Table of contents

Rho, Integrins and the Extracellular Matrix

This study investigates how intracellular signaling modifies the structure of the extracellular matrix. Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work has demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid. C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. In a study of the mechanism by which Rho promotes fibronectin polymerization, C3 was used to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly, only small changes in the state of integrin activation are detected. However, several inhibitors of cellular contractility that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, a monoclonal antibody, L8, was found that stains fibronectin matrices differentially depending on the state of cell contractility. L8 inhibits fibronectin matrix assembly. When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. The consequences of mechanically stretching fibronectin were investigated in the absence of cells. Applying a 30-35% stretch to immobilized fibronectin induces binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension (Zhong, 1998).

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, an examination was made of the roles of Cdc42-, Rac-, and Rho-dependent signaling pathways in the regulation of cytoskeleton during integrin-mediated adhesion and cell spreading, using the dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, some phosphorylation is observed of the focal adhesion kinase (FAK) and Src, which occurs independent of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Rho dependence is found for the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity is dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior (Clark, 1998).

Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTP-binding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rho-dependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing between changes in Rho activity and the structural contributions of ECM and the formation of focal adhesions. To address these questions, an assay was establised for GTP-bound cellular Rho. Plating Swiss 3T3 cells on fibronectin-coated dishes elicits a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase is greatly enhanced by serum. In serum-starved adherent cells, LPA induces transient Rho activation, whereas in suspended cells, Rho activation is sustained. Furthermore, in the presence of serum, suspended cells show higher Rho activity than adherent cells. These data indicate the existence of an adhesion-dependent negative-feedback loop. Both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. These results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity. It is proposed that under serum-free condition, Rho activity is decreased and integrins are not significantly clustered; therefore, focal adhesions are not readily detectable. Adding LPA or serum activates Rho within minutes to generate contractile force, leading to clustering of integrins and formation of focal adhesions and stress fibers. Newly formed focal adhesions generate signals to downregulate Rho to prevent excessive formation of focal adhesions. Downregulation of Rho may also play a role in disassembly of focal adhesions required for cell migration. The actin cytoskeleton and microtubules suppress Rho activity, possibly by controlling the localization of guanine nucleotide exchange factors or GAPs (Ren, 1999).

alphaß1 integrins have been implicated in the survival, spreading, and migration of cells and tissues. To explore the underlying biology, conditions were identified where primary ß1 null keratinocytes adhere, proliferate, and display robust alphavß6 integrin-induced, peripheral focal contacts associated with elaborate stress fibers. Mechanistically, this appears to be due to reduced FAK and Src and elevated RhoA and Rock activities. Visualization on a genetic background of GFPactin shows that ß1 null keratinocytes spread, but do so aberrantly, and when induced to migrate from skin explants in vitro, the cells are not able to rapidly reorient their actin cytoskeleton toward the polarized movement. As judged by RFPzyxin/GFPactin videomicroscopy, the alphavß6-actin network does not undergo efficient turnover. Without the ability to remodel their integrin-actin network efficiently, alphaß1-deficient keratinocytes cannot respond dynamically to their environment and polarize movements (Raghavan, 2003).

The results underscore a novel and distinct role for alphaß1 integrins in regulating this equilibrium in focal adhesion dynamics. Not surprisingly, three well-known regulators of focal contacts, FAK, RhoA, and Rock, appear to be at the heart of this regulation. As judged by immunofluorescence with purportedly specific phospho-FAK Abs, activated FAK localizes to the focal contacts of ß1 null keratinocytes. By this criterion, the underlying defects in focal contact turnover and in overall FAK and Src activities are not attributable to a defect in targeting FAK to alphavß6 focal contacts, and indeed, ligand-engaged alphavß6 can bind and activate FAK. Rather, in the absence of ß1, alphavß6 appears unable on its own to activate FAK to the threshold levels needed to properly control focal adhesion-actin cytoskeletal dynamics. Irrespective of the precise underlying mechanism, the consequences to this imbalance are excessive adhesion and inefficient spreading (Raghavan, 2003).

