ridley, Anne J

    2015-01-01

    Cells migrate in multiple different ways depending on their environment, which includes the extracellular matrix composition, interactions with other cells, and chemical stimuli. For all types of cell migration, Rho GTPases play a central role, although the relative contribution of each Rho GTPase depends on the environment and cell type. Here, I review recent advances in our understanding of how Rho GTPases contribute to different types of migration, comparing lamellipodium-driven versus bleb-driven migration modes. I also describe how cells migrate across the endothelium. In addition to Rho, Rac and Cdc42, which are well known to regulate migration, I discuss the roles of other less-well characterized members of the Rho family. PMID:26363959

  4. Molecular characterisation of the small GTPase CDC42 in the ectomycorrhizal fungus Tuber borchii Vittad.

    PubMed

    Menotta, M; Amicucci, A; Basili, G; Rivero, F; Polidori, E; Sisti, D; Stocchi, V

    2007-01-01

    The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii. PMID:17762910

  5. Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics

    PubMed Central

    Martin, Katrin; Reimann, Andreas; Fritz, Rafael D.; Ryu, Hyunryul; Jeon, Noo Li; Pertz, Olivier

    2016-01-01

    The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility. PMID:26912264

  6. Approaches of targeting Rho GTPases in cancer drug discovery

    PubMed Central

    Lin, Yuan; Zheng, Yi

    2016-01-01

    Introduction Rho GTPases are master regulators of actomyosin structure and dynamics and play pivotal roles in a variety of cellular processes including cell morphology, gene transcription, cell cycle progression and cell adhesion. Because aberrant Rho GTPase signaling activities are widely associated with human cancer, key components of Rho GTPase signaling pathways have attracted increasing interest as potential therapeutic targets. Similar to Ras, Rho GTPases themselves were, until recently, deemed “undruggable” because of structure-function considerations. Several approaches to interfere with Rho GTPase signaling have been explored and show promise as new ways for tackling cancer cells. Areas covered This review focuses on the recent progress in targeting the signaling activities of three prototypical Rho GTPases, i.e. RhoA, Rac1, and Cdc42. The authors describe the involvement of these Rho GTPases, their key regulators and effectors in cancer. Furthermore, the authors discuss the current approaches for rationally targeting aberrant Rho GTPases along their signaling cascades, upstream and downstream of Rho GTPases and posttranslational modifications at a molecular level. Expert opinion To date, while no clinically effective drugs targeting Rho GTPase signaling for cancer treatment are available, tool compounds and lead drugs that pharmacologically inhibit Rho GTPase pathways have shown promise. Small molecule inhibitors targeting Rho GTPase signaling may add new treatment options for future precision cancer therapy, particularly in combination with other anti-cancer agents. PMID:26087073

  7. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

    PubMed

    Lv, Qi; Zhu, Xian-Yang; Xia, Yu-Feng; Dai, Yue; Wei, Zhi-Feng

    2015-11-01

    Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK. PMID:26614458

  8. Distinct roles of Rho1, Cdc42, and Cyk3 in septum formation and abscission during yeast cytokinesis

    PubMed Central

    Onishi, Masayuki; Ko, Nolan; Nishihama, Ryuichi

    2013-01-01

    In yeast and animal cytokinesis, the small guanosine triphosphatase (GTPase) Rho1/RhoA has an established role in formation of the contractile actomyosin ring, but its role, if any, during cleavage-furrow ingression and abscission is poorly understood. Through genetic screens in yeast, we found that either activation of Rho1 or inactivation of another small GTPase, Cdc42, promoted secondary septum (SS) formation, which appeared to be responsible for abscission. Consistent with this hypothesis, a dominant-negative Rho1 inhibited SS formation but not cleavage-furrow ingression or the concomitant actomyosin ring constriction. Moreover, Rho1 is temporarily inactivated during cleavage-furrow ingression; this inactivation requires the protein Cyk3, which binds Rho1-guanosine diphosphate via its catalytically inactive transglutaminase-like domain. Thus, unlike the active transglutaminases that activate RhoA, the multidomain protein Cyk3 appears to inhibit activation of Rho1 (and thus SS formation), while simultaneously promoting cleavage-furrow ingression through primary septum formation. This work suggests a general role for the catalytically inactive transglutaminases of fungi and animals, some of which have previously been implicated in cytokinesis. PMID:23878277

  9. Formins as effector proteins of Rho GTPases

    PubMed Central

    Kühn, Sonja; Geyer, Matthias

    2014-01-01

    Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells. PMID:24914801

  10. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  11. The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae

    PubMed Central

    Chen, Guang-Chao; Kim, Yung-Jin; Chan, Clarence S.M.

    1997-01-01

    BEM2 of Saccharomyces cerevisiae encodes a Rho-type GTPase-activating protein that is required for proper bud site selection at 26°C and for bud emergence at elevated temperatures. We show here that the temperature-sensitive growth phenotype of bem2 mutant cells can be suppressed by increased dosage of the GIC1 gene. The Gic1 protein, together with its structural homolog Gic2, are required for cell size and shape control, bud site selection, bud emergence, actin cytoskeletal organization, mitotic spindle orientation/positioning, and mating projection formation in response to mating pheromone. Each protein contains a CRIB (Cdc42/Rac-interactive binding) motif and each interacts in the two-hybrid assay with the GTP-bound form of the Rho-type Cdc42 GTPase, a key regulator of polarized growth in yeast. The CRIB motif of Gic1 and the effector domain of Cdc42 are required for this association. Genetic experiments indicate that Gic1 and Gic2 play positive roles in the Cdc42 signal transduction pathway, probably as effectors of Cdc42. Subcellular localization studies with a functional green fluorescent protein–Gic1 fusion protein indicate that this protein is concentrated at the incipient bud site of unbudded cells, at the bud tip and mother-bud neck of budded cells, and at cortical sites on large-budded cells that may delimit future bud sites in the two progeny cells. The ability of Gic1 to associate with Cdc42 is important for its function but is apparently not essential for its subcellular localization. PMID:9367979

  12. miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

    PubMed Central

    Franke, Kristin; Otto, Wolfgang; Johannes, Sascha; Baumgart, Jan; Nitsch, Robert; Schumacher, Stefan

    2012-01-01

    The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching. PMID:22588079

  13. miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling.

    PubMed

    Franke, Kristin; Otto, Wolfgang; Johannes, Sascha; Baumgart, Jan; Nitsch, Robert; Schumacher, Stefan

    2012-06-29

    The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching. PMID:22588079

  14. Locally excitable Cdc42 signals steer cells during chemotaxis

    PubMed Central

    Meyer, Tobias

    2016-01-01

    Neutrophils and other amoeboid cells chemotax by steering their front towards chemoattractant. While Ras, Rac, Cdc42, and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, preexisting local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement upon uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, while Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals. PMID:26689677

  15. Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases

    PubMed Central

    Shang, Xun; Marchioni, Fillipo; Sipes, Nisha; Evelyn, Chris R.; Jerabek-Willemsen, Moran; Duhr, Stefan; Seibel, William; Wortman, Matthew; Zheng, Yi

    2012-01-01

    SUMMARY Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho specific inhibitor, Rhosin. Rhosin contains two-aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase. PMID:22726684

  16. Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins

    PubMed Central

    Schlam, Daniel; Bagshaw, Richard D.; Freeman, Spencer A.; Collins, Richard F.; Pawson, Tony; Fairn, Gregory D.; Grinstein, Sergio

    2015-01-01

    Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large—but not small—phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles. PMID:26465210

  17. Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes.

    PubMed

    Kiwanuka, Elizabeth; Lee, Cameron Cy; Hackl, Florian; Caterson, Edward J; Junker, Johan Pe; Gerdin, Bengt; Eriksson, Elof

    2016-06-01

    Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration-promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basic mechanism of CCN2-induced cell migration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re-orientation of the Golgi. In vitro assays were used to show that CCN2-induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2-treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post-treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases. PMID:25185742

  18. Novel role of Cdc42 and RalA GTPases in TNF-α mediated secretion of CCL2

    PubMed Central

    Langert, Kelly A; Pervan, Cynthia L; Stubbs, Jr, Evan B

    2014-01-01

    Transendothelial migration of autoreactive leukocytes into peripheral nerves is an early pathological hallmark of acute inflammatory demyelinating polyneuropathy (AIDP), a North American and European variant of Guillain-Barré Syndrome. Whereas the clinical management of AIDP is currently limited to non-selective immune modulating therapies, recent experimental studies support selective targeting of leukocyte trafficking as a promising alternative therapeutic strategy. Here, using a combination of targeted siRNA knockdown and pharmacological inhibitors, we report a novel role of both Cdc42 and RalA GTPases in facilitating TNF-α mediated CCL2 trafficking and release from immortalized rat peripheral nerve microvascular endoneurial endothelial cells. These findings raise interest in Cdc42 and RalA GTPases as potential therapeutic targets for the management of autoimmune inflammatory peripheral nerve disease. PMID:24911990

  19. Therapeutic effects of the Rho GTPase modulator CNF1 in a model of Parkinson's disease.

    PubMed

    Musilli, Marco; Ciotti, Maria Teresa; Pieri, Massimo; Martino, Assunta; Borrelli, Sonia; Dinallo, Vincenzo; Diana, Giovanni

    2016-10-01

    Recent evidence suggests an early involvement of dopaminergic (DA) processes and terminals in Parkinson's disease (PD). The arborization of neurons depends on the actin cytoskeleton, which in turn is regulated by small GTPases of the Rho family, encompassing Rho, Rac and Cdc42 subfamilies. Indeed, some reports point to a role for Rac and Cdc42 signaling in the pathophysiology of inherited parkinsonisms. We thus investigated the potential therapeutic effect of the modulation of cerebral Rho GTPases in PD. Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa protein toxin produced by Escherichia coli, permanently activates RhoA, Rac1 and Cdc42 in intact cells. We report that the modulation of Rho GTPases by CNF1 results in hypertrophy of DA cell processes of cultured substantia nigra neurons, including increase in length, branching and varicosity. In vivo, the treatment corrects long-standing motor and biochemical asymmetries and restores degenerated nigrostriatal DA tissue after 6-hydroxydopamine lesion. We conclude that the pharmacological modulation of Rho GTPases shows neurorestorative potential and represents a promising avenue in the treatment PD. The study also suggests that naturally occurring molecules acting on Rho GTPase signaling, such as some bacterial protein toxins, might play a role in the development of PD. PMID:27350290

  20. Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure.

    PubMed

    Toda, Akiyuki; Tsurumura, Toshiharu; Yoshida, Toru; Tsumori, Yayoi; Tsuge, Hideaki

    2015-08-01

    C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD(+) to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3. PMID:26067270

  1. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration

    PubMed Central

    Stankiewicz, Trisha R.; Linseman, Daniel A.

    2014-01-01

    The Rho family of GTPases belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. The most extensively studied members are RhoA, Rac1, and Cdc42. In the last few decades, studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. Indeed, Rho GTPases mediate many diverse critical cellular processes, such as gene transcription, cell–cell adhesion, and cell cycle progression. However, Rho GTPases also play an essential role in regulating neuronal morphology. In particular, Rho GTPases regulate dendritic arborization, spine morphogenesis, growth cone development, and axon guidance. In addition, more recent efforts have underscored an important function for Rho GTPases in regulating neuronal survival and death. Interestingly, Rho GTPases can exert either a pro-survival or pro-death signal in neurons depending upon both the cell type and neurotoxic insult involved. This review summarizes key findings delineating the involvement of Rho GTPases and their effectors in the regulation of neuronal survival and death. Collectively, these results suggest that dysregulation of Rho family GTPases may potentially underscore the etiology of some forms of neurodegenerative disease such as amyotrophic lateral sclerosis. PMID:25339865

  2. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration.

    PubMed

    Stankiewicz, Trisha R; Linseman, Daniel A

    2014-01-01

    The Rho family of GTPases belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. The most extensively studied members are RhoA, Rac1, and Cdc42. In the last few decades, studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. Indeed, Rho GTPases mediate many diverse critical cellular processes, such as gene transcription, cell-cell adhesion, and cell cycle progression. However, Rho GTPases also play an essential role in regulating neuronal morphology. In particular, Rho GTPases regulate dendritic arborization, spine morphogenesis, growth cone development, and axon guidance. In addition, more recent efforts have underscored an important function for Rho GTPases in regulating neuronal survival and death. Interestingly, Rho GTPases can exert either a pro-survival or pro-death signal in neurons depending upon both the cell type and neurotoxic insult involved. This review summarizes key findings delineating the involvement of Rho GTPases and their effectors in the regulation of neuronal survival and death. Collectively, these results suggest that dysregulation of Rho family GTPases may potentially underscore the etiology of some forms of neurodegenerative disease such as amyotrophic lateral sclerosis. PMID:25339865

  3. Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling

    PubMed Central

    Zhan, Rixing; He, Weifeng; Wang, Fan; Yao, Zhihui; Tan, Jianglin; Xu, Rui; Zhou, Junyi; Wang, Yuzhen; Li, Haisheng; Wu, Jun; LUO, Gaoxing

    2016-01-01

    The migration and reepithelization of epidermal stem cells (ESCs) are the most critical processes in wound healing. The gaseous messenger nitric oxide (NO) has multiple biological effects, but its actions on ESCs are poorly understood. In this study, an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), was found to facilitate the in vitro migration of human ESCs (huESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases RhoA and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of cGMP, PKG, RhoA or Rac1, and by a specific siRNA of RhoA or Rac1, but not by a Cdc42 inhibitor or siRNA. Furthermore, the roles of NO in ESC migration via cGMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine (BrdU)-labelled cells in a superficial, partial-thickness scald mouse model. Thus, the present study demonstrated that the NO donor SNAP could promote huESC migration in vitro. Furthermore, NO was found to induce ESC migration via cGMP-Rho GTPase RhoA and Rac1 signalling, but not Cdc42 signalling, both in vivo and in vitro. PMID:27469024

  4. The interdependence of the Rho GTPases and apicobasal cell polarity

    PubMed Central

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. PMID:25469537

  5. The interdependence of the Rho GTPases and apicobasal cell polarity.

    PubMed

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. PMID:25469537

  6. Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1.

    PubMed

    Norden, Pieter R; Kim, Dae Joong; Barry, David M; Cleaver, Ondine B; Davis, George E

    2016-01-01

    A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network

  7. Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1

    PubMed Central

    Norden, Pieter R.; Kim, Dae Joong; Barry, David M.; Cleaver, Ondine B.; Davis, George E.

    2016-01-01

    A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network

  8. The Rho GTPase Family Genes in Bivalvia Genomes: Sequence, Evolution and Expression Analysis

    PubMed Central

    Li, Xue; Wang, Ruijia; Xun, Xiaogang; Jiao, Wenqian; Zhang, Mengran; Wang, Shuyue; Wang, Shi; Zhang, Lingling; Huang, Xiaoting; Hu, Xiaoli; Bao, Zhenmin

    2015-01-01

    Background Rho GTPases are important members of the Ras superfamily, which represents the largest signaling protein family in eukaryotes, and function as key molecular switches in converting and amplifying external signals into cellular responses. Although numerous analyses of Rho family genes have been reported, including their functions and evolution, a systematic analysis of this family has not been performed in Mollusca or in Bivalvia, one of the most important classes of Mollusca. Results In this study, we systematically identified and characterized a total set (Rho, Rac, Mig, Cdc42, Tc10, Rnd, RhoU, RhoBTB and Miro) of thirty Rho GTPase genes in three bivalve species, including nine in the Yesso scallop Patinopecten yessoensis, nine in the Zhikong scallop Chlamys farreri, and twelve in the Pacific oyster Crassostrea gigas. Phylogenetic analysis and interspecies comparison indicated that bivalves might possess the most complete types of Rho genes in invertebrates. A multiple RNA-seq dataset was used to investigate the expression profiles of bivalve Rho genes, revealing that the examined scallops share more similar Rho expression patterns than the oyster, whereas more Rho mRNAs are expressed in C. farreri and C. gigas than in P. yessoensis. Additionally, Rho, Rac and Cdc42 were found to be duplicated in the oyster but not in the scallops. Among the expanded Rho genes of C. gigas, duplication pairs with high synonymous substitution rates (Ks) displayed greater differences in expression. Conclusion A comprehensive analysis of bivalve Rho GTPase family genes was performed in scallop and oyster species, and Rho genes in bivalves exhibit greater conservation than those in any other invertebrate. This is the first study focusing on a genome-wide characterization of Rho GTPase genes in bivalves, and the findings will provide a valuable resource for a better understanding of Rho evolution and Rho GTPase function in Bivalvia. PMID:26633655

  9. Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

    PubMed Central

    Gong, Ting; Liao, Yuan; He, Fei; Yang, Yang; Yang, Dan-Dan; Chen, Xiang-Dong

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rhorho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rhorho4Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rhorho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4. PMID:23264647

  10. Silencing of CDC42 inhibits neuroblastoma cell proliferation and transformation

    PubMed Central

    Lee, Sora; Craig, Brian T.; Romain, Carmelle V.; Qiao, Jingbo; Chung, Dai H.

    2014-01-01

    Cell division cycle 42 (CDC42), a small GTPase of the Rho-subfamily, regulates diverse cellular functions including proliferation, cytoskeletal rearrangement and even promotes malignant transformation. Here, we found that increased expression of CDC42 correlated with undifferentiated neuroblastoma as compared to a more benign phenotype. CDC42 inhibition decreased cell growth and soft agar colony formation, and increased cell death in BE(2)-C and BE(2)-M17 cell lines, but not in SK-N-AS. In addition, silencing of CDC42 decreased expression of N-myc in BE(2)-C and BE(2)-M17 cells. Our findings suggest that CDC42 may play a role in the regulation of aggressive neuroblastoma behavior. PMID:25264923

  11. Structural Mechanisms and Drug Discovery Prospects of Rho GTPases

    PubMed Central

    Smithers, Cameron C.; Overduin, Michael

    2016-01-01

    Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies. PMID:27304967

  12. Epithelial junctions and Rho family GTPases: the zonular signalosome

    PubMed Central

    Citi, Sandra; Guerrera, Diego; Spadaro, Domenica; Shah, Jimit

    2014-01-01

    The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of “zonular signalosome” is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors. PMID:25483301

  13. Structural Mechanisms and Drug Discovery Prospects of Rho GTPases.

    PubMed

    Smithers, Cameron C; Overduin, Michael

    2016-01-01

    Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies. PMID:27304967

  14. Small rho GTPases mediate tumor-induced inhibition of endocytic activity of dendritic cells.

    PubMed

    Tourkova, Irina L; Shurin, Galina V; Wei, Sheng; Shurin, Michael R

    2007-06-15

    The generation, maturation, and function of dendritic cells (DC) have been shown to be markedly compromised in the tumor microenvironment in animals and humans. However, the molecular mechanisms and intracellular pathways involved in the regulation of the DC system in cancer are not yet fully understood. Recently, we have reported on the role of the small Rho GTPase family members Cdc42, Rac1, and RhoA in regulating DC adherence, motility, and Ag presentation. To investigate involvement of small Rho GTPases in dysregulation of DC function by tumors, we next evaluated how Cdc42, Rac1, and RhoA regulated endocytic activity of DC in the tumor microenvironment. We revealed a decreased uptake of dextran 40 and polystyrene beads by DC generated in the presence of different tumor cell lines, including RM1 prostate, MC38 colon, 3LL lung, and B7E3 oral squamous cell carcinomas in vitro and by DC prepared from tumor-bearing mice ex vivo. Impaired endocytic activity of DC cocultured with tumor cells was associated with decreased levels of active Cdc42 and Rac1. Transduction of DC with the dominant negative Cdc42 and Rac1 genes also led to reduced phagocytosis and receptor-mediated endocytosis. Furthermore, transduction of DC with the constitutively active Cdc42 and Rac1 genes restored endocytic activity of DC that was inhibited by the tumors. Thus, our results suggest that tumor-induced dysregulation of endocytic activity of DC is mediated by reduced activity of several members of the small Rho GTPase family, which might serve as new targets for improving the efficacy of DC vaccines. PMID:17548616

  15. A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction.

    PubMed

    Zhang, Wenwu; Huang, Youliang; Wu, Yidi; Gunst, Susan J

    2015-02-01

    Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist. PMID:25531582

  16. Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

    SciTech Connect

    Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie; Burridge, Keith; Siderovski, David P.

    2012-08-10

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

  17. Deregulation of Rho GTPases in cancer

    PubMed Central

    Porter, Andrew P.; Papaioannou, Alexandra; Malliri, Angeliki

    2016-01-01

    ABSTRACT In vitro and in vivo studies and evidence from human tumors have long implicated Rho GTPase signaling in the formation and dissemination of a range of cancers. Recently next generation sequencing has identified direct mutations of Rho GTPases in human cancers. Moreover, the effects of ablating genes encoding Rho GTPases and their regulators in mouse models, or through pharmacological inhibition, strongly suggests that targeting Rho GTPase signaling could constitute an effective treatment. In this review we will explore the various ways in which Rho signaling can be deregulated in human cancers. PMID:27104658

  18. Rho GTPases have diverse effects on the organization of the actin filament system.

    PubMed Central

    Aspenström, Pontus; Fransson, Asa; Saras, Jan

    2004-01-01

    The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases. PMID:14521508

  19. Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility.

