Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Adenovirus (Ad) endocytosis via α_v integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

Rac1‐Rab11‐FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T‐cell activation

The immunological synapse generation and function is the result of a T‐cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11‐positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1‐dependent manner, key morphological events, like T‐cell spreading and synapse symmetry. Finally, Rab11‐/FIP3‐mediated regulation is necessary for T‐cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T‐cell activation.     

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.     

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1.     

Rab35 is required for Wnt5a/Dvl2-induced Rac1 activation and cell migration in MCF-7 breast cancer cells

The small GTPases regulate many major biological processes in both tumorigenesis and tumor progression such as cell survival, actin cytoskeleton organization, cell polarity and movement. Wnt5a, a non-canonical Wnt family member, is implicated in the activation of small GTPases in breast cancer. We previously demonstrated that Wnt5a signaling stimulates the migration of breast cancer cells MDA-MB-231 via activating RhoA. However, we found here that RhoA activation was not enhanced by Wnt5a in breast cancer cells MCF-7. The conflicting results prompted us to further probe novel small GTPases in response to Wnt5a and investigate the mechanisms whereby cell migration is regulated. We showed here that Wnt5a dose dependently activated Dvl2, Rab35 and Rac1 and subsequently promoted the migration of MCF-7 cells, which was, however, abolished by knocking down Wnt5a expression via small interfering RNA (siRNA) transfection. Dvl2 siRNA significantly decreased background and Wnt5a-induced Rab35/Rac1 activation and, consequently, cell migration. Rab35 short hairpin RNA (shRNA) remarkably inhibited background and Wnt5a-induced Rac1 activation and cell migration. Additionally, blockade of Rac1 activation with Rac1 siRNA suppressed background and Wnt5a-induced cell migration. Co-immunoprecipitation and immunofluorescence assays showed that Dvl2 bound to Rab35 in mammalian cells. Taken together, we demonstrated that Wnt5a promotes breast cancer cell migration via the Dvl2/Rab35/Rac1 signaling pathway. These findings implicate Wnt5a signaling in regulating small GTPases, which could be targeted for manipulating breast cancer cell migration.     

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.     

Treponema denticola major outer sheath protein induces actin assembly at free barbed ends by a PIP2-dependent uncapping mechanism in fibroblasts

The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends.     

Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho

Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance [1-3]. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance [4-7]. Using yeast two-hybrid screening and in vitro interaction assays, we show that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induced similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. Our findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration.     

The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts

Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

SWAN-1, a Caenorhabditis elegans WD repeat protein of the AN11 family, is a negative regulator of Rac GTPase function

Rac GTPases are key regulators of cell shape and cytoskeletal organization. While some regulators of Rac activity are known, such as GTPase-activating proteins (GAPs) that repress Rac activity, other Rac regulators remain to be identified. The novel Caenorhabditis elegans WD-repeat protein SWAN-1 was identified in a yeast two-hybrid screen with the LIM domains of the Rac effector UNC-115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases. The swan-1(ok267) loss-of-function mutation suppressed defects caused by the hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and filopodia formation induced by constitutively active Rac in C. elegans neurons. Furthermore, SWAN-1(+) transgenic expression suppressed the effects of overactive Rac, including ectopic lamellipodia and filopodia formation in C. elegans neurons, ectopic lamellipodia formation in cultured mammalian fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular morphogenesis and cytoskeletal organization. While broadly conserved across species, SWAN-1 family members show no sequence similarity to previously known Rac inhibitors.     