Although tyrosine kinase inhibitors can block focal adhesion formation in some situations, a greater role for tyrosine phosphorylation has been found in focal adhesion turnover and cell motility. Thus, activated FAK negatively regulates RhoA activity, and FAK null fibroblasts express robust actin stress-fiber networks that can be dissipated by Rock inhibition. The ability of Rho and Rock inhibitors to disperse both stress fibers and associated focal contacts in ß1 null keratinocytes provides compelling evidence that a FAK-RhoA imbalance is at the root of the focal adhesion-cytoskeletal imbalance in these cells. Although more complicated mechanisms are possible, the data are consistent with a model whereby in the absence of alphaß1 integrins, FAK/Src activation is not fully achieved, thereby diminishing p190RhoGAP phosphorylation, and yielding elevated RhoA/Rock activities (Raghavan, 2003).

Rho and Cell Polarity

Polarization of cells during mouse preimplantation development first occurs within blastomeres at the eight-cell stage, as part of a process called compaction. Cell-cell contact mediated by the cell adhesion molecule uvomorulin (E-cadherin) and the activity of the microfilament cytoskeleton are important in the development of compaction, which is crucial for establishment of trophoblast and pluriblast (inner cell mass) lineages and for subsequent development. Members of the Rho family of p21 GTPases have been shown to regulate the organization of the actin cytoskeleton and adhesion in other cell types. The potential role of these proteins in compaction was investigated. Inhibition of Rho with Clostridium botulinum C3-transferase disturbs intercellular flattening at compaction and prevents cytocortical microfilament polarization of eight-cell blastomeres, in contrast to cytochalasin D, which inhibits only adhesion. Microinjection of a constitutively activated recombinant Rho protein into four-cell blastomeres induces cortical microfilament disruption and apical displacement of nuclei associated with polarized clustering of microtubules. Interblastomere adhesion is reduced and E-cadherin is aberrently clustered at remaining cell-cell contacts. Similarly, activated Cdc42 protein induces nuclear displacement with additional cytoplasmic actin bundle formation between nucleus and cell-cell contacts. The effects produced by both of the activated GTPase proteins are indicative of prematurely induced but aberrently organized polarity. These results suggest that Rho family GTPases are involved in the polarization of early mouse blastomeres (Clayton, 1999).

MDCK cells expressing RhoA or Rac1 mutants under control of the tetracycline repressible transactivator were used to examine short-term effects of known amounts of each mutant before, during, or after development of cell polarity. At low cell density, Rac1V12 cells have a flattened morphology and intact cell-cell contacts, whereas Rac1N17 cells are tightly compacted. Abnormal intracellular aggregates form between Rac1N17, F-actin, and E-cadherin in these nonpolarized cells. At all subsequent stages of polarity development, Rac1N17 and Rac1V12 colocalize with E-cadherin and F-actin in an unusual beaded pattern at lateral membranes. In polarized cells, intracellular aggregates form with Rac1V12, F-actin, and an apical membrane protein (GP135). At low cell density, RhoAV14 and RhoAN19 are localized in the cytoplasm, and cells are generally flattened and more fibroblastic than epithelial in morphology. In polarized RhoAV14 cells, F-actin is diffuse at lateral membranes and prominent in stress fibers on the basal membrane. GP135 is abnormally localized to the lateral membrane and in intracellular aggregates, but E-cadherin distribution appears normal. In RhoAN19 cells, F-actin, E-cadherin, and GP135 distributions are similar to those in controls. Expression of either RhoAV14 or RhoAN19 in Rac1V12 cells disrupts Rac1V12 distribution and causes cells to adopt the more fibroblastic, RhoA mutant phenotype. It is suggested that Rac1 and RhoA are involved in the transition of epithelial cells from a fibroblastic to a polarized structure and function by direct and indirect regulation of actin and actin-associated membrane protein organizations (Jou, 1998a).