    PubMed

    Friesland, Amy; Zhao, Yaxue; Chen, Yan-Hua; Wang, Lie; Zhou, Huchen; Lu, Qun

    2013-01-22

    Signaling through the Rho family of small GTPases has been intensely investigated for its crucial roles in a wide variety of human diseases. Although RhoA and Rac1 signaling pathways are frequently exploited with the aid of effective small molecule modulators, studies of the Cdc42 subclass have lagged because of a lack of such means. We have applied high-throughput in silico screening and identified compounds that are able to fit into the surface groove of Cdc42, which is critical for guanine nucleotide exchange factor binding. Based on the interaction between Cdc42 and intersectin (ITSN), a specific Cdc42 guanine nucleotide exchange factor, we discovered compounds that rendered ITSN-like interactions in the binding pocket. By using in vitro binding and imaging as well as biochemical and cell-based assays, we demonstrated that ZCL278 has emerged as a selective Cdc42 small molecule modulator that directly binds to Cdc42 and inhibits its functions. In Swiss 3T3 fibroblast cultures, ZCL278 abolished microspike formation and disrupted GM130-docked Golgi structures, two of the most prominent Cdc42-mediated subcellular events. ZCL278 reduces the perinuclear accumulation of active Cdc42 in contrast to NSC23766, a selective Rac inhibitor. ZCL278 suppresses Cdc42-mediated neuronal branching and growth cone dynamics as well as actin-based motility and migration in a metastatic prostate cancer cell line (i.e., PC-3) without disrupting cell viability. Thus, ZCL278 is a small molecule that specifically targets Cdc42-ITSN interaction and inhibits Cdc42-mediated cellular processes, thus providing a powerful tool for research of Cdc42 subclass of Rho GTPases in human pathogenesis, such as those of cancer and neurological disorders. PMID:23284167

  20. Rapid Remodeling of Invadosomes by Gi-coupled Receptors: DISSECTING THE ROLE OF Rho GTPases.

    PubMed

    Kedziora, Katarzyna M; Leyton-Puig, Daniela; Argenzio, Elisabetta; Boumeester, Anja J; van Butselaar, Bram; Yin, Taofei; Wu, Yi I; van Leeuwen, Frank N; Innocenti, Metello; Jalink, Kees; Moolenaar, Wouter H

    2016-02-26

    Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA1 receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca(2+) signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through Gi, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a Gi-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance. PMID:26740622

  1. Antipsychotic drugs induce cell cytoskeleton reorganization in glial and neuronal cells via Rho/Cdc42 signal pathway.

    PubMed

    Chen, Mao-Liang; Tsai, Fu-Ming; Lee, Ming-Cheng; Lin, Yi-Yin

    2016-11-01

    Long-term administration of antipsychotic drugs (APDs) has been theorized to effect drug-induced changes in protein expression in the brain. Our previous findings revealed that ADPs can regulate Rho GDP-dissociation inhibitor 1 (RhoGDI1) expression in glial cells. To reveal whether APDs (haloperidol, risperidone, and clozapine) might regulate cell functions in rat brain by affecting RhoGDI1, RhoGDI1 regulation, RhoGDI1-related Rho family protein, and also MLC2 in brain of 7-day APD treatment rat were examined. Increased expression of RhoGDI1 and RhoA and decreased expression of MLC2, p-MLC2 and ARP2/3 were found in the cortex of APD-treated rats. The activation of RhoA in APD-treated rat cortex was also found. The regulation of RhoGDI1-induced protein expression and its relation to intracellular stress filament production and cell migration were further examined in APD-treated C6 and B35 cells. APD-induced RhoA expression and activation in C6 cells and Cdc42 expression and activation in B35 cells were investigated. In C6 cells, ARP2/3, ROCK1, pMLC2, and PFN1 expressions were decreased, and N-WASP expression was increased by any of the three APDs. In B35 cells, haloperidol decreased ROCK1 expression, but risperidone increased ROCK1 expression. MLC2, p-MLC2, and PFN1 expressions were decreased in B35 cells treated with either risperidone or clozapine. N-WASP expression was decreased by haloperidol and clozapine. We also found all three APDs enhance C6 and B35 F-actin condensation and migration ability. PMID:27302765

  2. Regulating Rho GTPases and their regulators.

    PubMed

    Hodge, Richard G; Ridley, Anne J

    2016-08-01

    Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases. PMID:27301673

  3. RhoGDI-1 modulation of the activity of monomeric RhoGTPase RhoA regulates endothelial barrier function in mouse lungs.

    PubMed

    Gorovoy, Matvey; Neamu, Radu; Niu, Jiaxin; Vogel, Stephen; Predescu, Dan; Miyoshi, Jun; Takai, Yoshimi; Kini, Vidisha; Mehta, Dolly; Malik, Asrar B; Voyno-Yasenetskaya, Tatyana

    2007-07-01

    Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1(-/-) mice. We observed that basal endothelial permeability in lungs of RhoGDI-1(-/-) mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1(-/-) lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1-mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome. PMID:17525371

  4. Cdc42p Is Activated during Vacuole Membrane Fusion in a Sterol-dependent Subreaction of Priming*

    PubMed Central

    Jones, Lynden; Tedrick, Kelly; Baier, Alicia; Logan, Michael R.; Eitzen, Gary

    2010-01-01

    Cdc42p is a Rho GTPase that initiates signaling cascades at spatially defined intracellular sites for many cellular functions. We have previously shown that Cdc42p is localized to the yeast vacuole where it initiates actin polymerization during membrane fusion. Here we examine the activation cycle of Cdc42p during vacuole membrane fusion. Expression of either GTP- or GDP-locked Cdc42p mutants caused several morphological defects including enlarged cells and fragmented vacuoles. Stimulation of multiple rounds of fusion enhanced vacuole fragmentation, suggesting that cycles of Cdc42p activation, involving rounds of GTP binding and hydrolysis, are required to propagate Cdc42p signaling. We developed an assay to directly examine Cdc42p activation based on affinity to a probe derived from the p21-activated kinase effector, Ste20p. Cdc42p was rapidly activated during vacuole membrane fusion, which kinetically coincided with priming subreaction. During priming, Sec18p ATPase activity dissociates SNARE complexes and releases Sec17p, however, priming inhibitors such as Sec17p and Sec18p ligands did not block Cdc42p activation. Therefore, Cdc42p activation seems to be a parallel subreaction of priming, distinct from Sec18p activity. Specific mutants in the ergosterol synthesis pathway block both Sec17p release and Cdc42p activation. Taken together, our results define a novel sterol-dependent subreaction of vacuole priming that activates cycles of Cdc42p activity to facilitate membrane fusion. PMID:20007700

  5. Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in Yeast.

    PubMed

    Bender, L; Lo, H S; Lee, H; Kokojan, V; Peterson, V; Bender, A

    1996-05-01

    The src homology region 3 (SH3) domain-bearing protein Bem1p and the Rho-type GTPase Cdc42p are important for bud emergence in Saccharomyces cervisiae. Here, we present evidence that through its second SH3 domain, Bem1p binds to the structurally and functionally similar proteins Boi1p and Boi2p, each of which contain an SH3 and pleckstrin homology (PH) domain. Deletion of BOI1 and BO12 together leads to impaired morphogenesis and poor ability. A PH domain-bearing segment of Boi1p that lacks the Bem1p-binding site is necessary and sufficient for function. This segment of Boi1p displays a two-hybrid interaction with Cdc42p, suggesting that Boi1p either binds directly to or is part of a larger complex that contains Cdc42p. Consistent with these possibilities, overexpression of Boi1p inhibits bud emergence, but this inhibition is counteracted by cooverexpression of Cdc42p. Increased expression of the Rho-type GTPase Rho3p, which is implicated in bud growth defects of boil boi2 mutants, suggesting that Boi1p and Boi2p may also play roles in the activation or function of Rho3p. These findings provide an example of a tight coupling in function between PH domain-bearing proteins and both Rho-type GTPases and SH3 domain-containing proteins, and they raise the possibility that Boi1p and Boi2 play a role in linking the actions of Cdc42p and Rho3p. PMID:8666672

  6. Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma

    PubMed Central

    Kumar, Ambrish; Al-Sammarraie, Nadia; DiPette, Donald J.; Singh, Ugra S.

    2014-01-01

    Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway. PMID:25365944

  7. Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration

    PubMed Central

    2013-01-01

    Introduction The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast tumors compared to normal breast tissue. Cdc42 regulates key processes that are critical for mammary gland morphogenesis and become disrupted during the development, progression, and metastasis of breast cancer. However, the contribution of Cdc42 to normal and neoplastic mammary gland development in vivo remains poorly understood. We were therefore interested in investigating the effects of Cdc42 overexpression on mammary gland morphogenesis as a first step toward understanding how its overexpression may contribute to mammary tumorigenesis. Methods We developed a tetracycline-regulatable Cdc42 overexpression mouse model in which Cdc42 can be inducibly overexpressed in the developing mammary gland. The effects of Cdc42 overexpression during postnatal mammary gland development were investigated using in vivo and in vitro approaches, including morphometric analysis of wholemounted mammary glands, quantification of histological markers, and primary mammary epithelial cell (MEC) functional and biochemical assays. Results Analysis of Cdc42-overexpressing mammary glands revealed abnormal terminal end bud (TEB) morphologies, characterized by hyperbudding and trifurcation, and increased side branching within the ductal tree. Quantification of markers of proliferation and apoptosis suggested that these phenotypes were not due to increased cell proliferation or survival. Rather, Cdc42 overexpressing MECs were more migratory and contractile and formed dysmorphic, invasive acini in three-dimensional cultures. Cdc42 and RhoA activities, phosphorylated myosin light chain, and MAPK signaling, which contribute to migration and invasion, were markedly elevated in Cdc42 overexpressing MECs. Interestingly, Cdc42 overexpressing mammary glands displayed several features associated with altered epithelial-stromal interactions, which are known to regulate branching morphogenesis. These included increased

  8. CDC42 inhibition suppresses progression of incipient intestinal tumors

    PubMed Central

    Sakamori, Ryotaro; Yu, Shiyan; Zhang, Xiao; Hoffman, Andrew; Sun, Jiaxin; Das, Soumyashree; Vedula, Pavan; Li, Guangxun; Fu, Jiang; Walker, Francesca; Yang, Chung S.; Yi, Zheng; Hsu, Wei; Yu, Da-Hai; Shen, Lanlan; Rodriguez, Alexis J.; Taketo, Makoto M.; Bonder, Edward M.; Verzi, Michael P.; Gao, Nan

    2014-01-01

    Mutations in the APC or β-catenin genes are well established initiators of colorectal cancer (CRC), yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacological approaches in mouse CRC and human CRC xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or β-catenin mutations. Similarly, human CRC with relatively higher levels of CDC42 activity were particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem-cell-enriched Rho family exchange factor Arhgef4. Our results suggest that early-stage mutant intestinal epithelial cells must recruit the pleiotropic functions of Cdc42 for malignant progression, suggesting its relevance as a biomarker and therapeutic target for selective CRC intervention. PMID:25113996

  9. Cdc42 is critical for cartilage development during endochondral ossification.

    PubMed

    Suzuki, Wataru; Yamada, Atsushi; Aizawa, Ryo; Suzuki, Dai; Kassai, Hidetoshi; Harada, Takeshi; Nakayama, Mutsuko; Nagahama, Ryo; Maki, Koutaro; Takeda, Shu; Yamamoto, Matsuo; Aiba, Atsu; Baba, Kazuyoshi; Kamijo, Ryutaro

    2015-01-01

    Cdc42 is a widely expressed protein that belongs to the family of Rho GTPases and controls a broad variety of signal transduction pathways in a variety of cell types. To investigate the physiological functions of Cdc42 during cartilage development, we generated chondrocyte-specific inactivated Cdc42 mutant mice (Cdc42(fl/fl); Col2-Cre). The gross morphology of mutant neonates showed shorter limbs and body as compared with the control mice (Cdc42(fl/fl)). Skeletal preparations stained with alcian blue and alizarin red also revealed that the body and the long bone length of the mutants were shorter than those of the control mice. Furthermore, severe defects were found in growth plate chondrocytes in the femur sections of mutant mice, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes. The expression levels of chondrocyte marker genes, such as Col2, Col10, and Mmp13, in mutant mice were decreased as compared with the control mice. Mineralization of trabecular bones in the femur sections was also decreased in the mutants as compared with control mice, whereas osteoid volume was increased. Together these results suggested that chondrocyte proliferation and differentiation in growth plates in the present mutant mice were not normally organized, which contributed to abnormal bone formation. We concluded that Cdc42 is essential for cartilage development during endochondral bone formation. PMID:25343271

  10. Rho 1 GTPase activates the (1-3)beta-D-glucan synthase and is involved in Schizosaccharomyces pombe morphogenesis.

    PubMed Central

    Arellano, M; Durán, A; Pérez, P

    1996-01-01

    The Schizosaccharomyces pombe Cdc42 and Rho1 GTPases were tested for their ability to complement the cwg2-1 mutant phenotype of a decrease in (1-3)beta-D-glucan synthase activity when grown at the non-permissive temperature. Only Rho1 is able to partly complement the defect in glucan synthase associated with the cwg2-1 mutation. Moreover, overexpression of the rho1 gene in wild-type S.pombe cells causes aberrant morphology with loss of polarity and cells with several septa. Under this condition (1-3)beta-D-glucan synthase activity is increased four times, but is still dependent on GTP. When S.pombe is transformed with constitutively active rho1 mutant alleles (rho1-G15V or rho1-Q64L), cells stop growing and show a very thick cell wall with hardly any septum. Under this condition the level of (1-3)beta-D-glucan synthase activity is at least 20 times higher than wild-type and is independent of GTP. Neither cdc42+ nor the cdc42-V12G or cdc42-Q61L constitutively active mutant alleles affect (1-3)beta-D-glucan synthase activity when overexpressed in S.pombe. Cells overproducing Rho1 are hypersensitive to inhibitors of cell wall biosynthesis or to cell wall degrading enzymes. We conclude that Rho1 GTPase directly activates (1-3)beta-D-glucan synthase and regulates S.pombe morphogenesis. Images PMID:8887550

  11. Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway

    PubMed Central

    Huang, Z; Zhang, L; Chen, Y; Zhang, H; Zhang, Q; Li, R; Ma, J; Li, Z; Yu, C; Lai, Y; Lin, T; Zhao, X; Zhang, B; Ye, Z; Liu, S; Wang, W; Liang, X; Liao, R; Shi, W

    2016-01-01

    Podocyte apoptosis is a major mechanism that leads to proteinuria in many chronic kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. The Rho family of small GTPases has been shown to be required in maintaining podocyte structure and function. Recent studies have indicated that podocyte-specific deletion of Cdc42 in vivo, but not of RhoA or Rac1, leads to congenital nephrotic syndrome and glomerulosclerosis. However, the underlying cellular events in podocyte controlled by Cdc42 remain unclear. Here, we assessed the cellular mechanisms by which Cdc42 regulates podocyte apoptosis. We found that the expression of Cdc42 and its activity were significantly decreased in high glucose-, lipopolysaccharide- or adriamycin-injured podocytes. Reduced Cdc42 expression in vitro and in vivo by small interfering RNA and selective Cdc42 inhibitor ML-141, respectively, caused podocyte apoptosis and proteinuria. Our results further demonstrated that insufficient Cdc42 or Nwasp, its downstream effector, could decrease the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Moreover, our data indicated that the loss of stress fibers caused by Cdc42/Nwasp deficiency also decreased Yes-associated protein (YAP) mRNA and protein expression, and induced podocyte apoptosis. Podocyte apoptosis induced by Cdc42/Nwasp/stress fiber deficiency was significantly inhibited by overexpressing-active YAP. Thus, the Cdc42/Nwasp/stress fibers/YAP signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases. PMID:26986510

  12. Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae.

    PubMed

    Moskow, J J; Gladfelter, A S; Lamson, R E; Pryciak, P M; Lew, D J

    2000-10-01

    CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p. PMID:11003652

  13. miR-330 regulates the proliferation of colorectal cancer cells by targeting Cdc42

    SciTech Connect

    Li, Yuefeng; Zhu, Xiaolan; Xu, Wenlin; Wang, Dongqing; Yan, Jinchuan

    2013-02-15

    Highlights: ► miR-330 was inversely correlated with Cdc42 in colorectal cancer cells. ► Elevated miR-330 suppressed cell proliferation in vivo and in vitro. ► Elevated miR-330 mimicked the effect of Cdc42 knockdown. ► Restoration of Cdc42 could partially attenuate the effects of miR-330. -- Abstract: MicroRNAs are small non-coding RNA molecules that play important roles in the multistep process of colorectal carcinoma (CRC) development. However, the miRNA–mRNA regulatory network is far from being fully understood. The objective of this study was to investigate the expression and the biological roles of miR-330 in colorectal cancer cells. Cdc42, one of the best characterized members of the Rho GTPase family, was found to be up-regulated in several types of human tumors including CRC and has been implicated in cancer initiation and progression. In the present study, we identified miR-330, as a potential regulator of Cdc42, was found to be inversely correlated with Cdc42 expression in colorectal cancer cell lines. Ectopic expression of miR-330 down-regulated Cdc42 expression at both protein and mRNA level, mimicked the effect of Cdc42 knockdown in inhibiting proliferation, inducing G1 cell cycle arrest and apoptosis of the colorectal cancer cells, whereas restoration of Cdc42 could partially attenuate the effects of miR-330. In addition, elevated expression of miR-330 could suppress the immediate downstream effectors of Cdc42 and inhibit the growth of colorectal cancer cells in vivo. To sum up, our results establish a role of miR-330 in negatively regulating Cdc42 expression and colorectal cancer cell proliferation. They suggest that manipulating the expression level of Cdc42 by miR-330 has the potential to influence colorectal cancer progression.

  14. Role of Cdc42p in Pheromone-Stimulated Signal Transduction in Saccharomyces cerevisiae

    PubMed Central

    Moskow, John J.; Gladfelter, Amy S.; Lamson, Rachel E.; Pryciak, Peter M.; Lew, Daniel J.

    2000-01-01

    CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20ΔCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p. PMID:11003652

  15. Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p

    PubMed Central

    Chen, Hsin; Kuo, Chun-Chen; Kang, Hui; Howell, Audrey S.; Zyla, Trevin R.; Jin, Michelle; Lew, Daniel J.

    2012-01-01

    Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase–formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p–Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via “polarisome” components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton. PMID:22918946

  16. Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

    PubMed Central

    Adamo, Joan E.; Moskow, John J.; Gladfelter, Amy S.; Viterbo, Domenic; Lew, Daniel J.; Brennwald, Patrick J.

    2001-01-01

    The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth. PMID:11706050

  17. Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

    PubMed

    Adamo, J E; Moskow, J J; Gladfelter, A S; Viterbo, D; Lew, D J; Brennwald, P J

    2001-11-12

    The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth. PMID:11706050

  18. Spontaneous Cdc42 Polarization Independent of GDI-Mediated Extraction and Actin-Based Trafficking

    PubMed Central

    Bendezú, Felipe O.; Vincenzetti, Vincent; Vavylonis, Dimitrios; Wyss, Romain; Vogel, Horst; Martin, Sophie G.

    2015-01-01

    The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules. PMID:25837586

  19. Distinct predictive performance of Rac1 and Cdc42 in cell migration

    PubMed Central

    Yamao, Masataka; Naoki, Honda; Kunida, Katsuyuki; Aoki, Kazuhiro; Matsuda, Michiyuki; Ishii, Shin

    2015-01-01

    We propose a new computation-based approach for elucidating how signaling molecules are decoded in cell migration. In this approach, we performed FRET time-lapse imaging of Rac1 and Cdc42, members of Rho GTPases which are responsible for cell motility, and quantitatively identified the response functions that describe the conversion from the molecular activities to the morphological changes. Based on the identified response functions, we clarified the profiles of how the morphology spatiotemporally changes in response to local and transient activation of Rac1 and Cdc42, and found that Rac1 and Cdc42 activation triggers laterally propagating membrane protrusion. The response functions were also endowed with property of differentiator, which is beneficial for maintaining sensitivity under adaptation to the mean level of input. Using the response function, we could predict the morphological change from molecular activity, and its predictive performance provides a new quantitative measure of how much the Rho GTPases participate in the cell migration. Interestingly, we discovered distinct predictive performance of Rac1 and Cdc42 depending on the migration modes, indicating that Rac1 and Cdc42 contribute to persistent and random migration, respectively. Thus, our proposed predictive approach enabled us to uncover the hidden information processing rules of Rho GTPases in the cell migration. PMID:26634649

  20. Manipulation of small Rho GTPases is a pathogen-induced process detected by Nod1

    PubMed Central

    Keestra, A. Marijke; Winter, Maria G.; Auburger, Josef J.; Fräßle, Simon P.; Xavier, Mariana N.; Winter, Sebastian E.; Kim, Anita; Poon, Victor; Ravesloot, Mariëtta M.; Waldenmaier, Julian; Tsolis, Renée M.; Eigenheer, Richard A.; Bäumler, Andreas J.