A tale of three GTPases and a RIN in endothelial cell adhesion

Endothelial cell adhesion to the extracellular matrix regulates migration and outgrowth of blood vessels during angiogenesis. Cell adhesion is mediated by integrins, which transduce signals from the extracellular environment into the cell and, in turn, are regulated by intracellular signaling molecules. In a paper recently published in Cell Research, Sandri et al. show that RIN2 connects three GTPases, R-Ras, Rab5 and Rac1, to promote endothelial cell adhesion through the regulation of integrin internalization and Rac1 activation.The formation of the vascular tree during development requires the orderly growth of blood vessels to irrigate all organs and tissues. This process of blood vessel remodeling, termed angiogenesis, requires endothelial cell proliferation, adhesion, migration and tube formation¹. Pathological angiogenesis takes place during tumor growth as hypoxia within the tumor induces the release of pro-angiogenic mediators such as vascular endothelial growth factor (VEGF).Small GTPases are critical for the regulation of cell behavior and thus also play a central role in angiogenesis. Small GTPases are 20-25 kDa signaling proteins that cycle between an active GTP-bound and an inactive GDP-bound state. When active, GTPases associate with and activate diverse effector molecules that subsequently relay the signal to other molecules, ultimately leading to a specific cell response. Two classes of proteins facilitate GTPase cycling. Guanine exchange factors (GEFs) catalyze GDP unloading thereby promoting GTP binding and GTPase activation. Conversely, GTPase activating proteins (GAPs) enhance the intrinsic GTP hydrolysis activity of the GTPase leading to its inactivation. Small GTPases form a large superfamily with over 100 members in mammals. Based on structural and functional criteria, the GTPase superfamily is subdivided in Ras, Rab, Rho, Arf and Ran subfamilies, each of them generally, but not exclusively, specialized in the regulation of specific cellular events. For example, Rho GTPases primarily regulate cytoskeletal dynamics; Rab GTPases regulate intracellular membrane trafficking; and Ras GTPases function in the regulation of cell proliferation and survival. However, complex processes such as angiogenesis require the coordinated action of several GTPases. This is evidenced by the work of Sandri et al.² recently published in Cell Research. In their paper, Sandri et al. propose a mechanism for the regulation of endothelial cell adhesion and migration involving three GTPases belonging to different GTPase branches, R-Ras, Rab5 and Rac1. The protein RIN2 (Ras and Rab adaptor 2) brings together R-Ras and Rab5 to form a signaling module that orchestrates integrin trafficking and Rac1 activation, processes that are essential for cell adhesion and migration.Integrins are heterodimeric transmembrane extracellular matrix (ECM) receptors composed of one α and one β chain. In a process known as ''outside-in'' signaling, integrins transmit signals from the extracellular environment to intracellular adaptor and signaling molecules that regulate cell migration, survival and growth. Conversely, during ''inside-out'' signaling, integrins can be switched from an inactive to an active conformation by cytoplasmic signaling molecules leading to increased integrin affinity for the ECM.During 2D migration of adherent cells, nascent, highly dynamic focal contacts are formed at the leading edge lamellipodia where integrins mediate adhesion to the ECM. Some of these focal contacts disassemble and some mature into larger focal adhesions with a longer half-life. Failure in maintaining a dynamic assembly and disassembly of focal contacts will result in the inhibition of cell migration.Integrin-mediated adhesion can be regulated at different levels: (1) by changing integrin conformation and thus affinity for their ligand; (2) by modulating integrin avidity, i.e., by promoting integrin clustering on the plasma membrane; and (3) by changing the kinetics of integrin endocytosis and/or recycling³.The Ras GTPase R-Ras is primarily expressed in the vascular system (endothelial cells and vascular smooth muscle cells)⁴. Zhang et al.⁵ were the first to show that R-Ras is a potent regulator of cell adhesion when they reported that expression of active R-Ras was enough to induce ECM adhesion of suspension cells, whereas dominant negative R-Ras reduced adhesion of the adherent cell line CHO. Although R-Ras was shown to enhance integrin affinity⁵, this effect was not consistently observed^6,7. These contradictory findings could be explained by the fact that R-Ras may activate integrins indirectly through antagonizing H-Ras-mediated integrin inhibition⁶.Recent findings suggest that R-Ras stimulates adhesion through the regulation of integrin internalization into Rab11-positive endosomes⁸. Now, the data of Sandri et al.