In C. elegans one-cell embryos, polarity is conventionally defined along the anteroposterior axis by the segregation of partitioning-defective (PAR) proteins into anterior (PAR-3, PAR-6) and posterior (PAR-1, PAR-2) cortical domains. The establishment of PAR asymmetry is coupled with acto-myosin cytoskeleton rearrangements. The small GTPases RHO-1 and CDC-42 are key players in cytoskeletal remodeling and cell polarity in a number of different systems. This study investigated he roles of these two GTPases and the RhoGEF ECT-2 in polarity establishment in C. elegans embryos. CDC-42 is shown to be required to remove PAR-2 from the cortex at the end of meiosis and to localize PAR-6 to the cortex. By contrast, RHO-1 activity is required to facilitate the segregation of CDC-42 and PAR-6 to the anterior. Loss of RHO-1 activity causes defects in the early organization of the myosin cytoskeleton but does not inhibit segregation of myosin to the anterior. It is therefore proposed that RHO-1 couples the polarization of the acto-myosin cytoskeleton with the proper segregation of CDC-42, which, in turn, localizes PAR-6 to the anterior cortex (Schonegg, 2006).

C. elegans embryos establish cortical domains of PAR proteins of reproducible size before asymmetric cell division. The ways in which the size of these domains is set remain unknown. This study identified the GTPase-activating proteins (GAPs) RGA-3 and RGA-4, which regulate the activity of the small GTPase RHO-1. rga-3/4(RNAi) embryos have a hypercontractile cortex, and the initial relative size of their anterior and posterior PAR domains is altered. Thus, RHO-1 activity appears to control the level of cortical contractility and concomitantly the size of cortical domains. These data support the idea that in C. elegans embryos the initial size of the PAR domains is set by regulating the contractile activity of the acto-myosin cytoskeleton through the activity of RHO-1. RGA-3/4 have functions different from CYK-4, the other known GAP required for the first cell division, showing that different GAPs cooperate to control the activity of the acto-myosin cytoskeleton in the first cell division of C. elegans embryos (Schonegg, 2007).

Rho and exocytosis

Polarized exocytosis involves at least three stages. (1) Post-Golgi secretory vesicles are transported along cytoskeletal elements towards specific regions of the plasma membrane. In the budding yeast, actin cables direct vesicle movement in the cell and a class V myosin, Myo2, is thought to be important in this process. (2) After the vesicles are transported to the right region, they are then tethered and docked at specific domains of the plasma membrane. (3) Finally, the lipid bilayers of the vesicle membrane and plasma membrane fuse in a reaction catalysed by the interactions of integral membrane proteins (soluble N-ethylmaleimide-sensitive fusion [NSF] attachment protein ['SNAP'] receptors, or 'SNAREs') that reside in the secretory vesicle membrane (v-SNAREs) and the target membranes (t-SNAREs). The last step leads to the release of vesicle contents and the incorporation of membrane proteins at specific domains of the plasma membrane. The Rab family small GTP-binding proteins are important regulators of polarized exocytosis. They cycle between GTP-bound and GDP-bound states. GTP-bound Rab proteins interact with various downstream effectors that are thought to mediate distinct stages of vesicle traffic, including transport along the cytoskeleton, tethering to the target membrane and assembly of the SNARE complex (Guo, 2001 and references therein).

Studies have indicated that a multiprotein complex, termed the exocyst, is involved in vesicle targeting and docking at the plasma membrane. The exocyst consists of eight components, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. In contrast to the t-SNAREs, which are evenly distributed along the entire plasma membrane, the exocyst is specifically localized to sites of active exocytosis. In the budding yeast, the exocyst proteins are localized to regions of active cell-surface expansion: the bud tip at the beginning of the cell cycle, and the mother/daughter connection during cytokinesis. In developing neurons exocyst components are localized to growth cones and the tips of growing neurites, whereas in epithelial cells they are concentrated near the tight junction, a region of active basolateral membrane addition (Guo, 2001 and references therein).