    2013-01-01

    Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns (PAMPs) 1. However, all microbes produce PAMPs, regardless of their pathogenic potential. To distinguish virulent microbes from ones with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes 2, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that Nod1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of Rac1 and Cdc42 by bacterial delivery or ectopic expression of a Salmonella virulence factor, SopE, triggered the Nod1 signaling pathway with consequent Rip2-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the Nod1 signaling pathway by peptidoglycan required Rac1 activity. Furthermore, constitutively active forms of Rac1, Cdc42 and RhoA activated the Nod1 signaling pathway. Our data identify activation of small Rho GTPases as a pathogen-induced process sensed through the Nod1 signaling pathway (Fig. S1). PMID:23542589

  1. The GTPase-Activating Protein Rga1 Interacts with Rho3 GTPase and May Regulate Its Function in Polarized Growth in Budding Yeast

    PubMed Central

    He, Fei; Nie, Wen-Chao; Tong, Zongtian; Yuan, Si-Min; Gong, Ting; Liao, Yuan; Bi, Erfei; Gao, Xiang-Dong

    2015-01-01

    In budding yeast, Rga1 negatively regulates the Rho GTPase Cdc42 by acting as a GTPase-activating protein (GAP) for Cdc42. To gain insight into the function and regulation of Rga1, we overexpressed Rga1 and an N-terminally truncated Rga1-C538 (a.a. 538-1007) segment. Overexpression of Rga1-C538 but not full-length Rga1 severely impaired growth and cell morphology in wild-type cells. We show that Rga1 is phosphorylated during the cell cycle. The lack of phenotype for full-length Rga1 upon overexpression may result from a negative regulation by G1-specific Pho85, a cyclin-dependent kinase (CDK). From a high-copy suppressor screen, we isolated RHO3, SEC9, SEC1, SSO1, SSO2, and SRO7, genes involved in exocytosis, as suppressors of the growth defect caused by Rga1-C538 overexpression. Moreover, we detected that Rga1 interacts with Rho3 in two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Rga1 preferentially interacts with the GTP-bound form of Rho3 and the interaction requires the GAP domain and additional sequence upstream of the GAP domain. Our data suggest that the interaction of Rga1 with Rho3 may regulate Rho3’s function in polarized bud growth. PMID:25860339

  2. Rho GTPase protein expression and activation in murine monocytes/macrophages is not modulated by model biomaterial surfaces in serum-containing in vitro cultures.

    PubMed

    Godek, M L; Sampson, J A; Duchsherer, N L; McElwee, Q; Grainger, D W

    2006-01-01

    The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-)macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue culture polystyrene, polystyrene, poly-l-lactide and Teflon(®) AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue culture polystyrene and poly

  3. Rho GTPase protein expression and activation in murine monocytes/macrophages is not modulated by model biomaterial surfaces in serum-containing in vitro cultures

    PubMed Central

    GODEK, M. L.; SAMPSON, J. A.; DUCHSHERER, N. L.; McELWEE, Q.; GRAINGER, D. W.

    2006-01-01

    The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-)macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue culture polystyrene, polystyrene, poly-l-lactide and Teflon® AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue culture polystyrene and poly

  4. Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

    PubMed Central

    Kim, Seung Joon; Wan, Qiaoqiao; Cho, Eunhye; Han, Bumsoo; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Na, Sungsoo

    2014-01-01

    Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs. PMID:24393843

  5. The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

    PubMed Central

    Wang, Ning; Wang, Mo; Zhu, Yi-Hua; Grosel, Timothy W.; Sun, Daokun; Kudryashov, Dmitri S.; Wu, Jian-Qiu

    2015-01-01

    Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast. PMID:25411334

  6. GRP75 upregulates clathrin-independent endocytosis through actin cytoskeleton reorganization mediated by the concurrent activation of Cdc42 and RhoA.

    PubMed

    Chen, Hang; Gao, Zhihui; He, Changzheng; Xiang, Rong; van Kuppevelt, Toin H; Belting, Mattias; Zhang, Sihe

    2016-05-01

    Therapeutic macromolecules are internalized into the cell by either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). Although some chaperone proteins play an essential role in CME (e.g. Hsc70 in clathrin uncoating), relatively few of these proteins are functionally involved in CIE. We previously revealed a role for the mitochondrial chaperone protein GRP75 in heparan sulfate proteoglycan (HSPG)-mediated, membrane raft-associated macromolecule endocytosis. However, the mechanism underlying this process remains unclear. In this study, using a mitochondrial signal peptide-directed protein trafficking expression strategy, we demonstrate that wild-type GRP75 expression enhanced the uptakes of HSPG and CIE marker cholera toxin B subunit but impaired the uptake of CME marker transferrin. The endocytosis regulation function of GRP75 is largely mediated by its subcellular location in mitochondria and is essentially determined by its ATPase domain. Interestingly, the mitochondrial expression of GRP75 or its ATPase domain significantly stimulates increases in both RhoA and Cdc42 activation, remarkably induces stress fibers and enhances filopodia formation, which collectively results in the promotion of CIE, but the inhibition of CME. Furthermore, silencing of Cdc42 or RhoA impaired the ability of GRP75 overexpression to increase CIE. Therefore, these results suggest that endocytosis vesicle enrichment of GRP75 by mitochondria trafficking upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA. This finding provides novel insight into organelle-derived chaperone signaling and the regulation of different endocytosis pathways in cells. PMID:27090015

  7. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    SciTech Connect

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-07-05

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.

  8. Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1.

    PubMed

    Keestra, A Marijke; Winter, Maria G; Auburger, Josef J; Frässle, Simon P; Xavier, Mariana N; Winter, Sebastian E; Kim, Anita; Poon, Victor; Ravesloot, Mariëtta M; Waldenmaier, Julian F T; Tsolis, Renée M; Eigenheer, Richard A; Bäumler, Andreas J

    2013-04-11

    Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway. PMID:23542589

  9. Filamin A regulates monocyte migration through Rho small GTPases during osteoclastogenesis.

    PubMed

    Leung, Roland; Wang, Yongqiang; Cuddy, Karl; Sun, Chunxiang; Magalhaes, Joyce; Grynpas, Marc; Glogauer, Michael

    2010-05-01

    Osteoclastogenesis (OCG) results from the fusion of monocytes after stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Migration of monocytes into close proximity precedes critical fusion events that are required for osteoclast formation. Cellular migration requires leading-edge actin cytoskeleton assembly that drives cellular locomotion. Filamin A (FLNa) cross-links F-actin filaments in the leading edge of migrating cells and also has been shown to regulate signal transduction during cell migration. However, little is known about the possible role of FLNa in osteoclastogenesis. Our objective in this study was to investigate the role of FLNa in osteoclastogenesis. Bone marrow monocytes isolated from the tibiae and femora of wild type (WT) and Flna-null mice were cultured for 6 days with M-CSF and RANKL, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRACP) staining. The Flna-null mouse skeletal phenotype was characterized using dual-energy X-ray absorptiometry (DXA) to analyze the skeleton, as well as tests on blood chemistry. Osteoclast levels in vivo were quantified by counting of TRACP-stained histologic sections of distal femora. To elucidate the mechanisms by which Flna regulates osteoclastogenesis, migration, actin polymerization, and activation of Rho GTPases, Rac1, Cdc42, and RhoA were assessed in monocytes during in vitro OCG. Deficiencies in migration were rescued using constitutively active Rac1 and Cdc42 TAT fusion proteins. The RANKL signaling pathway was evaluated for activation by monitoring nuclear translocation of NF kappaB and c-jun and expression of key osteoclast genes using quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that Flna-null monocytes formed fewer osteoclasts in vitro, and those that were formed were smaller with fewer nuclei. Decreased OCG was reflected in vivo in TRACP-stained histologic bone sections. Flna

  10. Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1

    PubMed Central

    Francis, Monika K.; Holst, Mikkel R.; Vidal-Quadras, Maite; Henriksson, Sara; Santarella-Mellwig, Rachel; Sandblad, Linda; Lundmark, Richard

    2015-01-01

    ABSTRACT Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism. PMID:26446261

  11. Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

    PubMed Central

    Takahashi, Satoe

    2007-01-01

    The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP2-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase–dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein–protein and protein–membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex. PMID:17914055

  12. An Essential Role for Cdc42 in the Functioning of the Adult Mammary Gland.

    PubMed

    Druso, Joseph E; Endo, Makoto; Lin, Miao-Chong Joy; Peng, Xu; Antonyak, Marc A; Meller, Stephanie; Cerione, Richard A

    2016-04-22

    The Rho family small GTPase Cdc42 has been implicated in a wide range of cellular functions including the establishment of cell polarity and the remodeling of the actin cytoskeletal architecture, resulting in the tight regulation of cell growth and survival during developmental processes. The complete knock-out of Cdc42 in the mouse is embryonic-lethal, and its targeted deletion in various tissues has been shown to disrupt tissue homeostasis. Thus far, in most studies, the targeted deletion of Cdc42 occurred during embryogenesis. Here, we have used a conditional gene deletion strategy in mice to probe the specific role of Cdc42 during adult mammary gland function. Cdc42 conditional-knock-out females were unable to adequately nourish their pups, due to a disorganized epithelial compartment within their mammary glands. A closer examination showed that their mammary epithelial cells were not able to maintain functional alveolar lumens, due to an inability to establish normal apical/basal epithelial polarity, as well as proper cell-cell contacts. Loss of these essential epithelial characteristics led to a premature sloughing off of the Cdc42-null epithelial cells. Overall our findings demonstrate that Cdc42 plays essential roles in mammary gland function post pregnancy, where it helps to establish proper epithelial cell polarity and tissue homeostasis during lactation. PMID:26912661

  13. SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

    PubMed Central

    Bendris, Nawal; Williams, Karla C.; Reis, Carlos R.; Welf, Erik S.; Chen, Ping-Hung; Lemmers, Bénédicte; Hahne, Michael; Leong, Hon Sing; Schmid, Sandra L.

    2016-01-01

    Despite current advances in cancer research, metastasis remains the leading factor in cancer-related deaths. Here we identify sorting nexin 9 (SNX9) as a new regulator of breast cancer metastasis. We detect an increase in SNX9 expression in human breast cancer metastases compared with primary tumors and demonstrate that SNX9 expression in MDA-MB-231 breast cancer cells is necessary to maintain their ability to metastasize in a chick embryo model. Conversely, SNX9 knockdown impairs this process. In vitro studies using several cancer cell lines derived from a variety of human tumors reveal a role for SNX9 in cell invasion and identify mechanisms responsible for this novel function. We show that SNX9 controls the activation of RhoA and Cdc42 GTPases and also regulates cell motility via the modulation of well-known molecules involved in metastasis, namely RhoA-ROCK and N-WASP. In addition, we find that SNX9 is required for RhoGTPase-dependent, clathrin-independent endocytosis, and in this capacity can functionally substitute to the bona fide Rho GAP, GTPase regulator associated with focal adhesion kinase (GRAF1). Taken together, our data establish novel roles for SNX9 as a multifunctional protein scaffold that regulates, and potentially coordinates, several cellular processes that together can enhance cancer cell metastasis. PMID:26960793

  14. Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

    PubMed Central

    Mukherjee, Debarati; Sen, Arpita; Boettner, Douglas R.; Fairn, Gregory D.; Schlam, Daniel; Bonilla Valentin, Fernando J.; Michael McCaffery, J.; Hazbun, Tony; Staiger, Chris J.; Grinstein, Sergio; Lemmon, Sandra K.; Claudio Aguilar, R.

    2013-01-01

    Summary Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking. PMID:23943876

  15. Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

    PubMed

    Fediuk, Jena; Sikarwar, Anurag S; Nolette, Nora; Dakshinamurti, Shyamala

    2014-12-01

    In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms

  16. Swiprosin-1 stimulates cancer invasion and metastasis by increasing the Rho family of GTPase signaling.

    PubMed

    Huh, Yun Hyun; Oh, Sena; Yeo, Yu Ra; Chae, In Hee; Kim, So Hee; Lee, Ji Shin; Yun, Sook Jung; Choi, Kyu Yeong; Ryu, Je-Hwang; Jun, Chang-Duk; Song, Woo Keun

    2015-05-30

    Ectopic expression of Swiprosin-1, an actin-binding protein (also known as EF hand domain containing 2; EFHD2), enhanced motile protrusions associated with actin, such as lamellipodia and membrane ruffles. Swiprosin-1 levels were increased in various human cancer tissues, particularly at highly invasive stages of malignant melanoma. Expression of Swiprosin-1 was correlated with that of epidermal growth factor receptor (EGFR) and induced by EGF. In a mouse metastasis model, Swiprosin-1 overexpression induced pulmonary metastasis whereas its knockdown led to marked inhibition of metastasis of highly invasive melanoma cells. Swiprosin-1 at the lamellipodia and membrane ruffles controlled the direction of cell protrusion and enhanced migration velocity through activating the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Our collective findings support the potential utility of Swiprosin-1 as a therapeutic target to prevent cancer invasion and metastasis. PMID:26079945

  17. Defective Dendrite Elongation but Normal Fertility in Mice Lacking the Rho-Like GTPase Activator Dbl

    PubMed Central

    Hirsch, Emilio; Pozzato, Michela; Vercelli, Alessandro; Barberis, Laura; Azzolino, Ornella; Russo, Chiara; Vanni, Cristina; Silengo, Lorenzo; Eva, Alessandra; Altruda, Fiorella

    2002-01-01

    Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation. PMID:11940671

  18. Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology

    PubMed Central

    Bhattacharya, Kurchi; Swaminathan, Karthic; Peche, Vivek S.; Clemen, Christoph S.; Knyphausen, Philipp; Lammers, Michael; Noegel, Angelika A.; Rastetter, Raphael H.

    2016-01-01

    The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP ‘hyperactivity’ upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner. PMID:27143109

  19. Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology.

    PubMed

    Bhattacharya, Kurchi; Swaminathan, Karthic; Peche, Vivek S; Clemen, Christoph S; Knyphausen, Philipp; Lammers, Michael; Noegel, Angelika A; Rastetter, Raphael H

    2016-01-01

    The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP 'hyperactivity' upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner. PMID:27143109

  20. Dock10, a Cdc42 and Rac1 GEF, induces loss of elongation, filopodia, and ruffles in cervical cancer epithelial HeLa cells

    PubMed Central

    Ruiz-Lafuente, Natalia; Alcaraz-García, María-José; García-Serna, Azahara-María; Sebastián-Ruiz, Silvia; Moya-Quiles, María-Rosa; García-Alonso, Ana-María; Parrado, Antonio

    2015-01-01

    Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells. PMID:25862245

  1. Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

    PubMed Central

    Distel, Jesús S.; Aguilera, Milton O.; Colombo, María I.; Berón, Walter

    2015-01-01

    The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process. PMID:26674774

  2. Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity.

    PubMed

    Yeh, Yi-Chun; Wang, Chau-Zen; Tang, Ming-Jer

    2009-01-01

    Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation. PMID:18780290

  3. Thyroid bud morphogenesis requires CDC42- and SHROOM3-dependent apical constriction

    PubMed Central

    Loebel, David A. F.; Plageman, Timothy F.; Tang, Theresa L.; Jones, Vanessa J.; Muccioli, Maria; Tam, Patrick P. L.

    2016-01-01

    ABSTRACT Early development of the gut endoderm and its subsequent remodeling for the formation of organ buds are accompanied by changes to epithelial cell shape and polarity. Members of the Rho-related family of small GTPases and their interacting proteins play multiple roles in regulating epithelial morphogenesis. In this study we examined the role of Cdc42 in foregut development and organ bud formation. Ablation of Cdc42 in post-gastrulation mouse embryos resulted in a loss of apical-basal cell polarity and columnar epithelial morphology in the ventral pharyngeal endoderm, in conjunction with a loss of apical localization of the known CDC42 effector protein PARD6B. Cell viability but not proliferation in the foregut endoderm was impaired. Outgrowth of the liver, lung and thyroid buds was severely curtailed in Cdc42-deficient embryos. In particular, the thyroid bud epithelium did not display the apical constriction that normally occurs concurrently with the outgrowth of the bud into the underlying mesenchyme. SHROOM3, a protein that interacts with Rho GTPases and promotes apical constriction, was strongly expressed in the thyroid bud and its sub-cellular localization was disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutant embryos, the thyroid bud epithelium showed no apical constriction, while the bud continued to grow and protruded into the foregut lumen. Our findings indicate that Cdc42 is required for epithelial polarity and organization in the endoderm and for apical constriction in the thyroid bud. It is possible that the function of CDC42 is partly mediated by SHROOM3. PMID:26772200

  4. CDC42 Use in Viral Cell Entry Processes by RNA Viruses

    PubMed Central

    Swaine, Thomas; Dittmar, Matthias T.

    2015-01-01

    The cellular actin cytoskeleton presents a barrier that must be overcome by many viruses, and it has become increasingly apparent many viral species have developed a diverse repertoire of mechanisms to hijack cellular actin-regulating signalling pathways as part of their cell entry processes. The Rho family GTPase Cdc42 is appreciated as a key moderator of cellular actin dynamics, and the development of specific Cdc42-inhibiting agents has given us an unprecedented ability to investigate its individual role in signalling pathways. However, investigative use of said agents, and the subsequent characterisation of the role Cdc42 plays in viral entry processes has been lacking. Here, we describe the current literature on the role of Cdc42 in human immunodeficiency virus (HIV)-1 cell entry, which represents the most investigated instance of Cdc42 function in viral cell entry processes, and also review evidence of Cdc42 use in other RNA virus cell entries, demonstrating prime areas for more extensive research using similar techniques. PMID:26690467

  5. Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

    PubMed Central

    Burbage, Marianne; Keppler, Selina J.; Gasparrini, Francesca; Martínez-Martín, Nuria; Gaya, Mauro; Feest, Christoph; Domart, Marie-Charlotte; Brakebusch, Cord; Collinson, Lucy; Bruckbauer, Andreas

    2015-01-01

    The small Rho GTPase Cdc42, known to interact with Wiskott–Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell–intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology. PMID:25547673

  6. Cdc42 and Rac1 activity is reduced in human pheochromocytoma and correlates with FARP1 and ARHGEF1 expression.

    PubMed

    Croisé, Pauline; Houy, Sébastien; Gand, Mathieu; Lanoix, Joël; Calco, Valérie; Tóth, Petra; Brunaud, Laurent; Lomazzi, Sandra; Paramithiotis, Eustache; Chelsky, Daniel; Ory, Stéphane; Gasman, Stéphane

    2016-04-01

    Among small GTPases from the Rho family, Cdc42, RAC, and Rho are well known to mediate a large variety of cellular processes linked with cancer biology through their ability to cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, whereas the GTPase-activating proteins (GAPs) catalyze GTP hydrolysis, leading to the inactivated form. Modulation of Rho GTPase activity following altered expression of RHO-GEFs and/or RHO-GAPs has already been reported in various human tumors. However, nothing is known about the Rho GTPase activity or the expression of their regulators in human pheochromocytomas, a neuroendocrine tumor (NET) arising from chromaffin cells of the adrenal medulla. In this study, we demonstrate, through an ELISA-based activity assay, that Rac1 and Cdc42 activities decrease in human pheochromocytomas (PCCs) compared with the matched adjacent non-tumor tissue. Furthermore, through quantitative mass spectrometry (MS) approaches, we show that the expression of two RHO-GEF proteins, namely ARHGEF1 and FARP1, is significantly reduced in tumors compared with matched non-tumor tissue, whereas ARHGAP36 expression is increased. Moreover, siRNA-based knockdown of ARHGEF1 and FARP1 in PC12 cells leads to a significant inhibition of Rac1 and Cdc42 activities, respectively. Finally, a principal component analysis (PCA) of our dataset was able to discriminate PCC from non-tumor tissue and indicates a close correlation between Cdc42/Rac1 activity and FARP1/ARHGEF1 expression. Altogether, our findings reveal for the first time the importance of modulation of Rho GTPase activities and expression of their regulators in human PCCs. PMID:26911374

  7. Regulation of phagocytosis by Rho GTPases

    PubMed Central

    Mao, Yingyu; Finnemann, Silvia C

    2015-01-01

    Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review. PMID:25941749

  8. CdGAP/ARHGAP31, a Cdc42/Rac1 GTPase regulator, is critical for vascular development and VEGF-mediated angiogenesis.

    PubMed

    Caron, Christine; DeGeer, Jonathan; Fournier, Patrick; Duquette, Philippe M; Luangrath, Vilayphone; Ishii, Hidetaka; Karimzadeh, Fereshteh; Lamarche-Vane, Nathalie; Royal, Isabelle

    2016-01-01

    Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP(-/-) mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction. PMID:27270835

  9. CdGAP/ARHGAP31, a Cdc42/Rac1 GTPase regulator, is critical for vascular development and VEGF-mediated angiogenesis

    PubMed Central

    Caron, Christine; DeGeer, Jonathan; Fournier, Patrick; Duquette, Philippe M.; Luangrath, Vilayphone; Ishii, Hidetaka; Karimzadeh, Fereshteh; Lamarche-Vane, Nathalie; Royal, Isabelle

    2016-01-01

    Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP−/− mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction. PMID:27270835

  10. Cdc42: Role in Cancer Management.

    PubMed

    Qadir, Muhammad Imran; Parveen, Amna; Ali, Muhammad

    2015-10-01

    Contribution of Cdc42, a member of Rho family, has been characterized for the beginning of variety of cellular responses including cellular transformation, cell division, cell invasion, migration, invadopodia formation, enzyme activity, filopodia formation, and cell polarity in cells. Deregulation of Cdc42 can alter the normal functioning of the cells, responsible for the initiation of signaling pathways and is correlated with several pathogenic processes such as cancer. Therefore, maintaining the level of Cdc42 and its effectors in cells, tumor progression can be controlled. Therefore, it can be suggested that deeper understanding about the Cdc42 contribution in cancer cell progression at molecular level can approach to the development of Cdc42 inhibitors in cancer management. PMID:25777055

  11. δ-Catenin Activates Rho GTPase, Promotes Lymphangiogenesis and Growth of Tumor Metastases

    PubMed Central

    Lin, P. Charles

    2015-01-01

    δ-catenin, an adherens junctions protein, is not only involved in early development, cell-cell adhesion and cell motility in neuronal cells, but it also plays an important role in vascular endothelial cell motility and pathological angiogenesis. In this study, we report a new function of δ-catenin in lymphangiogenesis. Consistent with expression of δ-catenin in vascular endothelial cells, we detected expression of the gene in lymphatic endothelial cells (LECs). Ectopic expression of δ-catenin in LECs increased cell motility and lymphatic vascular network formation in vitro and lymphangiogenesis in vivo in a Matrigel plug assay. Conversely, knockdown of δ-catenin in LECs impaired lymphangiogenesis in vitro and in vivo. Biochemical analysis shows that δ-catenin regulates activation of Rho family small GTPases, key mediators in cell motility. δ-catenin activates Rac1 and Cdc42 but inhibits RhoA in LECs. Notably, blocking of Rac1 activation impaired δ-catenin mediated lymphangiogenesis in a Matrigel assay. Consistently, loss of δ-catenin in mice inhibited the growth of tumor metastases. Taken together, these findings identify a new function of δ-catenin in lymphangiogenesis and tumor growth/metastasis, likely through modulation of small Rho GTPase activation. Targeting δ-catenin may offer a new way to control tumor metastasis. PMID:25635825

  12. FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones

    PubMed Central

    Rappaz, Benjamin; Lai Wing Sun, Karen; Correia, James P.; Wiseman, Paul W.; Kennedy, Timothy E.