² support this model. The authors addressed the question on how R-Ras regulates cell adhesion of endothelial cells by performing a yeast-two-hybrid screen using constitutively-active R-Ras as bait. The screen revealed that RIN2 is a major R-Ras-interacting protein. RIN proteins (RIN1, 2 and 3) are downstream effectors of Ras GTPases that function as GEFs for Rab5⁹, a GTPase that regulates endocytosis. RIN1 was shown to mediate the stimulation of EGF receptor-mediated endocytosis by H-Ras through the activation of Rab5¹⁰. Surprisingly, Sandri et al. found that R-Ras dramatically impaired the Rab5 exchange activity of RIN2, while H-Ras had no effect. However, RIN2 was still able to specifically bind active Rab5. These data suggest that active R-Ras, RIN2 and active Rab5 form a signaling complex. Accordingly, Sandri et al. show that endogenous R-Ras, RIN2 and Rab5 are indeed found in a complex in endothelial cells. While active R-Ras and RIN2 colocalize at nascent focal contacts and on intracellular vesicles, colocalization with Rab5 takes place on endosomes. The deletion of either the Ras- or the Rab5-binding domains of RIN2 prevented the colocalization of the trio. Thus, RIN2 appears to facilitate the transport of active R-Ras to Rab5-positive endosomes. What is the functional relevance of these interactions? Sandri et al. show that silencing of endogenous RIN2 impaired the increase in adhesion induced by active R-Ras and by Rab5. A similar effect was obtained upon expression of RIN2 deletion mutants lacking Ras- or Rab5-binding domains. These data strongly suggest that the adaptor function of RIN2 in connecting R-Ras and Rab5 regulates endothelial cell adhesion to the ECM. But what is the mechanism? Previous work has shown that the pro-adhesive activity of active R-Ras is linked to its ability to regulate β1 integrin endocytosis⁸. Sandri et al. confirm these data by showing that silencing of R-Ras or RIN2 decreases the rate of endocytosis of active ECM-engaged β1 integrins. In addition, the authors set a step further as they show that the signaling complex R-Ras/RIN2/Rab5 mediates basal Rac1 GTPase activation. Rac1 regulates actin dynamics and ruffle formation at the leading edge of migrating cells and its activity is essential for cell adhesion and migration. TIAM-1-mediated activation of Rac1 on endosomes and subsequent polarized transport to the plasma membrane has been proposed as a way to restrict Rac activity to sites of membrane protrusion^11,12. In line with this model, Sandri et al. show that active R-Ras and RIN2 colocalize with Rac1 on endosomes and that the endosomal Rac GEF TIAM-1 is necessary for R-Ras- and RIN2-induced cell adhesion.Altogether, the data of Sandri et al. support a model in which, integrin-activated R-Ras recruits RIN2 to focal adhesions and induces RIN2 conversion from a Rab5 GEF to a Rab5-docking protein. Subsequently, the complex promotes the endocytosis of ECM-engaged integrins and moves to early endosomes where R-Ras activates the TIAM-1/Rac1 pathway¹³. Active Rac1 translocates to the plasma membrane where it promotes actin polymerization and formation of new focal contacts (Figure 1).Open in a separate windowFigure 1Model proposed by Sandri et al.² for the regulation of focal adhesion dynamics by R-Ras. (1) R-Ras is activated by ECM-engaged integrins, recruits RIN2 and converts it from a Rab5 GEF to a Rab5 adaptor; (2) RIN2 binding to active Rab5 mediates the endocytosis of integrins and the transport of active R-Ras to endosomes; (3) R-Ras contributes to the activation of the Rac1 GEF TIAM-1, which then activates Rac1; (4) Active Rac1 translocates to the plasma membrane and promotes actin polymerization and formation of new focal contacts.By bridging active R-Ras and Rab5, RIN2 combines two processes essential for cell adhesion: (1) focal contact dynamics through the internalization of ECM-engaged integrins; and (2) local Rac1 activation to ensure actin polymerization at lamellipodia. Similarly, RIN2 also connects H-Ras and Rab5 in the internalization of the epithelial cell-cell adhesion molecule E-cadherin¹⁴. Thus, RIN2 appears to be a universal effector of Ras-induced endocytosis of membrane receptors.Interestingly, the phenotype of a family with a homozygous mutation in RIN2 was recently described¹⁵. The affected individuals showed diverse abnormalities related to a defective connective tissue. Indeed, ultrastructural analysis of the skin showed an abnormal morphology of collagen fibrils. Collagen is a ligand for β1 integrins. Through simultaneous binding to collagen and to the intracellular cytoskeleton, integrins contribute to the assembly of the ECM by transmitting contraction forces from the cell to the ECM. It is tempting to speculate that the phenotype of the patients lacking RIN2 is due to a deficient β1 integrin function as found by Sandri et al. in their in vitro analysis. In addition, these patients bruise easily and present prolonged bleeding, which could be caused by deficient wound healing of blood vessels as a consequence of impaired R-Ras signaling.It should be noted, however, that R-Ras knockout mice have no major defects in vascular development but respond with increased angiogenesis to stress conditions such as tumor implantation⁴. On the contrary, the in vitro study by Sandri et al. suggests that R-Ras deficiency results in decreased endothelial cell migration. Further research is needed to clarify the role of R-Ras in angiogenesis. Likewise, it will be interesting to study vascular responses in RIN2-deficient mice in comparison to R-Ras knockout mice.     