In addition to the exocyst, many components of the yeast exocytic machinery are localized to sites of exocytosis. For example Sec4, the Rab protein required at this stage of transport, is localized to exocytic sites through its association with the secretory vesicles, which are concentrated there. Many components, such as Sec8 and Sec1, become localized to exocytic sites in response to the arrival and docking of secretory vesicles, whereas the exocyst subunit Sec3 is localized to sites of exocytosis, independent of actin and the continuing flux of membrane through the secretory pathway. Furthermore, the localization of Sec3 seems to be independent of the other subunits of the exocyst. On the basis of these observations, Sec3 has been proposed to represent a spatial landmark for polarized secretion. Another exocyst subunit, Sec15, associates with secretory vesicles and interacts with the GTP-bound form of the Rab protein, Sec4. Therefore, the assembly of the exocyst complex would link secretory vesicles, through a series of protein-protein interactions among the exocyst proteins, to the specific exocytic sites on the plasma membrane (Guo, 2001 and references therein).

Proteins that regulate the polarized localization of the exocyst have been sought in the budding yeast Saccharomyces cerevisiae. Certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted, independent of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity. It is proposed that Rho1 coordinates the actin cytoskeleton, polarized exocytosis and cell-wall construction/modification to ensure polarized growth of yeast (Guo, 2001).

Embryonic morphogenesis involves the coordinate behaviour of multiple cells and requires the accurate balance of forces acting within different cells through the application of appropriate brakes and throttles. In C. elegans, embryonic elongation is driven by Rho-binding kinase (ROCK) and actomyosin contraction in the epidermis. This study identified an evolutionary conserved, actin microfilament-associated RhoGAP (RGA-2) that behaves as a negative regulator of LET-502/ROCK. The small GTPase RHO-1 is the preferred target of RGA-2 in vitro, and acts between RGA-2 and LET-502 in vivo. Two observations show that RGA-2 acts in dorsal and ventral epidermal cells to moderate actomyosin tension during the first half of elongation: (1) time-lapse microscopy shows that loss of RGA-2 induces localised circumferentially oriented pulling on junctional complexes in dorsal and ventral epidermal cells; (2) specific expression of RGA-2 in dorsal/ventral, but not lateral, cells rescues the embryonic lethality of rga-2 mutants. It is proposed that actomyosin-generated tension must be moderated in two out of the three sets of epidermal cells surrounding the C. elegans embryo to achieve morphogenesis (Diogon, 2007).

Rho and Junctions

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. Roles of the RhoA and Rac1 GTPases were examined 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 perturb TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy reveals abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appear normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupt the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane are unaffected. Expression levels and protein complexes of occludin and ZO-1 appear normal in all mutant cells, although ZO-1 is 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 (Jou, 1998b).

Rho and mitosis

The role of the small GTP-binding protein Rho in cytokinesis has been investigated by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induces immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrate with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells shows a diffuse distribution throughout cell division. All treated, well-adherent cells undergo cleavage-like activities and most of them divided successfully. However, cytokinesis becomes abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis is blocked with nocodazole. The effects of C3 appear to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibit irregular cortical ingression only when cells start to increase attachment during respreading, but manage to complete cytokinesis. Poorly adherent HeLa cells show neither ectopic cleavage nor completion of cytokinesis. These results indicate that Rho does not simply activate actin-myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex (O'Connell, 1999).

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. The human ECT2 (Drosophila homolog: Pebble) is shown to be involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division (Tatsumoto, 1999).