    2016-01-01

    Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1. PMID:27482713

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

    PubMed Central

    Elbediwy, Ahmed; Zihni, Ceniz; Terry, Stephen J.; Clark, Peter

    2012-01-01

    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. PMID:22891260

  14. Dendritic spine geometry can localize GTPase signaling in neurons

    PubMed Central

    Ramirez, Samuel A.; Raghavachari, Sridhar; Lew, Daniel J.

    2015-01-01

    Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia. PMID:26337387

  15. The structure of FMNL2–Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation

    PubMed Central

    Kühn, Sonja; Erdmann, Constanze; Kage, Frieda; Block, Jennifer; Schwenkmezger, Lisa; Steffen, Anika; Rottner, Klemens; Geyer, Matthias

    2015-01-01

    Formins are actin polymerization factors that elongate unbranched actin filaments at the barbed end. Rho family GTPases activate Diaphanous-related formins through the relief of an autoregulatory interaction. The crystal structures of the N-terminal domains of human FMNL1 and FMNL2 in complex with active Cdc42 show that Cdc42 mediates contacts with all five armadillo repeats of the formin with specific interactions formed by the Rho-GTPase insert helix. Mutation of three residues within Rac1 results in a gain-of-function mutation for FMNL2 binding and reconstitution of the Cdc42 phenotype in vivo. Dimerization of FMNL1 through a parallel coiled coil segment leads to formation of an umbrella-shaped structure that—together with Cdc42—spans more than 15 nm in diameter. The two interacting FMNL–Cdc42 heterodimers expose six membrane interaction motifs on a convex protein surface, the assembly of which may facilitate actin filament elongation at the leading edge of lamellipodia and filopodia. PMID:25963737

  16. The structure of FMNL2-Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation

    NASA Astrophysics Data System (ADS)

    Kühn, Sonja; Erdmann, Constanze; Kage, Frieda; Block, Jennifer; Schwenkmezger, Lisa; Steffen, Anika; Rottner, Klemens; Geyer, Matthias

    2015-05-01

    Formins are actin polymerization factors that elongate unbranched actin filaments at the barbed end. Rho family GTPases activate Diaphanous-related formins through the relief of an autoregulatory interaction. The crystal structures of the N-terminal domains of human FMNL1 and FMNL2 in complex with active Cdc42 show that Cdc42 mediates contacts with all five armadillo repeats of the formin with specific interactions formed by the Rho-GTPase insert helix. Mutation of three residues within Rac1 results in a gain-of-function mutation for FMNL2 binding and reconstitution of the Cdc42 phenotype in vivo. Dimerization of FMNL1 through a parallel coiled coil segment leads to formation of an umbrella-shaped structure that--together with Cdc42--spans more than 15 nm in diameter. The two interacting FMNL-Cdc42 heterodimers expose six membrane interaction motifs on a convex protein surface, the assembly of which may facilitate actin filament elongation at the leading edge of lamellipodia and filopodia.

  17. The structure of FMNL2-Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation.

    PubMed

    Kühn, Sonja; Erdmann, Constanze; Kage, Frieda; Block, Jennifer; Schwenkmezger, Lisa; Steffen, Anika; Rottner, Klemens; Geyer, Matthias

    2015-01-01

    Formins are actin polymerization factors that elongate unbranched actin filaments at the barbed end. Rho family GTPases activate Diaphanous-related formins through the relief of an autoregulatory interaction. The crystal structures of the N-terminal domains of human FMNL1 and FMNL2 in complex with active Cdc42 show that Cdc42 mediates contacts with all five armadillo repeats of the formin with specific interactions formed by the Rho-GTPase insert helix. Mutation of three residues within Rac1 results in a gain-of-function mutation for FMNL2 binding and reconstitution of the Cdc42 phenotype in vivo. Dimerization of FMNL1 through a parallel coiled coil segment leads to formation of an umbrella-shaped structure that—together with Cdc42—spans more than 15 nm in diameter. The two interacting FMNL-Cdc42 heterodimers expose six membrane interaction motifs on a convex protein surface, the assembly of which may facilitate actin filament elongation at the leading edge of lamellipodia and filopodia. PMID:25963737

  18. Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

    PubMed Central

    Fortin, Shannon P.; Ennis, Matthew J.; Schumacher, Cassie A.; Zylstra-Diegel, Cassandra R.; Williams, Bart O.; Ross, Julianna T.D.; Winkles, Jeffrey A.; Loftus, Joseph C.; Symons, Marc H.; Tran, Nhan L.

    2012-01-01

    Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor–inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14–directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy. PMID:22571869

  19. Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases.

    PubMed

    Ren, Y; Li, R; Zheng, Y; Busch, H

    1998-12-25

    The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. Here we have identified a new member of the Dbl family, GEF-H1, by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the lfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules. PMID:9857026

  20. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

    PubMed

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-05-23

    RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs. PMID:23706742

  1. Intersectin-2L Regulates Caveola Endocytosis Secondary to Cdc42-mediated Actin Polymerization*

    PubMed Central

    Klein, Irene K.; Predescu, Dan N.; Sharma, Tiffany; Knezevic, Ivana; Malik, Asrar B.; Predescu, Sanda

    2009-01-01

    Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PHITSN-2L) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The “catalytically dead” DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PHITSN-2L-transfected cells compared with control and 1 μm jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane. PMID:19622753

  2. Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization.

    PubMed

    Klein, Irene K; Predescu, Dan N; Sharma, Tiffany; Knezevic, Ivana; Malik, Asrar B; Predescu, Sanda

    2009-09-18

    Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH(ITSN-2L)) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The "catalytically dead" DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH(ITSN-2L)-transfected cells compared with control and 1 microM jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane. PMID:19622753

  3. Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease

    PubMed Central

    Cook, Danielle R.; Rossman, Kent L.; Der, Channing J.

    2016-01-01

    The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly by indirect mechanisms in disease. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflect the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2, Tiam1, Vav and P-Rex1/2. PMID:24037532

  4. Tax-interacting protein 1 coordinates the spatiotemporal activation of Rho GTPases and regulates the infiltrative growth of human glioblastoma

    PubMed Central

    Wang, Hailun; Han, Miaojun; Whetsell, William; Wang, Jialiang; Rich, Jeremy; Hallahan, Dennis; Han, Zhaozhong

    2014-01-01

    PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. TIP-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as one novel TIP-1 interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector, and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas. PMID:23563176

  5. CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

    PubMed

    Lichius, Alexander; Goryachev, Andrew B; Fricker, Mark D; Obara, Boguslaw; Castro-Longoria, Ernestina; Read, Nick D

    2014-05-01

    Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth. PMID:24790223

  6. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting Rho GTPases and ERK signaling pathways

    PubMed Central

    Pang, Xiufeng; Yi, Tingfang; Yi, Zhengfang; Cho, Sung Gook; Qu, Weijing; Pinkaew, Decha; Fujise, Ken; Liu, Mingyao

    2009-01-01

    Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown anti-oxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness and metastasis. We demonstrated that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical endothelial cells (HUVECs) in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the rat aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we demonstrated that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases, but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/MEK/ERK pathway kinases without affecting VEGFR2 activity. Together, our results indicate that morelloflavone exerts anti-angiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action. PMID:19147565

  7. RhoA GTPase inhibition organizes contraction during epithelial morphogenesis.

    PubMed

    Mason, Frank M; Xie, Shicong; Vasquez, Claudia G; Tworoger, Michael; Martin, Adam C

    2016-08-29

    During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding. PMID:27551058

  8. Phosphorylation of Bem2p and Bem3p may contribute to local activation of Cdc42p at bud emergence

    PubMed Central

    Knaus, Michèle; Pelli-Gulli, Marie-Pierre; van Drogen, Frank; Springer, Sander; Jaquenoud, Malika; Peter, Matthias

    2007-01-01

    Site-specific activation of the Rho-type GTPase Cdc42p is critical for the establishment of cell polarity. Here we investigated the role and regulation of the GTPase-activating enzymes (GAPs) Bem2p and Bem3p for Cdc42p activation and actin polarization at bud emergence in Saccharomyces cerevisiae. Bem2p and Bem3p are localized throughout the cytoplasm and the cell cortex in unbudded G1 cells, but accumulate at sites of polarization after bud emergence. Inactivation of Bem2p results in hyperactivation of Cdc42p and polarization toward multiple sites. Bem2p and Bem3p are hyperphosphorylated at bud emergence most likely by the Cdc28p-Cln2p kinase. This phosphorylation appears to inhibit their GAP activity in vivo, as non-phosphorylatable Bem3p mutants are hyperactive and interfere with Cdc42p activation. Taken together, our results indicate that Bem2p and Bem3p may function as global inhibitors of Cdc42p activation during G1, and their inactivation by the Cdc28p/Cln kinase contributes to site-specific activation of Cdc42p at bud emergence. PMID:17914457

  9. Phosphorylation of Bem2p and Bem3p may contribute to local activation of Cdc42p at bud emergence.

    PubMed

    Knaus, Michèle; Pelli-Gulli, Marie-Pierre; van Drogen, Frank; Springer, Sander; Jaquenoud, Malika; Peter, Matthias

    2007-10-31

    Site-specific activation of the Rho-type GTPase Cdc42p is critical for the establishment of cell polarity. Here we investigated the role and regulation of the GTPase-activating enzymes (GAPs) Bem2p and Bem3p for Cdc42p activation and actin polarization at bud emergence in Saccharomyces cerevisiae. Bem2p and Bem3p are localized throughout the cytoplasm and the cell cortex in unbudded G1 cells, but accumulate at sites of polarization after bud emergence. Inactivation of Bem2p results in hyperactivation of Cdc42p and polarization toward multiple sites. Bem2p and Bem3p are hyperphosphorylated at bud emergence most likely by the Cdc28p-Cln2p kinase. This phosphorylation appears to inhibit their GAP activity in vivo, as non-phosphorylatable Bem3p mutants are hyperactive and interfere with Cdc42p activation. Taken together, our results indicate that Bem2p and Bem3p may function as global inhibitors of Cdc42p activation during G1, and their inactivation by the Cdc28p/Cln kinase contributes to site-specific activation of Cdc42p at bud emergence. PMID:17914457

  10. Kindlin-1 Is Required for RhoGTPase-Mediated Lamellipodia Formation in Keratinocytes

    PubMed Central

    Has, Cristina; Herz, Corinna; Zimina, Elena; Qu, Hai-Yan; He, Yinghong; Zhang, Zhi-Gang; Wen, Ting-Ting; Gache, Yannick; Aumailley, Monique; Bruckner-Tuderman, Leena

    2009-01-01

    Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with β1 integrin, α-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS. PMID:19762715

  11. Control of T lymphocyte morphology by the GTPase Rho

    NASA Technical Reports Server (NTRS)

    Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

    2003-01-01

    BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

  12. Polarity establishment requires localized activation of Cdc42

    PubMed Central

    Woods, Benjamin; Kuo, Chun-Chen; Wu, Chi-Fang; Zyla, Trevin R.

    2015-01-01

    Establishment of cell polarity in animal and fungal cells involves localization of the conserved Rho-family guanosine triphosphatase, Cdc42, to the cortical region destined to become the “front” of the cell. The high local concentration of active Cdc42 promotes cytoskeletal polarization through various effectors. Cdc42 accumulation at the front is thought to involve positive feedback, and studies in the budding yeast Saccharomyces cerevisiae have suggested distinct positive feedback mechanisms. One class of mechanisms involves localized activation of Cdc42 at the front, whereas another class involves localized delivery of Cdc42 to the front. Here we show that Cdc42 activation must be localized for successful polarity establishment, supporting local activation rather than local delivery as the dominant mechanism in this system. PMID:26459595

  13. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast

    PubMed Central

    1994-01-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine- nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

  14. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

    PubMed

    Peterson, J; Zheng, Y; Bender, L; Myers, A; Cerione, R; Bender, A

    1994-12-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

  15. Atypical Rho GTPases of the RhoBTB Subfamily: Roles in Vesicle Trafficking and Tumorigenesis

    PubMed Central

    Ji, Wei; Rivero, Francisco

    2016-01-01

    RhoBTB proteins constitute a subfamily of atypical Rho GTPases represented in mammals by RhoBTB1, RhoBTB2, and RhoBTB3. Their characteristic feature is a carboxyl terminal extension that harbors two BTB domains capable of assembling cullin 3-dependent ubiquitin ligase complexes. The expression of all three RHOBTB genes has been found reduced or abolished in a variety of tumors. They are considered tumor suppressor genes and recent studies have strengthened their implication in tumorigenesis through regulation of the cell cycle and apoptosis. RhoBTB3 is also involved in retrograde transport from endosomes to the Golgi apparatus. One aspect that makes RhoBTB proteins atypical among the Rho GTPases is their proposed mechanism of activation. No specific guanine nucleotide exchange factors or GTPase activating proteins are known. Instead, RhoBTB might be activated through interaction with other proteins that relieve their auto-inhibited conformation and inactivated through auto-ubiquitination and destruction in the proteasome. In this review we discuss our current knowledge on the molecular mechanisms of action of RhoBTB proteins and the implications for tumorigenesis and other pathologic conditions. PMID:27314390

  16. Regulation of the formin for3p by cdc42p and bud6p.

    PubMed

    Martin, Sophie G; Rincón, Sergio A; Basu, Roshni; Pérez, Pilar; Chang, Fred

    2007-10-01

    Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Delta cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition. PMID:17699595

  17. Rho GTPase Transcriptome Analysis Reveals Oncogenic Roles for Rho GTPase-Activating Proteins in Basal-like Breast Cancers.

    PubMed

    Lawson, Campbell D; Fan, Cheng; Mitin, Natalia; Baker, Nicole M; George, Samuel D; Graham, David M; Perou, Charles M; Burridge, Keith; Der, Channing J; Rossman, Kent L

    2016-07-01

    The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR. PMID:27216196

  18. AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes*

    PubMed Central

    Woolery, Andrew R.; Yu, Xiaobo; LaBaer, Joshua; Orth, Kim

    2014-01-01

    Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules. Herein, we demonstrate that AMPylation of Rho GTPases by VopS is a multifaceted virulence mechanism that counters several host immunity strategies. Activation of NFκB, Erk, and JNK kinase signaling pathways were inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. Phosphorylation and degradation of IKBα were inhibited in the presence of VopS as was nuclear translocation of the NFκB subunit p65. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex, potentially by inhibiting the interaction of Rac and p67. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. We conclude that AMPylation of Rho Family GTPases by VopS results in diverse inhibitory consequences during infection beyond the most obvious phenotype, the collapse of the actin cytoskeleton. PMID:25301945

  19. Novel peptide recognized by RhoA GTPase.

    PubMed

    Drulis-Fajdasz, Dominika; Jelen, Filip; Oleksy, Arkadiusz; Otlewski, Jacek

    2006-01-01

    A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein. PMID:17019437

  20. Axin Regulates Dendritic Spine Morphogenesis through Cdc42-Dependent Signaling

    PubMed Central

    Chen, Yu; Liang, Zhuoyi; Fei, Erkang; Chen, Yuewen; Zhou, Xiaopu; Fang, Weiqun; Fu, Wing-Yu; Fu, Amy K. Y.; Ip, Nancy Y.

    2015-01-01

    During development, scaffold proteins serve as important platforms for orchestrating signaling complexes to transduce extracellular stimuli into intracellular responses that regulate dendritic spine morphology and function. Axin (“axis inhibitor”) is a key scaffold protein in canonical Wnt signaling that interacts with specific synaptic proteins. However, the cellular functions of these protein–protein interactions in dendritic spine morphology and synaptic regulation are unclear. Here, we report that Axin protein is enriched in synaptic fractions, colocalizes with the postsynaptic marker PSD-95 in cultured hippocampal neurons, and interacts with a signaling protein Ca2+/calmodulin-dependent protein kinase II (CaMKII) in synaptosomal fractions. Axin depletion by shRNA in cultured neurons or intact hippocampal CA1 regions significantly reduced dendritic spine density. Intriguingly, the defective dendritic spine morphogenesis in Axin-knockdown neurons could be restored by overexpression of the small Rho-GTPase Cdc42, whose activity is regulated by CaMKII. Moreover, pharmacological stabilization of Axin resulted in increased dendritic spine number and spontaneous neurotransmission, while Axin stabilization in hippocampal neurons reduced the elimination of dendritic spines. Taken together, our findings suggest that Axin promotes dendritic spine stabilization through Cdc42-dependent cytoskeletal reorganization. PMID:26204446

  1. Regulation of Neurotrophin-Induced Axonal Responses via Rho GTPases

    PubMed Central

    HANDEÖZDINLER, P.; ERZURUMLU, REHA S.

    2014-01-01

    Nerve growth factor (NGF) and related neurotrophins induce differential axon growth patterns from embryonic sensory neurons. In wholemount explant cultures of embryonic rat trigeminal ganglion and brainstem or in dissociated cell cultures of the trigeminal ganglion, exogenous supply of NGF leads to axonal elongation, whereas neurotrophin-3 (NT-3) treatment leads to short branching and arborization. Axonal responses to neurotrophins might be mediated via the Rho GTPases. To investigate this possibility, we prepared wholemount trigeminal pathway cultures from E15 rats. We infected the ganglia with recombinant vaccinia viruses that express GFP-tagged dominant negative Rac, Rho, or constitutively active Rac or treated the cultures with lysophosphatitic acid (LPA) to activate Rho. We then examined axonal responses to NGF by use of the lipophilic tracer DiI. Rac activity induced longer axonal growth from the central trigeminal tract, whereas the dominant negative construct of Rac eliminated NGF-induced axon outgrowth. Rho activity also significantly reduced, and the Rho dominant negative construct increased, axon growth from the trigeminal tract. Similar alterations in axonal responses to NT-3 and brain-derived neurotrophic factor were also noted. Our results demonstrate that Rho GTPases play a major role in neurotrophin-induced axonal differentiation of embryonic trigeminal axons. PMID:11559894

  2. EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42

    PubMed Central

    Bulgin, Richard R; Arbeloa, Ana; Chung, Jade C S; Frankel, Gad

    2009-01-01

    Subversion of the eukaryotic cell cytoskeleton is a virulence strategy employed by many bacterial pathogens. Due to the pivotal role of Rho GTPases in actin dynamics they are common targets of bacterial effector proteins and toxins. IpgB1, IpgB2 (Shigella), SifA, SifB (Salmonella) and Map and EspM (attaching and effacing pathogens) constitute a family of type III secretion system effectors that subverts small GTPase signalling pathways. In this study we identified and characterized EspT from Citrobacter rodentium that triggers formation of lamellipodia on Swiss 3T3 and membrane ruffles on HeLa cells, which are reminiscent of the membrane ruffles induced by IpgB1. Ectopic expression of EspT and IpgB1, but not EspM, resulted in a mitochondrial localization. Using dominant negative constructs we found that EspT-induced actin remodelling is dependent on GTP-bound Rac-1 and Cdc42 but not ELMO or Dock180, which are hijacked by IpgB1 in order to form a Rac-1 specific guanine nucleotide exchange factor. Using pull-down assays with the Rac-1 and Cdc42 binding domains of Pak and WASP we demonstrate that EspT is capable of activating both Rac-1 and Cdc42. These results suggest that EspT modulates the host cell cytoskeleton through coactivation of Rac-1 and Cdc42 by a distinct mechanism. PMID:19016787

  3. Unique spatiotemporal activation pattern of Cdc42 by Gef1 and Scd1 promotes different events during cytokinesis.

    PubMed

    Wei, Bin; Hercyk, Brian S; Mattson, Nicholas; Mohammadi, Ahmad; Rich, Julie; DeBruyne, Erica; Clark, Mikayla M; Das, Maitreyi

    2016-04-15

    The Rho-family GTPase Cdc42 regulates cell polarity and localizes to the cell division site. Cdc42 is activated by guanine nucleotide exchange factors (GEFs). We report that Cdc42 promotes cytokinesis via a unique spatiotemporal activation pattern due to the distinct action of its GEFs, Gef1 and Scd1, in fission yeast. Before cytokinetic ring constriction, Cdc42 activation, is Gef1 dependent, and after ring constriction, it is Scd1 dependent. Gef1 localizes to the actomyosin ring immediately after ring assembly and promotes timely onset of ring constriction. Gef1 is required for proper actin organization during cytokinesis, distribution of type V myosin Myo52 to the division site, and timely recruitment of septum protein Bgs1. In contrast, Scd1 localizes to the broader region of ingressing membrane during cytokinetic furrowing. Scd1 promotes normal septum formation, andscd1Δcells display aberrant septa with reduced Bgs1 localization. Thus we define unique roles of the GEFs Gef1 and Scd1 in the regulation of distinct events during cytokinesis. Gef1 localizes first to the cytokinetic ring and promotes timely constriction, whereas Scd1 localizes later to the ingressing membrane and promotes septum formation. Our findings are consistent with reports that complexity in GTPase signaling patterns enables exquisite precision over the control of cellular processes. PMID:26941334

  4. Unique spatiotemporal activation pattern of Cdc42 by Gef1 and Scd1 promotes different events during cytokinesis

    PubMed Central

    Wei, Bin; Hercyk, Brian S.; Mattson, Nicholas; Mohammadi, Ahmad; Rich, Julie; DeBruyne, Erica; Clark, Mikayla M.; Das, Maitreyi

    2016-01-01

    The Rho-family GTPase Cdc42 regulates cell polarity and localizes to the cell division site. Cdc42 is activated by guanine nucleotide exchange factors (GEFs). We report that Cdc42 promotes cytokinesis via a unique spatiotemporal activation pattern due to the distinct action of its GEFs, Gef1 and Scd1, in fission yeast. Before cytokinetic ring constriction, Cdc42 activation, is Gef1 dependent, and after ring constriction, it is Scd1 dependent. Gef1 localizes to the actomyosin ring immediately after ring assembly and promotes timely onset of ring constriction. Gef1 is required for proper actin organization during cytokinesis, distribution of type V myosin Myo52 to the division site, and timely recruitment of septum protein Bgs1. In contrast, Scd1 localizes to the broader region of ingressing membrane during cytokinetic furrowing. Scd1 promotes normal septum formation, and scd1Δ cells display aberrant septa with reduced Bgs1 localization. Thus we define unique roles of the GEFs Gef1 and Scd1 in the regulation of distinct events during cytokinesis. Gef1 localizes first to the cytokinetic ring and promotes timely constriction, whereas Scd1 localizes later to the ingressing membrane and promotes septum formation. Our findings are consistent with reports that complexity in GTPase signaling patterns enables exquisite precision over the control of cellular processes. PMID:26941334

  5. The function of RhoGTPases in axon ensheathment and myelination

    PubMed Central

    Feltri, M. Laura; Suter, Ueli; Relvas, João B.

