Cholesterol oxides mediated changes in cytoskeletal organisation involves Rho GTPases small star, filled

The small GTPases Rho, Rac, and Cdc42 regulate the actin cytoskeleton in all eukaryotic cells. In this study we have evaluated the effect of cholesterol oxides (7-ketocholesterol and 25-hydroxycholesterol) on cell migration, cell adhesion, and cytoskeletal organisation of lens epithelial cells (LEC). Effects of cholesterol oxides on cytoskeleton were evaluated by immunofluorescence confocal microscopy. The 7-ketocholesterol induced cell arborisation, with bundling of vimentin and tubulin in the cell processes and formation of filopodia and stress fibres. Cells treated with 25-hydroxycholesterol showed a collapse of vimentin filaments towards the nucleus and formation of lamellipodia. In addition, cells treated with 7-ketocholesterol or 25-hydroxycholesterol showed decreased migration. The effects of cholesterol oxides on cytoskeletal proteins involve the activation of the small GTPases Rho, Rac, and Cdc42. Indeed, formation of both filopodia and stress fibres induced by 7-ketocholesterol is inhibited by overexpressing dominant negatives forms of Cdc42 and RhoA, respectively. Similarly, the collapse of vimentin intermediate filament network and the formation of lamellipodia, induced by 25-hydroxycholesterol, is inhibited by overexpressing dominant negatives forms of Rac1. The effects of cholesterol oxides described in this study for LEC are also observed for at least two other cell lines (H36CE and U373), suggesting that this may represent a general mechanism whereby cholesterol oxides induces cytoskeletal disorganisation.     

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

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.     

Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling

The Rho family of small GTPases (RhoA, Rac1 and Cdc42) controls signal-transduction pathways that influence many aspects of cell behaviour, including cytoskeletal dynamics. At the leading edge, Rac1 and Cdc42 promote cell motility through the formation of lamellipodia and filopodia, respectively. On the contrary, RhoA promotes the formation of contractile actin-myosin-containing stress fibres in the cell body and at the rear. Here, we identify synaptopodin, an actin-associated protein, as a novel regulator of RhoA signalling and cell migration in kidney podocytes. We show that synaptopodin induces stress fibres by competitive blocking of Smurf1-mediated ubiquitination of RhoA, thereby preventing the targeting of RhoA for proteasomal degradation. Gene silencing of synaptopodin in kidney podocytes causes the loss of stress fibres and the formation of aberrant non-polarized filopodia and impairment of cell migration. Together, these data show that synaptopodin is essential for the integrity of the podocyte actin cytoskeleton and for the regulation of podocyte cell migration.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements

Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.     

Rho GTPases与肿瘤

Rho GTPases参与调控细胞的多种关键生物学行为,特别是细胞的生长、细胞骨架的形成、转录调节等生物学过程. 在肿瘤的发生发展中Rho GTPases也扮演了重要的角色．本文将回顾Rho GTPases的调控（包括经典及非经典调控方式）及其关键成员（RhoA、Cdc42及Rac1）与临床肿瘤的研究进展,特别是它们参与调控肿瘤的增殖、迁移、侵袭、凋亡等恶性生物学行为,从而为研发靶向Rho GTPases的小分子/基因药物了奠定基础．     

Transforming growth factor beta activates Rac1 and Cdc42Hs GTPases and the JNK pathway in skeletal muscle cells

The transforming growth factor beta (TGFbeta) plays an important role in cell growth and differentiation. However, the intracellular signaling pathways through which TGFbeta inhibits skeletal myogenesis remain largely undefined. By measuring GTP-loading of Rho GTPases and the organization of the F-actin cytoskeleton and the plasma membrane, we analyzed the effect of TGFbeta addition on the activity of three GTPases, Rac1, Cdc42Hs and RhoA. We report that TGFbeta activates Rac1 and Cdc42Hs in skeletal muscle cells, two GTPases previously described to inhibit skeletal muscle cell differentiation whereas it inactivates RhoA, a positive regulator of myogenesis. We further show that TGFbeta activates the C-jun N-terminal kinases (JNK) pathway in myoblastic cells through Rac1 and Cdc42Hs GTPases. We propose that the activation of Rho family proteins Rac1 and Cdc42Hs which subsequently regulate JNK activity participates in the inhibition of myogenesis by TGFbeta.     