Rho GTPases are important regulators of cellular behavior through their effects on processes such as cytoskeletal organization. Interactions between Drosophila Rho1 and the adherens junction components alpha-catenin and p120ctn (Delta-catenin) are reported. While Rho1 protein is present throughout the cell, it accumulates apically, particularly at sites of cadherin-based adherens junctions. Cadherin and catenin localization is disrupted in Rho1 mutants, implicating Rho1 in their regulation. p120ctn has recently been suggested to inhibit Rho activity through an unknown mechanism (Anastasiadis, 2000). Rho1 accumulates in response to lowered p120ctn activity. Significantly, Rho1 binds directly to alpha-catenin and p120ctn in vitro, and these interactions map to distinct surface-exposed regions of the protein not previously assigned functions. In addition, both alpha-catenin and p120ctn co-immunoprecipitate with Rho1-containing complexes from embryo lysates. These observations suggest that alpha-catenin and p120ctn are key players in a mechanism of recruiting Rho1 to its sites of action (Magie, 2002).

Rho has been implicated in the regulation of cell adhesion through its effects on a number of different types of cellular junctions, including integrin-based focal adhesions and cadherin-based AJs. Rho1 localization was examined relative to DE-cadherin, a component of AJs located around the apical margin of cells and Neurexin, a component of septate junctions (SJs), which are thought to be analogous to tight junctions in mammalian cells. Since AJs and other cell-cell contacts are not yet fully formed at the cellular blastoderm stage, cells of the gut epithelium, which show a clear apical-basal polarity with AJs located at the apical end of the cell and SJs more basal, were examined. As in blastoderm embryos, Rho1 protein is ubiquitously cytoplasmic but concentrated apically. Significantly, the apical cytoplasmic accumulation of Rho1 protein overlaps with the sites of cadherin localization. Neurexin is localized basal to the apical accumulations of Rho1 and does not show substantial overlap (Magie, 2002).

Rho1 activity is required to properly localize DE-cadherin during development, consistent with data from mammalian cell culture experiments implicating Rho and Rac in cadherin assembly and maintenance. The defects observed in cadherin localization are most prevalent in and around the leading edge (LE) cells undergoing dorsal closure. Previously Rho1 had been implicated in dorsal closure via its regulation of the LE actin cytoskeleton in cells flanking the segment borders. However, the disruption observed in cadherin distribution suggests that regulation of cell-cell adhesion may play a role in the dorsal closure phenotype observed in these embryos. Thus Rho1's effects on cadherin localization could be the result of a direct role in DE-cadherin clustering, or an indirect effect on the cortical actin cytoskeleton. The process of AJ formation in keratinocytes has been shown to require actin polymerization and the interdigitation of filopodia from neighboring cells. A similar interdigitation of filopodia is seen during dorsal closure in Drosophila and is likely involved in forming adhesive contacts between the two epithelial fronts. Since Rho and Cdc42 have been shown to act antagonistically in the formation of cellular processes in neurons, it is possible that disrupting the balance of Rho1 and Cdc42 function in LE cells results in inappropriate regulation of filopodial extensions. This could partially explain the disruption of DE-cadherin localization observed in Rho1 mutants. Alternatively, Rho1's primary role could be in directly regulating the adhesion of cells near the LE, with Rac and Cdc42 acting as the major organizers of the acto-myosin network (Magie, 2002).

In addition to the accumulation of Rho1 protein at sites of cadherin localization, a direct physical interaction is observed between Rho1 and both p120ctn and alpha-catenin. The catenin family of proteins is important in regulating cadherin-based adhesion and linking cadherins to the actin cytoskeleton. ß-catenin binds to the catenin-binding domain of the cadherin molecule as well as to alpha-catenin. alpha-catenin, in turn, acts as a link to the actin cytoskeleton, either by directly binding actin filaments or through association with other actin-binding proteins. alpha-catenin also has been shown to bind spectrin, a major component of the membrane skeleton underlying the plasma membrane involved in stabilizing it and determining cell shape. Human colon carcinoma Clone A cells that contain mutant alpha-catenin have defects in spectrin assembly. Consistent with this, a breakdown of the alpha-spectrin cytoskeleton is observed in embryos injected with alpha-catenin dsRNA, especially in morphogenetically active cells early in gastrulation. alpha-catenin protein is enriched at adherens junctions, but is not as strictly localized to them as is DE-cadherin. Binding of alpha-catenin to Rho1 may be a general mechanism through which Rho1 is recruited to the plasma membrane (Magie, 2002).