    2008-01-01

    RhoGTPases are molecular switches that integrate extracellular signals to perform diverse cellular responses. This ability relies on the network of proteins regulating RhoGTPases activity and localization, and on the interaction of RhoGTPases with many different cellular effectors. Myelination is an ideal place for RhoGTPases regulation, as it is the result of fine orchestration of many stimuli from at least two cell types. Recent work has revealed that RhoGTPases are required for Schwann cells to sort, ensheath and myelinate axons. Here we will review recent advances showing the critical roles for RhoGTPases in various aspects of Schwann development and myelination, including the recent discovery of their involvement in Charcot-Marie-Tooth disease. Comparison with potential roles of RhoGTPases in central nervous system myelination will be drawn. PMID:18803320

  6. RhoGTPases and p53 Are Involved in the Morphological Appearance and Interferon-α Response of Hairy Cells

    PubMed Central

    Chaigne-Delalande, Benjamin; Deuve, Lynda; Reuzeau, Edith; Basoni, Caroline; Lafarge, David; Varon, Christine; Tatin, Florence; Anies, Guerric; Garand, Richard; Kramer, Ijsbrand; Génot, Elisabeth

    2006-01-01

    Hairy cell leukemia is an uncommon B-cell lymphoproliferative disease of unknown etiology in which tumor cells display characteristic microfilamentous membrane projections. Another striking feature of the disease is its exquisite sensitivity to interferon (IFN)-α. So far, none of the known IFN-α regulatory properties have explained IFN-α responsiveness nor have they taken into account the morphological characteristics of hairy cells. IFN-α profoundly alters cytoskeletal organization of hairy cells and causes reversion of the hairy appearance into a rounded morphology. Because cytoskeletal rearrangements are controlled by the Rho family of GTPases, we investigated the GTPase activation status in hairy cells and their regulation by IFN-α. Using immunolocalization techniques and biochemical assays, we demonstrate that hairy cells display high levels of active Cdc42 and Rac1 and that IFN-α down-regulates these activities. In sharp contrast, RhoA activity was low in hairy cells but was increased by IFN-α treatment. Finally, IFN-α-mediated morphological changes also implicated a p53-induced response. These observations shed light on the mechanism of action of IFN-α in hairy cell leukemia and are of potential relevance for the therapeutical applications of this cytokine. PMID:16436670

  7. Chronic lymphocytic leukemia cells induce defective LFA-1–directed T-cell motility by altering Rho GTPase signaling that is reversible with lenalidomide

    PubMed Central

    Evans, Rachel; Kiaii, Shahryar; Svensson, Lena; Hogg, Nancy; Gribben, John G.

    2013-01-01

    T lymphocytes have an essential role in adaptive immunity and rely on the activation of integrin lymphocyte function–associated antigen-1 (LFA-1) to mediate cell arrest and migration. In cancer, malignant cells modify the immune microenvironment to block effective host antitumor responses. We show for the first time that CD4 and CD8 T cells from patients with chronic lymphocytic leukemia (CLL) exhibit globally impaired LFA-1–mediated migration and that this defect is mediated by direct tumor cell contact. We show that following the coculture of previously healthy T cells with CLL cells, subsequent LFA-1 engagement leads to altered Rho GTPase activation signaling by downregulating RhoA and Rac1, while upregulating Cdc42. Of clinical relevance, repair of this T-cell defect was demonstrated using the immunomodulatory drug lenalidomide, which completely rescued adhesion and motility function by restoring normal Rho GTPase activation signaling. Our report identifies a novel cancer immune evasion mechanism whereby tumor cells induce Rho GTPase signaling defects in T cells that prevent appropriate LFA-1 activation and motility. We believe these findings identify important biomarkers and highlight the clinical utility of immunotherapy to rescue normal T-cell function in CLLs that are likely to have relevance in other cancers. PMID:23325833

  8. Rho GTPase RhoJ is Associated with Gastric Cancer Progression and Metastasis

    PubMed Central

    Kim, Chan; Yang, Hannah; Park, Intae; Chon, Hong Jae; Kim, Joo Hoon; Kwon, Woo Sun; Lee, Won Suk; Kim, Tae Soo; Rha, Sun Young

    2016-01-01

    Rho GTPases play a pivotal role in tumor progression by regulating tumor cell migration and invasion. However, the role of Rho GTPases in gastric cancer (GC) remains unexplored. This study aimed to investigate the clinical implications of RhoJ, which is an uncharted member of Rho family. RhoJ expression in human GC cell lines and surgical specimens from GC patients were analyzed. Moreover, in vitro gain-of-function analysis was performed to evaluate the malignant phenotypes of RhoJ-overexpressing GC cells. The extent of RhoJ expression varied among GC cell lines and GC patients. YCC-9 cell line displayed the strongest expression, while YCC-10, -11, and -16 showed scant expressions. Of the 70 GC patients, 34 (48.6%) had RhoJ expression in their GC tissue, and patients with high RhoJ expression had more diffuse type GC (73.5% vs. 41.7%), were at more advanced stages (stage III, IV: 85.3% vs. 58.4%), and had more frequent metastasis (47.1% vs. 11.1%), denoting that RhoJ has a potential role in GC progression and metastasis. High RhoJ expression significantly correlated with poor overall survival and recurrence-free survival after surgical resection of gastric cancer. Finally, In vitro gain-of-function experiments showed 41.3% enhanced motility and 60.4% enhanced invasiveness in RhoJ-overexpressing GC cells compared to control, with negligible difference in cell proliferation. Collectively, high RhoJ expression is an independent negative prognostic factor for the survival outcome of GC and correlated with the increased cell motility and invasiveness. PMID:27471571

  9. Modelling Rho GTPase biochemistry to predict collective cell migration

    NASA Astrophysics Data System (ADS)

    Merchant, Brian; Feng, James

    The collective migration of cells, due to individual cell polarization and intercellular contact inhibition of locomotion, features prominently in embryogenesis and metastatic cancers. Existing methods for modelling collectively migrating cells tend to rely either on highly abstracted agent-based models, or on continuum approximations of the group. Both of these frameworks represent intercellular interactions such as contact inhibition of locomotion as hard-coded rules defining model cells. In contrast, we present a vertex-dynamics framework which predicts polarization and contact inhibition of locomotion naturally from an underlying model of Rho GTPase biochemistry and cortical mechanics. We simulate the interaction between many such model cells, and study how modulating Rho GTPases affects migratory characteristics of the group, in the context of long-distance collective migration of neural crest cells during embryogenesis.

  10. The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns

    PubMed Central

    Fritz, Rafael Dominik; Pertz, Olivier

    2016-01-01

    Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity. PMID:27158467

  11. Targeting Rho-GTPases in immune cell migration and inflammation

    PubMed Central

    Biro, Maté; Munoz, Marcia A; Weninger, Wolfgang

    2014-01-01

    Leukocytes are unmatched migrators capable of traversing barriers and tissues of remarkably varied structural composition. An effective immune response relies on the ability of its constituent cells to infiltrate target sites. Yet, unwarranted mobilization of immune cells can lead to inflammatory diseases and tissue damage ranging in severity from mild to life-threatening. The efficacy and plasticity of leukocyte migration is driven by the precise spatiotemporal regulation of the actin cytoskeleton. The small GTPases of the Rho family (Rho-GTPases), and their immediate downstream effector kinases, are key regulators of cellular actomyosin dynamics and are therefore considered prime pharmacological targets for stemming leukocyte motility in inflammatory disorders. This review describes advances in the development of small-molecule inhibitors aimed at modulating the Rho-GTPase-centric regulatory pathways governing motility, many of which stem from studies of cancer invasiveness. These inhibitors promise the advent of novel treatment options with high selectivity and potency against immune-mediated pathologies. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24 PMID:24571448

  12. Rac1 and Cdc42 Play Important Roles in Arsenic Neurotoxicity in Primary Cultured Rat Cerebellar Astrocytes.

    PubMed

    An, Yuan; Liu, Tingting; Liu, Xiaona; Zhao, Lijun; Wang, Jing

    2016-03-01

    This study aimed to explore whether Rac1 and Cdc42, representative members of Ras homologue guanosine triphosphatases (Rho GTPases), are involved in neurotoxicity induced by arsenic exposure in rat nervous system. Expressions of Rac1 and Cdc42 in rat cerebellum and cerebrum exposed to different doses of NaAsO2 (Wistar rats drank 0, 2, 10, and 50 mg/L NaAsO2 water for 3 months) were examined. Both Rac1 and Cdc42 expressions increased significantly in a dose-dependent manner in cerebellum (P < 0.01) by Western blot and immunohistochemistry assay, but in cerebrum, Rac1 and Cdc42 expressions only in 2 mg/L exposure groups were significantly higher than those in control groups (P < 0.01). Five to 50 μM NaAsO2 decreased cell viability in a dose-dependent manner in primary cultured rat astrocytes, whereas 1 μM NaAsO2 increased the cell viability in these cells. Rac1 inhibitor, NSC23766, decreased NaAsO2-induced apoptosis and increased the cell viability in primary cultured rat cerebellar astrocytes exposed to 30 μM NaAsO2. Cdc42 inhibitor, ZCL278, increased cell viability in the cells exposed to 30 μM NaAsO2. Taken together, our current studies in vivo and in vitro indicate that activations of Rac1 and Cdc42 play a very important role in arsenic neurotoxicity in rat cerebellum, providing a new insight into arsenic neurotoxicity. PMID:26231544

  13. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways.

    PubMed

    Pang, Xiufeng; Yi, Tingfang; Yi, Zhengfang; Cho, Sung Gook; Qu, Weijing; Pinkaew, Decha; Fujise, Ken; Liu, Mingyao

    2009-01-15

    Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action. PMID:19147565

  14. A Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin.

    PubMed

    Swaminathan, Karthic; Müller-Taubenberger, Annette; Faix, Jan; Rivero, Francisco; Noegel, Angelika A

    2014-01-01

    The Cdc42- and Rac-interactive binding motif (CRIB) of coronin binds to Rho GTPases with a preference for GDP-loaded Rac. Mutation of the Cdc42- and Rac-interactive binding motif abrogates Rac binding. This results in increased 1evels of activated Rac in coronin-deficient Dictyostelium cells (corA(-)), which impacts myosin II assembly. corA(-) cells show increased accumulation of myosin II in the cortex of growth-phase cells. Myosin II assembly is regulated by myosin heavy chain kinase-mediated phosphorylation of its tail. Kinase activity depends on the activation state of the p21-activated kinase a. The myosin II defect of corA(-) mutant is alleviated by dominant-negative p21-activated kinase a. It is rescued by wild-type coronin, whereas coronin carrying a mutated Cdc42- and Rac-interactive binding motif failed to rescue the myosin defect in corA(-) mutant cells. Ectopically expressed myosin heavy chain kinases affinity purified from corA(-) cells show reduced kinase activity. We propose that coronin through its affinity for GDP-Rac regulates the availability of GTP-Rac for activation of downstream effectors. PMID:24347642

  15. Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity

    PubMed Central

    2013-01-01

    Background Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential. Methods We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197. Results In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo. Conclusions These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth. PMID:24279335

  16. Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

    SciTech Connect

    Delawary, Mina; Nakazawa, Takanobu; Tezuka, Tohru; Sawa, Mariko; Iino, Yuichi; Takenawa, Tadaomi; Yamamoto, Tadashi . E-mail: tyamamot@ims.u-tokyo.ac.jp

    2007-06-01

    The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.

  17. Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway

    PubMed Central

    Simon, Sylvia; Schell, Ursula; Heuer, Natalie; Hager, Dominik; Albers, Michael F.; Matthias, Jan; Fahrnbauer, Felix; Trauner, Dirk; Eichinger, Ludwig; Hedberg, Christian; Hilbi, Hubert

    2015-01-01

    Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9. PMID:26633832

  18. Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway

    PubMed Central

    Leung, Daisy W.; Otomo, Chinatsu; Chory, Joanne; Rosen, Michael K.

    2008-01-01

    General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs. PMID:18728185

  19. Neuronal Apoptosis Induced by Selective Inhibition of Rac GTPase versus Global Suppression of Rho Family GTPases Is Mediated by Alterations in Distinct Mitogen-activated Protein Kinase Signaling Cascades*

    PubMed Central

    Stankiewicz, Trisha R.; Ramaswami, Sai Anandi; Bouchard, Ron J.; Aktories, Klaus; Linseman, Daniel A.

    2015-01-01

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  20. Neuronal apoptosis induced by selective inhibition of Rac GTPase versus global suppression of Rho family GTPases is mediated by alterations in distinct mitogen-activated protein kinase signaling cascades.

    PubMed

    Stankiewicz, Trisha R; Ramaswami, Sai Anandi; Bouchard, Ron J; Aktories, Klaus; Linseman, Daniel A

    2015-04-10

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  1. RAB and RHO GTPases regulate intestinal crypt cell homeostasis and enterocyte function.

    PubMed

    Zhang, Xiao; Gao, Nan

    2016-04-01

    Recent human and mouse genetic studies have highlighted important contributions of several small GTPases, in particular Rab8a, (1) Cdc42, (2-4) and Rab11a, (5-8) to the proper morphogenesis and function of the mature intestinal epithelia. Additional insights about the involvement of these factors in maintaining intestinal stem cell homeostasis have also been obtained. (9,10) These studies suggest a conserved vesicular and membrane trafficking program utilized by the gastrointestinal tissue to support the rapid epithelial cell turnover and the highly sophisticated physiology of mature epithelial cells. PMID:27142493

  2. Modulation of osteoclast differentiation and bone resorption by Rho GTPases

    PubMed Central

    Touaitahuata, Heiani; Blangy, Anne; Vives, Virginie

    2014-01-01

    Bone is a dynamic tissue constantly renewed through a regulated balance between bone formation and resorption. Excessive bone degradation by osteoclasts leads to pathological decreased bone density characteristic of osteolytic diseases such as post-menopausal osteoporosis or bone metastasis. Osteoclasts are multinucleated cells derived from hematopoietic stem cells via a complex differentiation process. Their unique ability to resorb bone is dependent on the formation of the actin-rich sealing zone. Within this adhesion structure, the plasma membrane differentiates into the ruffled border where protons and proteases are secreted to demineralize and degrade bone, respectively. On the bone surface, mature osteoclasts alternate between stationary resorptive and migratory phases. These are associated with profound actin cytoskeleton reorganization, until osteoclasts die of apoptosis. In this review, we highlight the role of Rho GTPases in all the steps of osteoclasts differentiation, function, and death and conclude on their interest as targets for treatment of osteolytic pathologies. PMID:24614674

  3. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.

    PubMed

    Zhou, Wu; Li, Xiaobo; Premont, Richard T

    2016-05-15

    The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease. PMID:27182061

  4. Interaction of Anesthetics with the Rho GTPase Regulator Rho GDP Dissociation Inhibitor†

    PubMed Central

    Ho, Cojen; Shanmugasundararaj, Sivananthaperumal; Miller, Keith W.; Malinowski, Steve A.; Cook, Anthony C.; Slater, Simon J.

    2015-01-01

    The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599–601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIα), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIα was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short range and W192 and W194 of the RhoGDIα are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols (n<6) also reversed tryptophan quenching, suggesting that RhoGDIα may also bind n-alkanols. Consistent with this, E193 was photo-labeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photo-labeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIα, whereas alcohols bind to a distal site that interacts allosterically with this pocket. PMID:18702520

  5. Cdc42p GDP/GTP Cycling Is Necessary for Efficient Cell Fusion during Yeast Mating

    PubMed Central

    Barale, Sophie; McCusker, Derek

    2006-01-01

    The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP–GTP cycling is critical for efficient cell fusion. PMID:16571678

  6. Plexin-B2 negatively regulates macrophage motility, Rac, and Cdc42 activation.

    PubMed

    Roney, Kelly E; O'Connor, Brian P; Wen, Haitao; Holl, Eda K; Guthrie, Elizabeth H; Davis, Beckley K; Jones, Stephen W; Jha, Sushmita; Sharek, Lisa; Garcia-Mata, Rafael; Bear, James E; Ting, Jenny P-Y

    2011-01-01

    Plexins are cell surface receptors widely studied in the nervous system, where they mediate migration and morphogenesis though the Rho family of small GTPases. More recently, plexins have been implicated in immune processes including cell-cell interaction, immune activation, migration, and cytokine production. Plexin-B2 facilitates ligand induced cell guidance and migration in the nervous system, and induces cytoskeletal changes in overexpression assays through RhoGTPase. The function of Plexin-B2 in the immune system is unknown. This report shows that Plexin-B2 is highly expressed on cells of the innate immune system in the mouse, including macrophages, conventional dendritic cells, and plasmacytoid dendritic cells. However, Plexin-B2 does not appear to regulate the production of proinflammatory cytokines, phagocytosis of a variety of targets, or directional migration towards chemoattractants or extracellular matrix in mouse macrophages. Instead, Plxnb2(-/-) macrophages have greater cellular motility than wild type in the unstimulated state that is accompanied by more active, GTP-bound Rac and Cdc42. Additionally, Plxnb2(-/-) macrophages demonstrate faster in vitro wound closure activity. Studies have shown that a closely related family member, Plexin-B1, binds to active Rac and sequesters it from downstream signaling. The interaction of Plexin-B2 with Rac has only been previously confirmed in yeast and bacterial overexpression assays. The data presented here show that Plexin-B2 functions in mouse macrophages as a negative regulator of the GTPases Rac and Cdc42 and as a negative regulator of basal cell motility and wound healing. PMID:21966369

  7. Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

    PubMed Central

    Pulver, Rebecca; Heisel, Timothy; Gonia, Sara; Robins, Robert; Norton, Jennifer; Haynes, Paula

    2013-01-01

    The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. PMID:23223038

  8. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  9. A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis

    PubMed Central

    Abraham, Sabu; Scarcia, Margherita; Bagshaw, Richard D.; McMahon, Kathryn; Grant, Gary; Harvey, Tracey; Yeo, Maggie; Esteves, Filomena O.G.; Thygesen, Helene H.; Jones, Pamela F.; Speirs, Valerie; Hanby, Andrew M.; Selby, Peter J.; Lorger, Mihaela; Dear, T. Neil; Pawson, Tony; Marshall, Christopher J.; Mavria, Georgia

    2015-01-01

    During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis. PMID:26129894

  10. The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFY) Enhances Inflammation and Yop Delivery during Infection by Activation of Rho GTPases

    PubMed Central

    Schweer, Janina; Kulkarni, Devesha; Kochut, Annika; Pezoldt, Joern; Pisano, Fabio; Pils, Marina C.; Genth, Harald; Huehn, Jochen; Dersch, Petra

    2013-01-01

    Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection. PMID:24244167

  11. Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans

    PubMed Central

    Shen, Gui; Zhou, Erxun; Alspaugh, J. Andrew

    2012-01-01

    Human Wiskott-Aldrich syndrome protein (WASP) is a scaffold linking upstream signals to the actin cytoskeleton. In response to intersectin ITSN1 and Rho GTPase Cdc42, WASP activates the Arp2/3 complex to promote actin polymerization. The human pathogen Cryptococcus neoformans contains the ITSN1 homolog Cin1 and the WASP homolog Wsp1, which share more homology with human proteins than those of other fungi. Here we demonstrate that Cin1, Cdc42/Rac1, and Wsp1 function in an effector pathway similar to that of mammalian models. In the cin1 mutant, expression of the autoactivated Wsp1-B-GBD allele partially suppressed the mutant defect in endocytosis, and expression of the constitutively active CDC42Q61L allele restored normal actin cytoskeleton structures. Similar phenotypic suppression can be obtained by the expression of a Cdc42-green fluorescent protein (GFP)-Wsp1 fusion protein. In addition, Rac1, which was found to exhibit a role in early endocytosis, activates Wsp1 to regulate vacuole fusion. Rac1 interacted with Wsp1 and depended on Wsp1 for its vacuolar membrane localization. Expression of the Wsp1-B-GBD allele restored vacuolar membrane fusion in the rac1 mutant. Collectively, our studies suggest novel ways in which this pathogenic fungus has adapted conserved signaling pathways to control vesicle transport and actin organization, likely benefiting survival within infected hosts. PMID:22327008

  12. Grandparental stem cells in leech segmentation: differences in CDC42 expression are correlated with an alternating pattern of blast cell fates

    PubMed Central

    Zhang, Shaobing O.; Kuo, Dian-Han; Weisblat, David A.