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans ; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans . Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.     

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

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

The small GTPases RhoA and Rac1 regulate cerebellar development by controlling cell morphogenesis,migration and foliation

The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology.     

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

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

Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.     

Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells

Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by endothelial actin cytoskeletal and adherens junction remodeling, but the mechanisms involved are uncertain. We therefore measured the activities of the Rho GTPases Rac1, RhoA, and Cdc42 during hypoxia/reoxygenation and correlated them with changes in endothelial permeability, remodeling of the actin cytoskeleton and adherens junctions, and production of ROS. Dominant negative forms of Rho GTPases were introduced into cells by adenoviral gene transfer and transfection, and inhibitors of NADPH oxidase, PI3 kinase, and Rho kinase were used to characterize the signaling pathways involved. In some experiments constitutively activated forms of RhoA and Rac1 were also used. We show for the first time that hypoxia/reoxygenation-induced changes in endothelial permeability result from coordinated actions of the Rho GTPases Rac1 and RhoA. Rac1 and RhoA rapidly respond to changes in oxygen tension, and their activity depends on NADPH oxidase- and PI3 kinase-dependent production of ROS. Rac1 acts upstream of RhoA, and its transient inhibition by acute hypoxia leads to activation of RhoA followed by stress fiber formation, dispersion of adherens junctions, and increased endothelial permeability. Reoxygenation strongly activates Rac1 and restores cortical localization of F-actin and VE-cadherin. This effect is a result of Rac1-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA, and lysophosphatidic acid. Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodeling, junctional integrity, or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function.     

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.     

Cdc42: an important regulator of neuronal morphology

Regulation of neuronal morphology and activity-dependent synaptic modifications involves reorganization of the actin cytoskeleton. Dynamic changes of the actin cytoskeleton in many cell types are controlled by small GTPases of the Rho family, such as RhoA, Rac1 and Cdc42. As key regulators of both actin and microtubule cytoskeleton, Rho GTPases have also emerged as important regulators of dendrite and spine structural plasticity. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and growth cone protrusion, while the activation of RhoA induces stress fiber formation, leading to growth cone collapse and neurite retraction. This review focuses on recent advances in our understanding of the molecular mechanisms underlying physiological and pathological functions of Cdc42 in the nervous system. We also discuss application of different FRET-based biosensors as a powerful approach to examine the dynamics of Cdc42 activity in living cells.     

A Role for Cdc42 in Macrophage Chemotaxis

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

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

     

Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like,proline-rich,and GTPase-activating protein domains of a novel Rho GTPase-activating protein,BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.     

Roles of Rho-family GTPases in cell polarisation and directional migration

Polarised cell migration is a tightly regulated process that occurs in tissue development, chemotaxis and wound healing. Rho-family GTPases, including Cdc42, Rac1 and RhoA, play a central role in establishing cell polarisation, which requires asymmetric and ordered distribution of the signalling molecules and the cytoskeleton. Recent advances reveal that Rho GTPases, together with phosphatidylinositol 3-kinase, contribute to asymmetric phosphatidylinositol 3,4,5-trisphosphate distribution via a positive-feedback loop. Phosphatidylinositol 3,4,5-trisphosphate thereby activates the signalling cascades to the cytoskeleton as a second messenger. Rho GTPases also capture and stabilise microtubules through their effectors (e.g. IQGAP1, mDia and Par6) near the cell cortex, leading to polarised cell morphology and directional cell migration. Thus, elucidation of the signal transduction cascades from receptors to Rho GTPases and, subsequently, from Rho GTPases to microtubules has begun.