p120ctn regulates the adhesive properties of cadherin complexes through its binding to the juxtamembrane domain of the cadherin molecule, although the precise mechanisms underlying this function are not known. p120ctn also acts in the cytoplasm where it has been proposed to negatively regulate Rho activation in a manner similar to the GDI proteins, which prevent Rho from exchanging GDP for GTP, although it shares no sequence homology with them (Anastasiadis, 2000). The binding of p120ctn to cadherins and its effects on Rho function have been shown to be mutually exclusive, such that once p120ctn binds a cadherin molecule, it is no longer capable of inhibiting Rho activity or function (Anastasiadis, 2000). Rho would then be accessible to activating regulatory proteins such as GEFs, and could carry out its downstream functions. The physical interaction observed between Rho1 and p120ctn suggests that this negative regulation of Rho1 is due to direct binding of p120ctn to GDP-Rho1. Interestingly, this is the same face of the Rho protein that has been shown to bind to classical GDIs, consistent with the idea that despite the lack of sequence homology, p120ctn may be acting in a similar way. Overexpression of p120ctn in mammalian cells leads to an inhibition of Rho activity (Anastasiadis, 2000; Noren, 2000). Overexpression of p120ctn enhances the Rho1 mutant phenotype, as would be expected for a negative regulator. Embryos homozygous for a deficiency uncovering the p120ctn locus show an accumulation of Rho1 protein at the leading edge and exhibit a severe dorsal open phenotype. A similar accumulation of Rho1 protein is observed in embryos injected with p120ctn dsRNA. A positive feedback mechanism may be functioning whereby the relief of p120ctn-mediated regulation in those cells results in the upregulation of Rho1 protein or an increase in Rho1 stability. It has recently been shown that overexpression of a RhoGDI in the hearts of mouse embryos results in the upregulation of RhoA expression: this suggests the existence of a negative feedback mechanism in the regulation of RhoA levels -- there are no other instances in which a positive feedback mechanism has been linked to Rho expression. Excess Rho activity disrupts cellular migration; cells at the leading edge in embryos that lack p120ctn function remain cuboidal, rather than elongating as they would during normal dorsal closure, suggesting that Rho1 may be involved in regulating these cell shape changes. Alternatively, p120ctn has been suggested to activate Rac and Cdc42 in the cytoplasm through an interaction with the GEF Vav2, and this could account for some of its effects on cell morphology (Noren, 2000). The observation that Rho1 can bind both p120ctn and alpha-catenin and that their binding sites are not overlapping suggests that either could be involved in recruiting Rho1 to AJs or the plasma membrane in general. The data indicating that overexpression of alpha-catenin enhances the Rho1 mutant phenotype to a greater degree than p120ctn suggests an important role for alpha-catenin in Rho1 function, perhaps as a factor generally involved in localizing Rho1 to its sites of action, while p120ctn plays a more specific role at AJs (Magie, 2002).

The data suggest a model in which p120ctn or alpha-catenin or both are involved in recruiting Rho1 to sites of cadherin localization, where it can then be activated and carry out its functions, including proper AJ formation. If Rho1 is not recruited properly, as in the case of a Rho1 mutant, this results in mislocalization of AJ components. The binding of p120ctn to Rho1, either in the cytoplasm or while Rho1 is tethered at AJs through its interaction with alpha-catenin, inhibits the exchange of GDP for GTP and keeps Rho1 in an inactive state. The binding of p120ctn to the juxtamembrane domain may release Rho1, allowing it to be activated by GEFs. GTP-Rho1 could then bind its downstream effectors and either directly regulate DE-cadherin assembly or maintenance, or indirectly affect AJ formation through its effects on the actin cytoskeleton. Rho1 localization at AJs could then be mediated either through continued association with alpha-catenin or through isoprenylation and insertion into the plasma membrane. Mutational analysis aimed at distinguishing between these models will provide further insight into this important feature of Rho1 function during morphogenesis (Magie, 2002).