    2009-01-01

    Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the H. robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells. PMID:19747476

  13. Bacterial factors exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host

    PubMed Central

    Popoff, Michel R

    2014-01-01

    Actin cytoskeleton is a main target of many bacterial pathogens. Among the multiple regulation steps of the actin cytoskeleton, bacterial factors interact preferentially with RhoGTPases. Pathogens secrete either toxins which diffuse in the surrounding environment, or directly inject virulence factors into target cells. Bacterial toxins, which interfere with RhoGTPases, and to some extent with RasGTPases, catalyze a covalent modification (ADPribosylation, glucosylation, deamidation, adenylation, proteolysis) blocking these molecules in their active or inactive state, resulting in alteration of epithelial and/or endothelial barriers, which contributes to dissemination of bacteria in the host. Injected bacterial virulence factors preferentially manipulate the RhoGTPase signaling cascade by mimicry of eukaryotic regulatory proteins leading to local actin cytoskeleton rearrangement, which mediates bacterial entry into host cells or in contrast escape to phagocytosis and immune defense. Invasive bacteria can also manipulate RhoGTPase signaling through recognition and stimulation of cell surface receptor(s). Changes in RhoGTPase activation state is sensed by the innate immunity pathways and allows the host cell to adapt an appropriate defense response. PMID:25203748

  14. The Regulation of Cellular Responses to Mechanical Cues by Rho GTPases

    PubMed Central

    Hoon, Jing Ling; Tan, Mei Hua; Koh, Cheng-Gee

    2016-01-01

    The Rho GTPases regulate many cellular signaling cascades that modulate cell motility, migration, morphology and cell division. A large body of work has now delineated the biochemical cues and pathways, which stimulate the GTPases and their downstream effectors. However, cells also respond exquisitely to biophysical and mechanical cues such as stiffness and topography of the extracellular matrix that profoundly influence cell migration, proliferation and differentiation. As these cellular responses are mediated by the actin cytoskeleton, an involvement of Rho GTPases in the transduction of such cues is not unexpected. In this review, we discuss an emerging role of Rho GTPase proteins in the regulation of the responses elicited by biophysical and mechanical stimuli. PMID:27058559

  15. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

    PubMed Central

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal

    2016-01-01

    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. PMID:26728857

  16. Elevated Intraocular Pressure Induces Rho GTPase Mediated Contractile Signaling in the Trabecular Meshwork

    PubMed Central

    Pattabiraman, Padmanabhan P; Inoue, Toshihiro; Rao, P. Vasantha

    2015-01-01

    Rho GTPase regulated contractile signaling in the trabecular meshwork (TM) has been shown to modulate aqueous humor (AH) outflow and intraocular pressure (IOP). To explore whether elevated IOP, a major risk factor for primary open angle glaucoma (POAG) influences Rho GTPase signaling in the TM, we recorded AH outflow in enucleated contralateral porcine eyes perfused for 4–5 hours at either 15 mm or 50 mm Hg pressure. After perfusion, TM tissue extracted from perfused eyes was evaluated for the activation status of Rho GTPase, myosin light chain (MLC), myosin phosphatase target substrate 1 (MYPT1), myristoylated alanine-rich C-kinase substrate (MARCKS) and paxillin. Eyes perfused at 50 mm Hg exhibited a significant decrease in AH outflow facility compared with those perfused at 15 mm Hg. Additionally, TM tissue from eyes perfused at 50 mm Hg revealed significantly increased levels of activated RhoA and phosphorylated MLC, MYPT1, MARCKS and paxillin compared to TM tissue derived from eyes perfused at 15 mm Hg. Taken together, these observations indicate that elevated IOP-induced activation of Rho GTPase-dependent contractile signaling in the TM is associated with increased resistance to AH outflow through the trabecular pathway, and demonstrate the sensitivity of Rho GTPase signaling to mechanical force in the AH outflow pathway. PMID:25956210

  17. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling.

    PubMed

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal; Pertz, Olivier

    2016-01-01

    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. PMID:26728857

  18. Conserved roles for yeast Rho1 and mammalian RhoA GTPases in clathrin-independent endocytosis

    PubMed Central

    Prosser, Derek C.; Wendland, Beverly

    2012-01-01

    Eukaryotic cells use numerous endocytic pathways for nutrient uptake, protein turnover and response to the extracellular environment. While clathrin-mediated endocytosis (CME) has been extensively studied in yeast and mammalian cells, recent studies in higher eukaryotes have described multiple clathrin-independent endocytic pathways that depend upon Rho family GTPases and their effector proteins. In contrast, yeast cells have been thought to rely solely on CME. In a recent study, we used CME-defective yeast cells lacking clathrin-binding endocytic adaptor proteins in a genetic screen to identify novel factors involved in endocytosis. This approach revealed the existence of a clathrin-independent endocytic pathway involving the GTPase Rho1, which is the yeast homolog of RhoA. Further characterization of the yeast Rho1-mediated endocytic pathway suggested that the Rho1 pathway requires additional proteins that appear to play conserved roles in RhoA-dependent, clathrin-independent endocytic pathways in mammalian cells. Here, we discuss the parallels between the yeast Rho1-dependent and mammalian RhoA-dependent endocytic pathways, as well as the applications of yeast as a model for studying clathrin-independent endocytosis in higher eukaryotes. PMID:23238351

  19. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38γ) MAP kinase pathway

    PubMed Central

    Marinissen, Maria Julia; Chiariello, Mario; Gutkind, J. Silvio

    2001-01-01

    Small GTP-binding proteins of the Rho-family, Rho, Rac, and Cdc42, have been traditionally linked to the regulation of the cellular actin-based cytoskeleton. Rac and Cdc42 can also control the activity of JNK, thus acting in a molecular pathway transmitting extracellular signals to the nucleus. Interestingly, Rho can also regulate gene expression, albeit by a not fully understood mechanism. Here, we found that activated RhoA can stimulate c-jun expression and the activity of the c-jun promoter. As the complexity of the signaling pathways controlling the expression of c-jun has begun to be unraveled, this finding provided a unique opportunity to elucidate the biochemical routes whereby RhoA regulates nuclear events. We found that RhoA can initiate a linear kinase cascade leading to the activation of ERK6 (p38γ), a recently identified member of the p38 family of MAPKs. Furthermore, we present evidence that RhoA, PKN, MKK3/MKK6, and ERK6 (p38γ) are components of a novel signal transduction pathway involved in the regulation of gene expression and cellular transformation. PMID:11238375

  20. Investigation of the Interaction between Cdc42 and Its Effector TOCA1

    PubMed Central

    Watson, Joanna R.; Fox, Helen M.; Nietlispach, Daniel; Gallop, Jennifer L.; Owen, Darerca

    2016-01-01

    Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. PMID:27129201

  1. RhoA GTPase interacts with beta-catenin signaling in clinorotated osteoblasts

    PubMed Central

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2014-01-01

    Bone is a dynamic tissue under constant remodeling in response to various signals including mechanical loading. A lack of proper mechanical loading induces disuse osteoporosis that reduces bone mass and structural integrity. β-catenin signaling together with a network of GTPases is known to play a primary role in load-driven bone formation, but little is known about potential interactions of β-catenin signaling and GTPases in bone loss. In this study, we addressed a question: Does unloading suppress an activation level of RhoA GTPase and β-catenin signaling in osteoblasts? If yes, what is the role of RhoA GTPase and actin filaments in osteoblasts in regulating β-catenin signaling? Using a fluorescence resonance energy transfer (FRET) technique with a biosensor for RhoA together with a fluorescent T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter, we examined the effects of clinostat-driven simulated unloading. The results revealed that both RhoA activity and TCF/LEF activity were downregulated by unloading. Reduction in RhoA activity was correlated to a decrease in cytoskeletal organization of actin filaments. Inhibition of β-catenin signaling blocked unloading-induced RhoA suppression, and dominant negative RhoA inhibited TCF/LEF suppression. On the other hand, a constitutively active RhoA enhanced unloading-induced reduction of TCF/LEF activity. The TCF/LEF suppression by unloading was enhanced by co-culture with osteocytes, but it was independent on organization of actin filaments, myosin II activity, or a myosin light chain kinase. Collectively, the results suggest that β-catenin signaling is required for unloading-driven regulation of RhoA, and RhoA, but not actin cytoskeleton or intracellular tension, mediates the responsiveness of β-catenin signaling to unloading. PMID:23529802

  2. Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension.

    PubMed

    Simões, Sérgio de Matos; Mainieri, Avantika; Zallen, Jennifer A

    2014-02-17

    Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila melanogaster, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. We show that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation. PMID:24535826

  3. The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins

    PubMed Central

    Chen, Shuyi; Sun, Chunli; Wang, Haiying; Wang, Jufang

    2015-01-01

    Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of C. difficile, are the major virulence factors of CDI. The common mode of action of TcdA and TcdB is elicited by specific glucosylation of Rho-GTPase proteins in the host cytosol using UDP-glucose as a co-substrate, resulting in the inactivation of Rho proteins. Rho proteins are the key members in many biological processes and signaling pathways, inactivation of which leads to cytopathic and cytotoxic effects and immune responses of the host cells. It is supposed that Rho GTPases play an important role in the toxicity of C. difficile toxins. This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB. PMID:26633511

  4. Analysis of a minimal Rho-GTPase circuit regulating cell shape

    NASA Astrophysics Data System (ADS)

    Holmes, William R.; Edelstein-Keshet, Leah

    2016-08-01

    Networks of Rho-family GTPases regulate eukaryotic cell polarization and motility by controlling assembly and contraction of the cytoskeleton. The mutually inhibitory Rac–Rho circuit is emerging as a central, regulatory hub that can affect the shape and motility phenotype of eukaryotic cells. Recent experimental manipulation of the amounts of Rac and Rho or their regulators (guanine nucleotide-exchange factors, GTPase-activating proteins, guanine nucleotide dissociation inhibitors) have been shown to bias the prevalence of these different states and promote transitions between them. Here we show that part of this data can be understood in terms of inherent Rac–Rho mutually inhibitory dynamics. We analyze a spatio-temporal mathematical model of Rac–Rho dynamics to produce a detailed set of predictions of how parameters such as GTPase rates of activation and total amounts affect cell decisions (such as Rho-dominated contraction, Rac-dominated spreading, and spatially segregated Rac–Rho polarization). We find that in some parameter regimes, a cell can take on any of these three fates depending on its environment or stimuli. We also predict how experimental manipulations (corresponding to parameter variations) can affect cell shapes observed. Our methods are based on local perturbation analysis (a kind of nonlinear stability analysis), and an approximation of nonlinear feedback by sharp switches. We compare the Rac–Rho model to an even simpler single-GTPase (‘wave-pinning’) model and demonstrate that the overall behavior is inherent to GTPase properties, rather than stemming solely from network topology.

  5. Analysis of a minimal Rho-GTPase circuit regulating cell shape.

    PubMed

    Holmes, William R; Edelstein-Keshet, Leah

    2016-01-01

    Networks of Rho-family GTPases regulate eukaryotic cell polarization and motility by controlling assembly and contraction of the cytoskeleton. The mutually inhibitory Rac-Rho circuit is emerging as a central, regulatory hub that can affect the shape and motility phenotype of eukaryotic cells. Recent experimental manipulation of the amounts of Rac and Rho or their regulators (guanine nucleotide-exchange factors, GTPase-activating proteins, guanine nucleotide dissociation inhibitors) have been shown to bias the prevalence of these different states and promote transitions between them. Here we show that part of this data can be understood in terms of inherent Rac-Rho mutually inhibitory dynamics. We analyze a spatio-temporal mathematical model of Rac-Rho dynamics to produce a detailed set of predictions of how parameters such as GTPase rates of activation and total amounts affect cell decisions (such as Rho-dominated contraction, Rac-dominated spreading, and spatially segregated Rac-Rho polarization). We find that in some parameter regimes, a cell can take on any of these three fates depending on its environment or stimuli. We also predict how experimental manipulations (corresponding to parameter variations) can affect cell shapes observed. Our methods are based on local perturbation analysis (a kind of nonlinear stability analysis), and an approximation of nonlinear feedback by sharp switches. We compare the Rac-Rho model to an even simpler single-GTPase ('wave-pinning') model and demonstrate that the overall behavior is inherent to GTPase properties, rather than stemming solely from network topology. PMID:27434017

  6. Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration.

    PubMed

    O'Neill, Patrick R; Kalyanaraman, Vani; Gautam, N

    2016-05-01

    Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses. PMID:26941336

  7. Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration

    PubMed Central

    O’Neill, Patrick R.; Kalyanaraman, Vani; Gautam, N.

    2016-01-01

    Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses. PMID:26941336

  8. RhoGTPases as Key Players in Mammalian Cell Adaptation to Microgravity

    PubMed Central

    Deroanne, Christophe; Nusgens, Betty; Vico, Laurence; Guignandon, Alain

    2015-01-01

    A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions. PMID:25649831

  9. RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

    SciTech Connect

    Rousseau, Matthieu; Gaugler, Marie-Helene; Rodallec, Audrey; Bonnaud, Stephanie; Paris, Francois; Corre, Isabelle

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We explore the role of RhoA in endothelial cell response to ionizing radiation. Black-Right-Pointing-Pointer RhoA is rapidly activated by single high-dose of radiation. Black-Right-Pointing-Pointer Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. Black-Right-Pointing-Pointer Radiation-induced apoptosis does not require the RhoA/ROCK pathway. Black-Right-Pointing-Pointer Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial

  10. Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets.

    PubMed

    Beck, S; Fotinos, A; Lang, F; Gawaz, M; Elvers, M

    2013-01-01

    Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that - beside the GAP domain - the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation. PMID:22975681

  11. ATP8B1-mediated spatial organization of Cdc42 signaling maintains singularity during enterocyte polarization

    PubMed Central

    Bruurs, Lucas J.M.; Donker, Lisa; Zwakenberg, Susan; Zwartkruis, Fried J.; Begthel, Harry; Knisely, A.S.; Posthuma, George; van de Graaf, Stan F.J.; Paulusma, Coen C.

    2015-01-01

    During yeast cell polarization localization of the small GTPase, cell division control protein 42 homologue (Cdc42) is clustered to ensure the formation of a single bud. Here we show that the disease-associated flippase ATPase class I type 8b member 1 (ATP8B1) enables Cdc42 clustering during enterocyte polarization. Loss of this regulation results in increased apical membrane size with scattered apical recycling endosomes and permits the formation of more than one apical domain, resembling the singularity defect observed in yeast. Mechanistically, we show that to become apically clustered, Cdc42 requires the interaction between its polybasic region and negatively charged membrane lipids provided by ATP8B1. Disturbing this interaction, either by ATP8B1 depletion or by introduction of a Cdc42 mutant defective in lipid binding, increases Cdc42 mobility and results in apical membrane enlargement. Re-establishing Cdc42 clustering, by tethering it to the apical membrane or lowering its diffusion, restores normal apical membrane size in ATP8B1-depleted cells. We therefore conclude that singularity regulation by Cdc42 is conserved between yeast and human and that this regulation is required to maintain healthy tissue architecture. PMID:26416959

  12. Opposing Roles for Actin in Cdc42p PolarizationD⃞

    PubMed Central

    Irazoqui, Javier E.; Howell, Audrey S.; Theesfeld, Chandra L.; Lew, Daniel J.

    2005-01-01

    In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site. PMID:15616194

  13. Rho GTPase activity in the honey bee mushroom bodies is correlated with age and foraging experience

    PubMed Central

    Dobrin, Scott E.; Fahrbach, Susan E.

    2011-01-01

    Foraging experience is correlated with structural plasticity of the mushroom bodies of the honey bee brain. While several neurotransmitter and intracellular signaling pathways have been previously implicated as mediators of these structural changes, none interact directly with the cytoskeleton, the ultimate effector of changes in neuronal morphology. The Rho family of GTPases are small, monomeric G proteins that, when activated, initiate a signaling cascade that reorganizes the neuronal cytoskeleton. In this study, we measured activity of two members of the Rho family of GTPases, Rac and RhoA, in the mushroom bodies of bees with different durations of foraging experience. A transient increase in Rac activity coupled with a transient decrease in RhoA activity was found in honey bees with 4 days foraging experience compared with same-aged new foragers. These observations are in accord with previous reports based on studies of other species of a growth supporting role for Rac and a growth opposing role for RhoA. This is the first report of Rho GTPase activation in the honey bee brain. PMID:22108023

  14. Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

    PubMed Central

    Bretou, Marine; Jouannot, Ouardane; Fanget, Isabelle; Pierobon, Paolo; Larochette, Nathanaël; Gestraud, Pierre; Guillon, Marc; Emiliani, Valentina; Gasman, Stéphane; Desnos, Claire; Lennon-Duménil, Ana-Maria; Darchen, François

    2014-01-01

    Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force. PMID:25143404

  15. Control of postnatal apoptosis in the neocortex by RhoA-subfamily GTPases determines neuronal density.

    PubMed

    Sanno, Hitomi; Shen, Xiao; Kuru, Nilgün; Bormuth, Ingo; Bobsin, Kristin; Gardner, Humphrey A R; Komljenovic, Dorde; Tarabykin, Victor; Erzurumlu, Reha S; Tucker, Kerry L

    2010-03-24

    Apoptosis of neurons in the maturing neocortex has been recorded in a wide variety of mammals, but very little is known about its effects on cortical differentiation. Recent research has implicated the RhoA GTPase subfamily in the control of apoptosis in the developing nervous system and in other tissue types. Rho GTPases are important components of the signaling pathways linking extracellular signals to the cytoskeleton. To investigate the role of the RhoA GTPase subfamily in neocortical apoptosis and differentiation, we have engineered a mouse line in which a dominant-negative RhoA mutant (N19-RhoA) is expressed from the Mapt locus, such that all neurons of the developing nervous system are expressing the N19-RhoA inhibitor. Postnatal expression of N19-RhoA led to no major changes in neocortical anatomy. Six layers of the neocortex developed and barrels (whisker-related neural modules) formed in layer IV. However, the density and absolute number of neurons in the somatosensory cortex increased by 12-26% compared with wild-type littermates. This was not explained by a change in the migration of neurons during the formation of cortical layers but rather by a large decrease in the amount of neuronal apoptosis at postnatal day 5, the developmental maximum of cortical apoptosis. In addition, overexpression of RhoA in cortical neurons was seen to cause high levels of apoptosis. These results demonstrate that RhoA-subfamily members play a major role in developmental apoptosis in postnatal neocortex of the mouse but that decreased apoptosis does not alter cortical cytoarchitecture and patterning. PMID:20335457

  16. Control of postnatal apoptosis in the neocortex by RhoA-subfamily GTPases determines neuronal density

    PubMed Central

    Sanno, Hitomi; Shen, Xiao; Kuru, Nilgün; Bormuth, Ingo; Bobsin, Kristin; Komljenovic, Dorde; Tarabykin, Victor; Erzurumlu, Reha S.; Tucker, Kerry L.

    2010-01-01

    Apoptosis of neurons in the maturing neocortex has been recorded in a wide variety of mammals, but very little is known about its effects on cortical differentiation. Recent research has implicated the RhoA GTPase subfamily in the control of apoptosis in the developing nervous system and in other tissue types. Rho GTPases are important components of the signaling pathways linking extracellular signals to the cytoskeleton. To investigate the role of the RhoA GTPase subfamily in neocortical apoptosis and differentiation, we have engineered a mouse line in which a dominant-negative RhoA mutant (N19-RhoA) is expressed from the Mapt locus, such that all neurons of the developing nervous system are expressing the N19-RhoA inhibitor. Postnatal expression of N19-RhoA led to no major changes in neocortical anatomy. Six layers of the neocortex developed and barrels (whisker-related neural modules) formed in layer IV. However, the density and absolute number of neurons in the somatosensory cortex increased by 12 - 26%, as compared to wildtype littermates. This was not explained by a change in the migration of neurons during the formation of cortical layers, but rather by a large decrease in the amount of neuronal apoptosis at P5, the developmental maximum of cortical apoptosis. In addition, overexpression of RhoA in cortical neurons was seen to cause high levels of apoptosis. These results demonstrate that RhoA-subfamily members play a major role in developmental apoptosis in postnatal neocortex of the mouse, but that decreased apoptosis does not alter cortical cytoarchitecture and patterning. PMID:20335457

  17. Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

    PubMed Central

    Schulz, Anna M.; Stutte, Susanne; Hogl, Sebastian; Luckashenak, Nancy; Dudziak, Diana; Leroy, Céline; Forné, Ignasi; Imhof, Axel; Müller, Stephan A.; Brakebusch, Cord H.; Lichtenthaler, Stefan F.