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.     

ARF1 controls Rac1 signaling to regulate migration of MDA-MB-231 invasive breast cancer cells

ADP-ribosylation factors (ARFs) are monomeric G proteins that regulate many cellular processes such as reorganization of the actin cytoskeleton. We have previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced migration. In this study, we propose to define the molecular mechanism by which ARF1 regulates this complex cellular response by investigating the role of this ARF GTPase on the activation process of Rac1, a Rho GTPase, associated with lamellipodia formation during cell migration. Here, we first show that inhibition of ARF1 or Rac1 expression markedly impacts the ability of MDA-MB-231 cells to migrate upon EGF stimulation. However, the effect of ARF1 depletion can be reversed by overexpression of the Rac1 active mutant, Rac1 Q⁶¹L. Depletion of ARF1 also impairs the ability of EGF stimulation to promote GTP-loading of Rac1. To further investigate the possible cross-talk between ARF1 and Rac1, we next examined whether they could form a complex. We observed that the two GTPases could directly interact independently of the nature of the nucleotide bound to them. EGF treatment however resulted in the association of Rac1 with its effector IRSp53, which was completely abrogated in ARF1 depleted cells. We present evidences that this ARF isoform is responsible for the plasma membrane targeting of both Rac1 and IRSp53, a step essential for lamellipodia formation. In conclusion, this study provides a new mechanism by which ARF1 regulates cell migration and identifies this GTPase as a promising pharmacological target to reduce metastasis formation in breast cancer patients.     

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.     

p19Arf-p53 tumor suppressor pathway regulates cell motility by suppression of phosphoinositide 3-kinase and Rac1 GTPase activities

The p19(Arf)-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis, whereas Rho family small GTPases are key regulators of actin structure and cell motility. By using primary mouse embryonic fibroblasts that lack Arf, p53, or both, we studied the involvement of the p19(Arf)-p53 pathway in the regulation of cell motility and its relationship with Rho GTPases. Deletion of Arf and/or p53 led to actin cytoskeleton reorganization and a significant increase in cell motility. The endogenous phosphoinositide (PI) 3- kinase and Rac1 activities were elevated in Arf(-/-) and p53(-/-) cells, and these activities are required for p19(Arf)- and p53-regulated migration. Reintroduction of the wild type Arf or p53 genes into Arf(-/-) or p53(-/-) cells reversed the PI 3-kinase and Rho GTPase activities as well as the migration phenotype. These results suggest a functional relationship between an established tumor suppressor pathway and a signaling module that controls actin structure and cell motility and show that p19(Arf) and p53 negatively regulate cell migration by suppression of PI 3-kinase and Rac1 activities.     

Cortactin Promotes Migration and Platelet-derived Growth Factor-induced Actin Reorganization by Signaling to Rho-GTPases

Dynamic actin rearrangements are initiated and maintained by actin filament nucleators, including the Arp2/3-complex. This protein assembly is activated in vitro by distinct nucleation-promoting factors such as Wiskott-Aldrich syndrome protein/Scar family proteins or cortactin, but the relative in vivo functions of each of them remain controversial. Here, we report the conditional genetic disruption of murine cortactin, implicated previously in dynamic actin reorganizations driving lamellipodium protrusion and endocytosis. Unexpectedly, cortactin-deficient cells showed little changes in overall cell morphology and growth. Ultrastructural analyses and live-cell imaging studies revealed unimpaired lamellipodial architecture, Rac-induced protrusion, and actin network turnover, although actin assembly rates in the lamellipodium were modestly increased. In contrast, platelet-derived growth factor-induced actin reorganization and Rac activation were impaired in cortactin null cells. In addition, cortactin deficiency caused reduction of Cdc42 activity and defects in random and directed cell migration. Reduced migration of cortactin null cells could be restored, at least in part, by active Rac and Cdc42 variants. Finally, cortactin removal did not affect the efficiency of receptor-mediated endocytosis. Together, we conclude that cortactin is fully dispensable for Arp2/3-complex activation during lamellipodia protrusion or clathrin pit endocytosis. Furthermore, we propose that cortactin promotes cell migration indirectly, through contributing to activation of selected Rho-GTPases.     

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