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Actin filaments are shown to be required for cytokinesis in S. cerevisiae, and the actin ring is shown to be a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, the formins are shown to not be required for localization of Cyk1/Iqg1, an IQGAP-like protein that is required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring (Tolliday, 2002).

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), it has been shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. In Rat1A cells RhoA activity remains suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibits cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibits cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 is suppressed during cytokinesis and starts increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression is essential for cytokinesis because a constitutively active mutant of Rac1 induces a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was demonstrated by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delays the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells. It is concluded that RoA acitivity during cytokinesis exhibits cell type specificity (Yoshizaki, 2004).

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 (see Drosophila RhoGEF2) and MgcRacGAP (Drosophila homolog: RacGAP50C) have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. The inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells are treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis is induced precociously before chromosomal separation. Cytokinesis is also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody results in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibits BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induces excessive membrane blebbing, which appears to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolishes both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduces both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. It is proposed that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis (Niiya, 2005).

The epithelial cell transforming gene 2 protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. This study shows that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. An in vitro Cdk1 phosphorylation site (T412) has been identified in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding is inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, does not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displays a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant causes cortical hyperactivity in U2OS cells during cell division, this activity is not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert (Niiya, 2006).

During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol, it was shown that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. Both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. Centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins (Nishimura, 2006).

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors. Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. This study shows that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis (Piekny, 2008).

Various organisms might employ distinct mechanisms to recruit anillin, which executes its conserved function as a scaffold protein in the contractile ring. In both S. pombe and animal cells, anillin-like proteins are early markers of the nascent cleavage furrow. In most systems, anillin is not required for furrow formation. C. elegans have three anillin-like proteins, and ani-1 is required for contractile events in the early embryo, such as membrane ruffling and pseudocleavage, but is not strictly required for cytokinesis. Drosophila anillin is required for cellularization in early embryos and promotes late cytokinetic events in S2 cells. Fission yeast express two anillin-related proteins, Mid1p and Mid2p, which regulate proper positioning of the division plane and septation, respectively. Whereas in human cells anillin localizes via its C-terminal Rho binding domain, in S. pombe mid1p localization involves a C-terminal amphipathic helix. Interestingly, the AHD is not well conserved in mid1p, nor is there compelling evidence that RhoA triggers contractile-ring formation in S. pombe (Piekny, 2008).

Rho, oocyte maturation, and meiosis

Vertebrate oocyte maturation is an extreme form of asymmetric cell division, producing a mature egg alongside a diminutive polar body. Critical to this process is the attachment of one spindle pole to the oocyte cortex prior to anaphase. This study reports that asymmetric spindle pole attachment and anaphase initiation are required for localized cortical activation of Cdc42, which in turn defines the surface of the impending polar body. The Cdc42 activity zone overlaps with dynamic F-actin and is circumscribed by a RhoA-based actomyosin contractile ring. During cytokinesis, constriction of the RhoA contractile ring is accompanied by Cdc42-mediated membrane outpocketing such that one spindle pole and one set of chromosomes are pulled into the Cdc42 enclosure. Unexpectedly, the guanine nucleotide exchange factor Ect2, which is necessary for contractile ring formation, does not colocalize with active RhoA. Polar body emission thus requires a classical RhoA contractile ring and Cdc42-mediated membrane protrusion (K. Zhang, 2008).