    2015-01-01

    Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface. PMID:26553928

  18. R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis.

    PubMed

    Guo, Yuna; Kenney, S Ray; Muller, Carolyn Y; Adams, Sarah; Rutledge, Teresa; Romero, Elsa; Murray-Krezan, Cristina; Prekeris, Rytis; Sklar, Larry A; Hudson, Laurie G; Wandinger-Ness, Angela

    2015-10-01

    Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans. PMID:26206334

  19. Isolation and Characterization of Effector-Loop Mutants of CDC42 in Yeast

    PubMed Central

    Gladfelter, Amy S.; Moskow, John J.; Zyla, Trevin R.; Lew, Daniel J.

    2001-01-01

    The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of “effector-loop” mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors. PMID:11359919

  20. Isolation and characterization of effector-loop mutants of CDC42 in yeast.

    PubMed

    Gladfelter, A S; Moskow, J J; Zyla, T R; Lew, D J

    2001-05-01

    The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors. PMID:11359919

  1. Two Pathways through Cdc42 Couple the N-Formyl Receptor to Actin Nucleation in Permeabilized Human Neutrophils

    PubMed Central

    Glogauer, M.; Hartwig, J.; Stossel, T.

    2000-01-01

    We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends. PMID:10953003

  2. Localized RhoA GTPase activity regulates dynamics of endothelial monolayer integrity

    PubMed Central

    Szulcek, Robert; Beckers, Cora M.L.; Hodzic, Jasmina; de Wit, Jelle; Chen, Zhenlong; Grob, Tim; Musters, Rene J.P.; Minshall, Richard D.; van Hinsbergh, Victor W.M.; van Nieuw Amerongen, Geerten P.

    2013-01-01

    Aims Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith connected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are associated with distinct localized RhoA activities at the subcellular level. Methods and results Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility. Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for this protrusive activity and maintenance of barrier restoration. Conclusion Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter-endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re-annealing of endothelial junctions. PMID:23536606

  3. RhoA GTPase controls cytokinesis and programmed necrosis of hematopoietic progenitors

    PubMed Central

    Zhou, Xuan; Florian, Maria Carolina; Arumugam, Paritha; Chen, Xiaoyi; Cancelas, Jose A.; Lang, Richard; Malik, Punam; Geiger, Hartmut

    2013-01-01

    Hematopoietic progenitor cells (HPCs) are central to hematopoiesis as they provide large numbers of lineage-defined blood cells necessary to sustain blood homeostasis. They are one of the most actively cycling somatic cells, and their precise control is critical for hematopoietic homeostasis. The small GTPase RhoA is an intracellular molecular switch that integrates cytokine, chemokine, and adhesion signals to coordinate multiple context-dependent cellular processes. By using a RhoA conditional knockout mouse model, we show that RhoA deficiency causes a multilineage hematopoietic failure that is associated with defective multipotent HPCs. Interestingly, RhoA−/− hematopoietic stem cells retained long-term engraftment potential but failed to produce multipotent HPCs and lineage-defined blood cells. This multilineage hematopoietic failure was rescued by reconstituting wild-type RhoA into the RhoA−/− Lin−Sca-1+c-Kit+ compartment. Mechanistically, RhoA regulates actomyosin signaling, cytokinesis, and programmed necrosis of the HPCs, and loss of RhoA results in a cytokinesis failure of HPCs manifested by an accumulation of multinucleated cells caused by failed abscission of the cleavage furrow after telophase. Concomitantly, the HPCs show a drastically increased death associated with increased TNF–RIP-mediated necrosis. These results show that RhoA is a critical and specific regulator of multipotent HPCs during cytokinesis and thus essential for multilineage hematopoiesis. PMID:24101377

  4. Extracting Diffusive States of Rho GTPase in Live Cells: Towards In Vivo Biochemistry

    PubMed Central

    Sabanaygam, Chandran R.; van Golen, Kenneth L.; Mochrie, Simon G. J.

    2015-01-01

    Resolving distinct biochemical interaction states when analyzing the trajectories of diffusing proteins in live cells on an individual basis remains challenging because of the limited statistics provided by the relatively short trajectories available experimentally. Here, we introduce a novel, machine-learning based classification methodology, which we call perturbation expectation-maximization (pEM), that simultaneously analyzes a population of protein trajectories to uncover the system of diffusive behaviors which collectively result from distinct biochemical interactions. We validate the performance of pEM in silico and demonstrate that pEM is capable of uncovering the proper number of underlying diffusive states with an accurate characterization of their diffusion properties. We then apply pEM to experimental protein trajectories of Rho GTPases, an integral regulator of cytoskeletal dynamics and cellular homeostasis, in vivo via single particle tracking photo-activated localization microcopy. Remarkably, pEM uncovers 6 distinct diffusive states conserved across various Rho GTPase family members. The variability across family members in the propensities for each diffusive state reveals non-redundant roles in the activation states of RhoA and RhoC. In a resting cell, our results support a model where RhoA is constantly cycling between activation states, with an imbalance of rates favoring an inactive state. RhoC, on the other hand, remains predominantly inactive. PMID:26512894

  5. RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42

    PubMed Central

    Misselwitz, Benjamin; Dilling, Sabrina; Vonaesch, Pascale; Sacher, Raphael; Snijder, Berend; Schlumberger, Markus; Rout, Samuel; Stark, Manuel; Mering, Christian von; Pelkmans, Lucas; Hardt, Wolf-Dietrich

    2011-01-01

    The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these ‘hits' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs. PMID:21407211

  6. The N-BAR Domain Protein, Bin3, Regulates Rac1- and Cdc42-Dependent Processes in Myogenesis

    PubMed Central

    Simionescu-Bankston, Adriana; Leoni, Giovanna; Wang, Yanru; Pham, Peter P.; Ramalingam, Arivudainambi; DuHadaway, James B.; Faundez, Victor; Nusrat, Asma; Prendergast, George C.; Pavlath, Grace K.

    2013-01-01

    Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair. PMID:23872330

  7. Proper Regulation of Cdc42 Activity is Required for Tight Actin Concentration at the Equator during Cytokinesis in Adherent Mammalian Cells

    PubMed Central

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2012-01-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells. PMID:21763307

  8. A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

    PubMed Central

    Ageta-Ishihara, Natsumi; Yamazaki, Maya; Konno, Kohtarou; Nakayama, Hisako; Abe, Manabu; Hashimoto, Kenji; Nishioka, Tomoki; Kaibuchi, Kozo; Hattori, Satoko; Miyakawa, Tsuyoshi; Tanaka, Kohichi; Huda, Fathul; Hirai, Hirokazu; Hashimoto, Kouichi; Watanabe, Masahiko; Sakimura, Kenji; Kinoshita, Makoto

    2015-01-01

    The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4−/− mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1–5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance. PMID:26657011

  9. Cdc42 explores the cell periphery for mate selection in fission yeast.

    PubMed

    Bendezú, Felipe O; Martin, Sophie G

    2013-01-01

    How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification. PMID:23200991

  10. A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance.

    PubMed

    Ageta-Ishihara, Natsumi; Yamazaki, Maya; Konno, Kohtarou; Nakayama, Hisako; Abe, Manabu; Hashimoto, Kenji; Nishioka, Tomoki; Kaibuchi, Kozo; Hattori, Satoko; Miyakawa, Tsuyoshi; Tanaka, Kohichi; Huda, Fathul; Hirai, Hirokazu; Hashimoto, Kouichi; Watanabe, Masahiko; Sakimura, Kenji; Kinoshita, Makoto

    2015-01-01

    The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance. PMID:26657011

  11. Cdc42 and formin activity control non-muscle myosin dynamics during Drosophila heart morphogenesis

    PubMed Central

    Vogler, Georg; Liu, Jiandong; Iafe, Timothy W.; Migh, Ede; Mihály, József

    2014-01-01

    During heart formation, a network of transcription factors and signaling pathways guide cardiac cell fate and differentiation, but the genetic mechanisms orchestrating heart assembly and lumen formation remain unclear. Here, we show that the small GTPase Cdc42 is essential for Drosophila melanogaster heart morphogenesis and lumen formation. Cdc42 genetically interacts with the cardiogenic transcription factor tinman; with dDAAM which belongs to the family of actin organizing formins; and with zipper, which encodes nonmuscle myosin II. Zipper is required for heart lumen formation, and its spatiotemporal activity at the prospective luminal surface is controlled by Cdc42. Heart-specific expression of activated Cdc42, or the regulatory formins dDAAM and Diaphanous caused mislocalization of Zipper and induced ectopic heart lumina, as characterized by luminal markers such as the extracellular matrix protein Slit. Placement of Slit at the lumen surface depends on Cdc42 and formin function. Thus, Cdc42 and formins play pivotal roles in heart lumen formation through the spatiotemporal regulation of the actomyosin network. PMID:25267295

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

    PubMed

    Virag, Aleksandra; Lee, Maurice P; Si, Haoyu; Harris, Steven D

    2007-12-01

    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. PMID:18005099

  13. RhoA activation promotes transendothelial migration of monocytes via ROCK.

    PubMed

    Honing, Henk; van den Berg, Timo K; van der Pol, Susanne M A; Dijkstra, Christine D; van der Kammen, Rob A; Collard, John G; de Vries, Helga E

    2004-03-01

    Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells. PMID:14634067

  14. CLN3 deficient cells display defects in the ARF1-Cdc42 pathway and actin-dependent events.

    PubMed

    Schultz, Mark L; Tecedor, Luis; Stein, Colleen S; Stamnes, Mark A; Davidson, Beverly L

    2014-01-01

    Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development. PMID:24792215

  15. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases.

    PubMed

    Jou, T S; Schneeberger, E E; Nelson, W J

    1998-07-13

    Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane. PMID:9660866

  16. Investigation of the Interaction between Cdc42 and Its Effector TOCA1: HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES.

    PubMed

    Watson, Joanna R; Fox, Helen M; Nietlispach, Daniel; Gallop, Jennifer L; Owen, Darerca; Mott, Helen R

    2016-06-24

    Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. PMID:27129201

  17. Regulation of cerebral cortex development by Rho GTPases: insights from in vivo studies

    PubMed Central

    Azzarelli, Roberta; Kerloch, Thomas; Pacary, Emilie

    2015-01-01

    The cerebral cortex is the site of higher human cognitive and motor functions. Histologically, it is organized into six horizontal layers, each containing unique populations of molecularly and functionally distinct excitatory projection neurons and inhibitory interneurons. The stereotyped cellular distribution of cortical neurons is crucial for the formation of functional neural circuits and it is predominantly established during embryonic development. Cortical neuron development is a multiphasic process characterized by sequential steps of neural progenitor proliferation, cell cycle exit, neuroblast migration and neuronal differentiation. This series of events requires an extensive and dynamic remodeling of the cell cytoskeleton at each step of the process. As major regulators of the cytoskeleton, the family of small Rho GTPases has been shown to play essential functions in cerebral cortex development. Here we review in vivo findings that support the contribution of Rho GTPases to cortical projection neuron development and we address their involvement in the etiology of cerebral cortex malformations. PMID:25610373

  18. Role of Rho small GTPases in meniscus cells.

    PubMed

    Kanazawa, Tomoko; Furumatsu, Takayuki; Matsumoto-Ogawa, Emi; Maehara, Ami; Ozaki, Toshifumi

    2014-11-01

    We previously reported that mechanical stretch regulates Sry-type HMG box (SOX) 9-dependent α1(II) collagen (COL2A1) expression in inner meniscus cells. This study examined the role of the small Rho guanosine 5' triphosphatase Rac1 and Rho-associated kinase (ROCK) in the regulation of stretch-induced SOX9 gene expression in cultured human inner meniscus cells. COL2A1 and SOX9 gene expression was assessed by real-time PCR after application of uni-axial cyclic tensile strain (CTS) in the presence or absence of ROCK and Rac1 inhibitors. The subcellular localization of SOX9 and the Rac1 effector cyclic AMP response element-binding protein (CREB), the phosphorylation state of SOX9, Rac1 activation, and the binding of CREB to the SOX9 promoter were assessed. CTS increased the expression of COL2A1 and SOX9, which was suppressed by inhibition of Rac1. ROCK inhibition enhanced COL2A1 and SOX9 gene expression in the absence of CTS. CTS stimulated the nuclear translocation and phosphorylation of SOX9, and increased Rac1 activation. CTS also increased the binding of CREB to the SOX9 promoter. The results suggest that mechanical stretch-dependent upregulation of SOX9 by CREB in inner meniscus cells depends on the antagonistic activities of ROCK and Rac1. PMID:25130858

  19. RhoGTPases--NODes for effector-triggered immunity in animals.

    PubMed

    Stuart, Lynda M; Boyer, Laurent

    2013-08-01

    A recent study published in Nature by Keestra and colleagues addresses how the immune system detects the pathogenic potential of microbes and provides evidence that one strategy involves NOD1, which monitors the activation state of the RhoGTPases that are targeted by virulence effectors produced by pathogenic microbes. Interestingly, their findings reveal striking similarities with previous observations made in flies and plants, establishing the evolutionary conservation of this detection system in the innate immune arsenal in many taxa. PMID:23689278

  20. Platelet Rho GTPases–a focus on novel players, roles and relationships

    PubMed Central

    Goggs, Robert; Williams, Christopher M.; Mellor, Harry; Poole, Alastair W.

    2015-01-01

    Rho GTPases are critical for platelet function. Although the roles of RhoA, Rac and Cdc42 are characterized, platelets express other Rho GTPases, whose activities are less well understood. This review summarizes our understanding of the roles of platelet Rho GTPases and focuses particularly on the functions of Rif and RhoG. In human platelets, Rif interacts with cytoskeleton regulators including formins mDia1 and mDia3, whereas RhoG binds SNARE-complex proteins and cytoskeletal regulators ELMO and DOCK1. Knockout mouse studies suggest that Rif plays no critical functions in platelets, likely due to functional overlap with other Rho GTPases. In contrast, RhoG is essential for normal granule secretion downstream of the collagen receptor GPVI. The central defect in RhoG−/− platelets is reduced dense granule secretion, which impedes integrin activation and aggregation and limits platelet recruitment to growing thrombi under shear, translating into reduced thrombus formation in vivo. Potential avenues for future work on Rho GTPases in platelets are also highlighted, including identification of the key regulator for platelet filopodia formation and investigation of the role of the many Rho GTPase regulators in platelet function in both health and disease. PMID:25748676

  1. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

    PubMed Central

    Leberer, E; Wu, C; Leeuw, T; Fourest-Lieuvin, A; Segall, J E; Thomas, D Y

    1997-01-01

    Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins. PMID:9009270

  2. A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation.

    PubMed

    Aubert, Daniel F; Xu, Hao; Yang, Jieling; Shi, Xuyan; Gao, Wenqing; Li, Lin; Bisaro, Fabiana; Chen, She; Valvano, Miguel A; Shao, Feng

    2016-05-11

    Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome. PMID:27133449

  3. Small Rho GTPases and Cholesterol Biosynthetic Pathway Intermediates in African Swine Fever Virus Infection

    PubMed Central

    Quetglas, Jose I.; Hernáez, Bruno; Galindo, Inmaculada; Muñoz-Moreno, Raquel; Cuesta-Geijo, Miguel A.

    2012-01-01

    The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection. PMID:22114329

  4. MicroRNA-132 Interact with p250GAP/Cdc42 Pathway in the Hippocampal Neuronal Culture Model of Acquired Epilepsy and Associated with Epileptogenesis Process.

    PubMed

    Yuan, Jinxian; Huang, Hao; Zhou, Xin; Liu, Xi; Ou, Shu; Xu, Tao; Li, Ruohan; Ma, Limin; Chen, Yangmei

    2016-01-01

    Increasing evidence suggests that epilepsy is the result of synaptic reorganization and pathological excitatory loop formation in the central nervous system; however, the mechanisms that regulate this process are not well understood. We proposed that microRNA-132 (miR-132) and p250GAP might play important roles in this process by activating the downstream Rho GTPase family. We tested this hypothesis using a magnesium-free medium-induced epileptic model of cultured hippocampal neurons. We investigated whether miR-132 regulates GTPase activity through p250GAP and found that Cdc42 was significantly activated in our experimental model. Silencing miR-132 inhibited the electrical excitability level of cultured epileptic neurons, whereas silencing p250GAP had an opposite effect. In addition, we verified the effect of miR-132 in vivo and found that silencing miR-132 inhibited the aberrant formation of dendritic spines and chronic spontaneous seizure in a lithium-pilocarpine-induced epileptic mouse model. Finally, we confirmed that silencing miR-132 has a neuroprotective effect on cultured epileptic neurons; however, this effect did not occur through the p250GAP pathway. Generally, silencing miR-132 may suppress spontaneous seizure activity through the miR-132/p250GAP/Cdc42 pathway by regulating the morphology and electrophysiology of dendritic spines; therefore, miR-132 may serve as a potential target for the development of antiepileptic drugs. PMID:27579184

  5. MicroRNA-132 Interact with p250GAP/Cdc42 Pathway in the Hippocampal Neuronal Culture Model of Acquired Epilepsy and Associated with Epileptogenesis Process

    PubMed Central

    Huang, Hao; Zhou, Xin; Liu, Xi; Xu, Tao; Ma, Limin

    2016-01-01

    Increasing evidence suggests that epilepsy is the result of synaptic reorganization and pathological excitatory loop formation in the central nervous system; however, the mechanisms that regulate this process are not well understood. We proposed that microRNA-132 (miR-132) and p250GAP might play important roles in this process by activating the downstream Rho GTPase family. We tested this hypothesis using a magnesium-free medium-induced epileptic model of cultured hippocampal neurons. We investigated whether miR-132 regulates GTPase activity through p250GAP and found that Cdc42 was significantly activated in our experimental model. Silencing miR-132 inhibited the electrical excitability level of cultured epileptic neurons, whereas silencing p250GAP had an opposite effect. In addition, we verified the effect of miR-132 in vivo and found that silencing miR-132 inhibited the aberrant formation of dendritic spines and chronic spontaneous seizure in a lithium-pilocarpine-induced epileptic mouse model. Finally, we confirmed that silencing miR-132 has a neuroprotective effect on cultured epileptic neurons; however, this effect did not occur through the p250GAP pathway. Generally, silencing miR-132 may suppress spontaneous seizure activity through the miR-132/p250GAP/Cdc42 pathway by regulating the morphology and electrophysiology of dendritic spines; therefore, miR-132 may serve as a potential target for the development of antiepileptic drugs. PMID:27579184

  6. Sialylation and glycosylation modulate cell adhesion and invasion to extracellular matrix in human malignant lymphoma: Dependency on integrin and the Rho GTPase family

    PubMed Central

    SUZUKI, OSAMU; ABE, MASAFUMI; HASHIMOTO, YUKO

    2015-01-01

    To determine the biological roles of cell surface glycosylation, we modified the surface glycosylation of human malignant lymphoma cell lines using glycosylation inhibitors. The O-glycosylation inhibitor, benzyl-α-GalNAc (BZ) enhanced the fibronectin adhesion of HBL-8 cells, a human Burkitt's lymphoma cell line, and of H-ALCL cells, a human anaplastic large cell lymphoma cell line, both of which were established in our laboratory. The N-glycosylation inhibitor, tunicamycin (TM) inhibited the surface expression of Phaseolus vulgaris leukoagglutinating (L-PHA) lectin- and Canavalia ensiformis (ConA) lectin-reactive oligosaccharides in the HBL-8 cell line. Assay of the adhesion of HBL-8 cells to fibronectin showed that fibronectin adhesion is mediated by the integrin very late antigen (VLA)-4 and that not only BZ but also TM treatment enhanced HBL-8 cell adhesion to fibronectin. Furthermore, although BZ treatment also enhanced H-ALCL cell adhesion to fibronectin, this effect was not mediated by VLA-5 or the RGD sequence of fibronectin. We also showed that H-ALCL cell adhesion to galectin-3 was enhanced by pre-treatment with neuraminidase, which cleaves cell surface sialic acid. Additionally, H-ALCL cell adhesion to galectin-3 was inhibited by pre-treatment with the RGD peptide suggesting that cell adhesion to galectin-3 is mediated by integrin (VLA-5). Furthermore, H-ALCL cell invasion of galectin-1 and galectin-3 was inhibited by pre-treatment with the RGD peptide. Therefore, cell adhesion to and invasion of galectin-1 and galectin-3 are integrin-dependent. In addition to these findings, cell adhesion to galectin-3 was markedly inhibited by treatment with β-lactose compared to treatment with sucrose. Therefore, interactions between integrins and galectin-3 may be mediated through β-galactose that is linked to glycans of integrins. AZA1, an inhibitor of Ras homolog oncoprotein (Rho) GTPase family proteins, RAS-related C3 botulinus toxin substrate 1 (Rac 1) and Cell

  7. Sialylation and glycosylation modulate cell adhesion and invasion to extracellular matrix in human malignant lymphoma: Dependency on integrin and the Rho GTPase family.

    PubMed

    Suzuki, Osamu; Abe, Masafumi; Hashimoto, Yuko

    2015-12-01

    To determine the biological roles of cell surface glycosylation, we modified the surface glycosylation of human malignant lymphoma cell lines using glycosylation inhibitors. The O-glycosylation inhibitor, benzyl-α-GalNAc (BZ) enhanced the fibronectin adhesion of HBL-8 cells, a human Burkitt's lymphoma cell line, and of H-ALCL cells, a human anaplastic large cell lymphoma cell line, both of which were established in our laboratory. The N-glycosylation inhibitor, tunicamycin (TM) inhibited the surface expression of Phaseolus vulgaris leukoagglutinating (L-PHA) lectin- and Canavalia ensiformis (ConA) lectin-reactive oligosaccharides in the HBL-8 cell line. Assay of the adhesion of HBL-8 cells to fibronectin showed that fibronectin adhesion is mediated by the integrin very late antigen (VLA)-4 and that not only BZ but also TM treatment enhanced HBL-8 cell adhesion to fibronectin. Furthermore, although BZ treatment also enhanced H-ALCL cell adhesion to fibronectin, this effect was not mediated by VLA-5 or the RGD sequence of fibronectin. We also showed that H-ALCL cell adhesion to galectin-3 was enhanced by pre-treatment with neuraminidase, which cleaves cell surface sialic acid. Additionally, H-ALCL cell adhesion to galectin-3 was inhibited by pre‑treatment with the RGD peptide suggesting that cell adhesion to galectin-3 is mediated by integrin (VLA-5). Furthermore, H-ALCL cell invasion of galectin-1 and galectin-3 was inhibited by pre-treatment with the RGD peptide. Therefore, cell adhesion to and invasion of galectin-1 and galectin-3 are integrin-dependent. In addition to these findings, cell adhesion to galectin-3 was markedly inhibited by treatment with β-lactose compared to treatment with sucrose. Therefore, interactions between integrins and galectin-3 may be mediated through β-galactose that is linked to glycans of integrins. AZA1, an inhibitor of Ras homolog oncoprotein (Rho) GTPase family proteins, RAS-related C3 botulinus toxin substrate 1 (Rac 1) and

  8. Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

    PubMed Central

    Narayan, Monisha; Chou, Ching-Shan; Park, Hay-Oak

    2013-01-01

    Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model. PMID:23437206

  9. The Yersinia pseudotuberculosis cytotoxic necrotizing factor (CNFY) selectively activates RhoA.

    PubMed

    Hoffmann, Claudia; Pop, Marius; Leemhuis, Jost; Schirmer, Jörg; Aktories, Klaus; Schmidt, Gudula

    2004-04-16

    The cytotoxic necrotizing factors (CNF)1 and CNF2 from pathogenic Escherichia coli strains activate RhoA, Rac1, and Cdc42 by deamidation of Gln63 (RhoA) or Gln61 (Rac and Cdc42). Recently, a novel cytotoxic necrotizing factor termed CNFY was identified in Yersinia pseudotuberculosis strains (Lockman, H. A., Gillespie, R. A., Baker, B. D., and Shakhnovich, E. (2002) Infect. Immun. 70, 2708-2714). We amplified the cnfy gene from genomic DNA of Y. pseudotuberculosis, cloned and expressed the recombinant protein, and studied its activity. Recombinant GST-CNFY induced morphological changes in HeLa cells and caused an upward shift of RhoA in SDS-PAGE, as is known for GST-CNF1 and GST-CNF2. Mass spectrometric analysis of GST-CNFY-treated RhoA confirmed deamidation at Glu63. Treatment of RhoA, Rac1, and Cdc42 with GST-CNFY decreased their GTPase activities, indicating that all of these Rho proteins could serve as substrates for GST-CNFY in vitro. In contrast, RhoA, but not Rac or Cdc42, was the substrate of GST-CNFY in culture cells. GST-CNFY caused marked stress fiber formation in HeLa cells after 2 h. In contrast to GST-CNF1, formation of filopodia or lamellipodia was not induced with GST-CNFY. Accordingly, effector pull-down experiments with lysates of toxin-treated cells revealed strong activation of RhoA but no activation of Rac1 or Cdc42 after 6 h of GST-CNFY-treatment. Moreover, in rat hippocampal neurons, GST-CNFY results in the retraction of neurites, indicating RhoA activation. In contrast, no activation of Rac or Cdc42 was found. Altogether, our data suggest that CNFY from Y. pseudotuberculosis is a strong, selective activator of RhoA, which can be used as a powerful tool for constitutive RhoA activation without concomitant activation of Rac1 or Cdc42. PMID:14761941

  10. Dynamics of Cdc42 network embodies a Turing-type mechanism of yeast cell polarity.

    PubMed

    Goryachev, Andrew B; Pokhilko, Alexandra V

    2008-04-30

    Complex biochemical networks can be understood by identifying their principal regulatory motifs and mode of action. We model the early phase of budding yeast cellular polarization and show that the biochemical processes in the presumptive bud site comprise a Turing-type mechanism. The roles of the prototypical activator and substrate are played by GTPase Cdc42 in its active and inactive states, respectively. We demonstrate that the nucleotide cycling of Cdc42 converts cellular energy into a stable cluster of activated Cdc42. This energy drives a continuous membrane-cytoplasmic exchange of the cluster components to counteract diffusive spread of the cluster. This exchange explains why only one bud forms per cell cycle, because the winner-takes-all competition of candidate sites inevitably selects a single site. PMID:18381072

  11. RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

    PubMed Central

    Hertens, Fréderic; Parys, Jan B.; Leybaert, Luc; Vereecke, Johan; Himpens, Bernard; Bultynck, Geert

    2012-01-01

    ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress. PMID:22860057

  12. RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking.

    PubMed

    Mukherjee, Debarati; Sen, Arpita; Aguilar, R Claudio

    2014-01-01

    Cell polarity, the asymmetric distribution of proteins and lipids, is essential for a variety of cellular functions. One mechanism orchestrating cell polarity is polarized vesicle trafficking; whereby cargo loaded secretory vesicles are specifically transported to predetermined areas of the cell. The evolutionarily conserved exocyst complex and its small GTPase regulators play crucial roles in spatiotemporal control of polarized vesicle trafficking. In studies on neuronal membrane remodeling and synaptic plasticity, conserved mechanisms of exocyst regulation and cargo recycling during polarized vesicle trafficking are beginning to emerge as well. Recently, our lab demonstrated that RhoGTPase-binding proteins in both yeast (Bem3) and mammals (Ocrl1) are also required for the efficient traffic of secretory vesicles to sites of polarized growth and signaling. Together with our studies, we highlight the evolutionary conservation of the basic elements essential for polarized vesicle traffic across different cellular functions and model systems. In conclusion, we emphasize that studies on RhoGTPase-binding proteins in these processes should be included in the next level of investigation, for a more complete understanding of their hitherto unknown roles in polarized membrane traffic and exocyst regulation. PMID:24691289

  13. Control of Homeostasis and Dendritic Cell Survival by the GTPase RhoA.

    PubMed

    Li, Shuai; Dislich, Bastian; Brakebusch, Cord H; Lichtenthaler, Stefan F; Brocker, Thomas

    2015-11-01

    Tissues accommodate defined numbers of dendritic cells (DCs) in highly specific niches where different intrinsic and environmental stimuli control DC life span and numbers. DC homeostasis in tissues is important, because experimental changes in DC numbers influence immunity and tolerance toward various immune catastrophes and inflammation. However, the precise molecular mechanisms regulating DC life span and homeostasis are unclear. We report that the GTPase RhoA controls homeostatic proliferation, cytokinesis, survival, and turnover of cDCs. Deletion of RhoA strongly decreased the numbers of CD11b(-)CD8(+) and CD11b(+)Esam(hi) DC subsets, whereas CD11b(+)Esam(lo) DCs were not affected in conditional RhoA-deficient mice. Proteome analyses revealed a defective prosurvival pathway via PI3K/protein kinase B (Akt1)/Bcl-2-associated death promoter in the absence of RhoA. Taken together, our findings identify RhoA as a central regulator of DC homeostasis, and its deletion decreases DC numbers below critical thresholds for immune protection and homeostasis, causing aberrant compensatory DC proliferation. PMID:26408665

  14. Scambio, a novel guanine nucleotide exchange factor for Rho

    PubMed Central

    Curtis, Christina; Hemmeryckx, Bianca; Haataja, Leena; Senadheera, Dinithi; Groffen, John; Heisterkamp, Nora

    2004-01-01

    Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho. PMID:15107133

  15. Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

    PubMed Central

    Wheeler, David S.; Underhill, Suzanne M.; Stolz, Donna B.; Murdoch, Geoffrey H.; Thiels, Edda; Romero, Guillermo; Amara, Susan G.

    2015-01-01

    Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action. PMID:26553986

  16. Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine.

    PubMed

    Wheeler, David S; Underhill, Suzanne M; Stolz, Donna B; Murdoch, Geoffrey H; Thiels, Edda; Romero, Guillermo; Amara, Susan G

    2015-12-22

    Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH's effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action. PMID:26553986

  17. P-cadherin-mediated Rho GTPase regulation during collective cell migration

    PubMed Central

    Plutoni, Cédric; Bazellières, Elsa; Gauthier-Rouvière, Cécile

    2016-01-01

    ABSTRACT This commentary addresses the role of P-cadherin in collective cell migration (CCM), a cooperative and coordinated migration mode, used by cells during normal and pathological migration processes. We discuss how cadherin-mediated cell-cell junctions (CCJs) play a critical role in CCM through their ability to regulate Rho GTPase-dependent pathways and how this leads to the generation and orientation of mechanical forces. We will also highlight the key function of P-cadherin (a poor prognostic marker in several tumors) in promoting collective cell movement in epithelial and mesenchymal cells. PMID:27152729

  18. Suppression of Chemotaxis by SSeCKS via Scaffolding of Phosphoinositol Phosphates and the Recruitment of the Cdc42 GEF, Frabin, to the Leading Edge

    PubMed Central

    Ko, Hyun-Kyung; Guo, Li-wu; Su, Bing; Gao, Lingqiu; Gelman, Irwin H.

    2014-01-01

    Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the

  19. Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)

    SciTech Connect

    Wang, Hui; Hota, Prasanta K.; Tong, Yufeng; Li, Buren; Shen, Limin; Nedyalkova, Lyudmila; Borthakur, Susmita; Kim, SoonJeung; Tempel, Wolfram; Buck, Matthias; Park, Hee-Won

    2011-09-20

    Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD {center_dot} Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD {beta}3-{beta}4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

  20. Regulation of plasticity and fibrogenic activity of trabecular meshwork cells by Rho GTPase signaling.

    PubMed

    Pattabiraman, Padmanabhan P; Maddala, Rupalatha; Rao, Ponugoti Vasantha

    2014-07-01

    Glaucoma, a prevalent blinding disease is commonly associated with increased intraocular pressure due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although increased TM tissue contraction and stiffness in association with accumulation of extracellular matrix (ECM) are believed to be partly responsible for increased resistance to AH outflow, the extracellular cues and intracellular mechanisms regulating TM cell contraction and ECM production are not well defined. This study tested the hypothesis that sustained activation of Rho GTPase signaling induced by lysophosphatidic acid (LPA), TGF-β, and connective tissue growth factor (CTGF) influences TM cell plasticity and fibrogenic activity which may eventually impact resistance to AH outflow. Various experiments performed using human TM cells revealed that constitutively active RhoA (RhoAV14), TGF-β2, LPA, and CTGF significantly increase the levels and expression of Fibroblast Specific Protein-1 (FSP-1), α-smooth muscle actin (αSMA), collagen-1A1 and secretory total collagen, as determined by q-RT-PCR, immunofluorescence, immunoblot, flow cytometry and the Sircol assay. Significantly, these changes appear to be mediated by Serum Response Factor (SRF), myocardin-related transcription factor (MRTF-A), Slug, and Twist-1, which are transcriptional regulators known to control cell plasticity, myofibroblast generation/activation and fibrogenic activity. Additionally, the Rho kinase inhibitor-Y27632 and anti-fibrotic agent-pirfenidone were both found to suppress the TGF-β2-induced expression of αSMA, FSP-1, and collagen-1A1. Taken together, these observations demonstrate the significance of RhoA/Rho kinase signaling in regulation of TM cell plasticity, fibrogenic activity, and myofibroblast activation, events with potential implications for the pathobiology of elevated intraocular pressure in glaucoma patients. PMID:24318513

  1. The Rho-GTPase binding protein IQGAP2 is required for the glomerular filtration barrier.

    PubMed

    Sugano, Yuya; Lindenmeyer, Maja T; Auberger, Ines; Ziegler, Urs; Segerer, Stephan; Cohen, Clemens D; Neuhauss, Stephan C F; Loffing, Johannes

    2015-11-01

    Podocyte dysfunction impairs the size selectivity of the glomerular filter, leading to proteinuria, hypoalbuminuria, and edema, clinically defined as nephrotic syndrome. Hereditary forms of nephrotic syndrome are linked to mutations in podocyte-specific genes. To identify genes contributing to podocyte dysfunction in acquired nephrotic syndrome, we studied human glomerular gene expression data sets for glomerular-enriched gene transcripts differentially regulated between pretransplant biopsy samples and biopsies from patients with nephrotic syndrome. Candidate genes were screened by in situ hybridization for expression in the zebrafish pronephros, an easy-to-use in vivo assay system to assess podocyte function. One glomerulus-enriched product was the Rho-GTPase binding protein, IQGAP2. Immunohistochemistry found a strong presence of IQGAP2 in normal human and zebrafish podocytes. In zebrafish larvae, morpholino-based knockdown of iqgap2 caused a mild foot process effacement of zebrafish podocytes and a cystic dilation of the urinary space of Bowman's capsule upon onset of urinary filtration. Moreover, the glomerulus of zebrafish morphants showed a glomerular permeability for injected high-molecular-weight dextrans, indicating an impaired size selectivity of the glomerular filter. Thus, IQGAP2 is a Rho-GTPase binding protein, highly abundant in human and zebrafish podocytes, which controls normal podocyte structure and function as evidenced in the zebrafish pronephros. PMID:26154927

  2. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis

    PubMed Central

    Xu, Tongda; Wen, Mingzhang; Nagawa, Shingo; Fu, Ying; Chen, Jin-Gui; Wu, Ming-Jing; Perrot-Rechenmann, Catherine; Friml, Jiří; Jones, Alan M.; Yang, Zhenbiao

    2010-01-01

    Summary Auxin is a multi-functional hormone essential for plant development and pattern formation. A nuclear auxin signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling as in its coordination of cell polarization is unexplored. We found a cytoplasmic auxin signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 seconds and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based novel auxin signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells. PMID:20887895

  3. Involvement of geranylgeranylation of Rho and Rac GTPases in adipogenic and RANKL expression, which was inhibited by simvastatin.

    PubMed

    Baba, T T; Ohara-Nemoto, Y; Miyazaki, T; Nemoto, T K

    2013-12-01

    Simvastatin suppresses myoblast differentiation via inhibition of Rac GTPase, which is involved in the mevalonic acid pathway that produces cholesterol. Statins also inhibit adipogenic differentiation and receptor activator of NFκB ligand (RANKL) expression, possibly through the mevalonic acid pathway, although the involvement of that pathway and effector proteins in these cellular events has not been fully clarified. In the present study, we aimed to elucidate the mechanism of the effects of simvastatin on adipogenic differentiation and calcitriol-induced RANKL expression in bone marrow stromal ST2 cells. Adipogenesis and mRNA up-regulation of peroxisome proliferator-activated receptor γ and adipocyte fatty acid-binding protein were induced by troglitazone, and those events were efficiently inhibited by simvastatin. In addition, RANKL expression induced by calcitriol was abrogated by simvastatin in ST2 cells. The inhibitory effects of simvastatin were adequately compensated by the addition of either mevalonic acid or an intermediate of the mevalonic acid pathway, geranylgeranyl pyrophosphate, but not by another intermediate, farnesyl pyrophosphate. These findings suggest that protein geranylgeranylation is related to cellular differentiation in those two directions. Furthermore, inhibitor analysis demonstrated that Rac GTPase is involved in adipogenic differentiation, whereas Rho GTPase was found to be involved in RANKL expression. Taken together, the present findings suggest that geranylgeranylation of Rho family GTPase is involved in both adipogenesis and RANKL expression of stromal cells, while Rac GTPase is involved in adipogenesis and Rho GTPase in RANKL expression. PMID:23339033

  4. Dynamic and thermodynamic response of the Ras protein Cdc42Hs upon association with the effector domain of PAK3.

    PubMed

    Moorman, Veronica R; Valentine, Kathleen G; Bédard, Sabrina; Kasinath, Vignesh; Dogan, Jakob; Love, Fiona M; Wand, A Joshua

    2014-10-23

    Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs-PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately -10kcalmol(-1) at 298K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding. PMID:25109462

  5. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

    PubMed

    Guignandon, Alain; Faure, Céline; Neutelings, Thibaut; Rattner, Aline; Mineur, Pierre; Linossier, Marie-Thérèse; Laroche, Norbert; Lambert, Charles; Deroanne, Christophe; Nusgens, Betty; Demets, René; Colige, Alain; Vico, Laurence

    2014-09-01

    Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects. PMID:24903274

  6. Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination

    PubMed Central

    Chauhan, Bharesh K.; Disanza, Andrea; Choi, Sue-Yeon; Faber, Sonya C.; Lou, Ming; Beggs, Hilary E.; Scita, Giorgio; Zheng, Yi; Lang, Richard A.

    2009-01-01

    Summary The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. Here, we show that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5 - the lens pit stage - there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates. PMID:19820184

  7. The Fer tyrosine kinase regulates interactions of Rho GDP-Dissociation Inhibitor α with the small GTPase Rac

    PubMed Central

    2010-01-01

    Background RhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood. Results We report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes. Conclusions These results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac. PMID:21122136

  8. Inhibition of the RhoA GTPase Activity Increases Sensitivity of Melanoma Cells to UV Radiation Effects

    PubMed Central

    Espinha, Gisele; Osaki, Juliana Harumi; Costa, Erico Tosoni; Forti, Fabio Luis

    2016-01-01

    Ultraviolet radiation is the main cause of DNA damage to melanocytes and development of melanoma, one of the most lethal human cancers, which leads to metastasis due to uncontrolled cell proliferation and migration. These phenotypes are mediated by RhoA, a GTPase overexpressed or overactivated in highly aggressive metastatic tumors that plays regulatory roles in cell cycle progression and cytoskeleton remodeling. This work explores whether the effects of UV on DNA damage, motility, proliferation, and survival of human metastatic melanoma cells are mediated by the RhoA pathway. Mutant cells expressing dominant-negative (MeWo-RhoA-N19) or constitutively active RhoA (MeWo-RhoA-V14) were generated and subjected to UV radiation. A slight reduction in migration and invasion was observed in MeWo and MeWo-RhoA-V14 cells but not in MeWo-RhoA-N19 cells, which presented inefficient motility and invasiveness associated with stress fibers fragmentation. Proliferation and survival of RhoA-deficient cells were drastically reduced by UV compared to cells displaying normal or high RhoA activity, suggesting increased sensitivity to UV. Loss of RhoA activity also caused less efficient DNA repair, with elevated levels of DNA lesions such as strand breaks and cyclobutane pyrimidine dimers (CPDs). Thus, RhoA mediates genomic stability and represents a potential target for sensitizing metastatic tumors to genotoxic agents. PMID:26823948

  9. Regulation of the endothelial barrier function: a filum granum of cellular forces, Rho-GTPase signaling and microenvironment.

    PubMed

    Amado-Azevedo, Joana; Valent, Erik T; Van Nieuw Amerongen, Geerten P

    2014-03-01

    Although the endothelium is an extremely thin single-cell layer, it performs exceedingly well in preventing blood fluids from leaking into the surrounding tissues. However, specific pathological conditions can affect this cell layer, compromising the integrity of the barrier. Vascular leakage is a hallmark of many cardiovascular diseases and despite its medical importance, no specialized therapies are available to prevent it or reduce it. Small guanosine triphosphatases (GTPases) of the Rho family are known to be key regulators of various aspects of cell behavior and studies have shown that they can exert both positive and negative effects on endothelial barrier integrity. Moreover, extracellular matrix stiffness has now been implicated in the regulation of Rho-GTPase signaling, which has a direct impact on the integrity of endothelial junctions. However, knowledge about both the precise mechanism of this regulation and the individual contribution of the specific regulatory proteins remains fragmentary. In this review, we discuss recent findings concerning the balanced activities of Rho-GTPases and, in particular, aspects of the regulation of the endothelial barrier. We highlight the role of Rho-GTPases in the intimate relationships between biomechanical forces, microenvironmental influences and endothelial intercellular junctions, which are all interwoven in a beautiful filigree-like fashion. PMID:24633925

  10. Optimization and stabilization of Rho small GTPase proteins for solution NMR studies: The case of Rnd1.

    PubMed

    Cao, Shufen; Buck, Matthias

    2011-11-01

    Rho GTPases of the Ras superfamily have important roles in regulating the organization of the actin filament system, morphogenesis and migration of cells. Structural details for these proteins are still emerging, and information on their dynamics in solution is much needed to understand the mechanisms underlying their signaling functions. This report reviews conditions for solution NMR studies of Rho GTPases and describes our optimization and stabilization of Rnd1 for such experiments. Rnd1 belongs to the Rnd protein subfamily branch of Rho small GTPases and functions in neurite outgrowth, dendrite development and in axon guidance. However, as we report here, solution NMR studies of this protein are challenging. Multiple methods have been employed to enhance the stability of Rnd1, including by cleavage of an N-terminal His expression tag and by addition of non-hydrolysable GMPPNP (β: γ-imidoguanosine 5'-triphosphate) nucleotide. Further stabilization of Rnd1 against aggregation was achieved through a structure informed point mutation while maintaining its conformation and binding affinity for a partner protein. The NMR spectrum of the optimized protein reveals significant improvement in NMR signal dispersion and intensity. This work paves the way for structural and protein-protein/protein-ligand interaction studies of Rnd1 by solution NMR and also provides a guide for optimization and stabilization of other Rho GTPases. PMID:22545226