Cdc42 activation is first observed at the spindle-cortex contact site shortly (within a few minutes) following anaphase initiation, suggesting that activation of Cdc42 might be temporarily and mechanistically coupled to anaphase. Attempts were made to inhibit anaphase without altering spindle assembly or the perpendicular spindle attachment to the oocyte cortex. The role of cyclin B degradation and the resultant loss of Cdk1 activity during amphibian meiosis is controversial. While it has been reported that stabilization of Cdk1 activity via prevention of cyclin B destruction does not prevent polar body formation, it has also been reported that experimental stabilization of Cdk1 by other means does block polar body emission and that this blockade can be relieved by Cdk1 inhibition. The role of cyclin B degradation on polar body emission was reinvestigated. A truncated form of cyclin B1 (ΔN cyclin B1 was employed that lacks the destruction box required for APC-targeted degradation. Injection of ΔN cyclin B1 mRNA efficiently eliminated the transient inactivation of MPF seen in control oocytes. Oocytes injected with ΔN cyclin B1 underwent progesterone-induced GVBD indistinguishably from control oocytes. However, oocytes injected with ΔN cyclin B1 mRNA failed to emit the first polar body. Similarly, injection of methylubiquitin effectively inhibited cyclin B degradation and inhibited first polar body emission (K. Zhang, 2008).

Time-lapse imaging (or 4D imaging) experiments were carried to determine the effect of ΔN cyclin B1 on Cdc42 activation in live oocytes, using eGFP-wGBD, a GFP fusion protein containing the GTPase-binding domain of WASP (wGBD) that binds only active (GTP-bound) Cdc42. Control oocytes exhibited Cdc42 activation approximately 2 hr after GVBD, emitted the first polar body, and then arrested in metaphase II indefinitely. In contrast, ΔN cyclin B1-injected oocytes failed to activate Cdc42, and the spindle remained intact and attached to the cortex for an extended period of time. Similarly, methylubiquitin-injected oocytes exhibited intact metaphase I spindles that remained asymmetrically attached to the cortex, with no Cdc42 activation (K. Zhang, 2008).

Given the controversy regarding whether the transient degradation of cyclin B is required for homolog separation in Xenopus oocytes, chromosome dynamics were observed in live oocytes injected with ΔN cyclin B1 mRNA. Fluorescent antibodies against Xenopus Aurora B were used to track endogenous Aurora B, a chromosome passenger kinase. Fluorescent anti-Aur B faithfully tracked chromosomes through the metaphase I (00:10) to metaphase II (00:52) transition. In addition, fluorescent anti-Aur B also clearly marked the central spindle or spindle midzone (MZ) at anaphase/telophase (arrows, 00:20–00:24), and the midbody following polar body emission. These data are consistent with previous conclusions based on staining of fixed oocytes of other species. It is noteworthy that in contrast to mitosis in which Aurora B is completely transferred to the central spindle at anaphase, in the oocytes Aurora B persisted with the segregated chromosome homologs. Presumably, Aurora B signal persisted at anaphase I due to the cohesion of sister centromeres, which only resolve at anaphase II upon fertilization (K. Zhang, 2008).

Having established the utility of fluorescent anti-Aur B in tracking chromosomes in live oocytes, the fate of chromosomes was followed in oocytes injected with ΔN cyclin B1. Metaphase I spindles remained asymmetrically positioned against the animal pole cortex for an extended period of time, with no homolog separation. Similarly, oocytes injected with methylubiquitin did not separate chromosome homologs. These data clearly indicate that cyclin B degradation is required for anaphase initiation and for Cdc42 activation (K. Zhang, 2008).

Rho and development

The Rho family of small GTPases regulates a variety of cellular functions, including the dynamics of the actin cytoskeleton, cell adhesion, transcription, cell growth and membrane trafficking. The first Xenopus homologs of the Rho-like GTPases, RhoA and Rnd1, have been isolated and their potential roles in early Xenopus development have been examined. Xenopus Rnd1 (XRnd1), a member of a newly discovered family of Rho GTPases) is expressed in tissues undergoing extensive morphogenetic changes, such as marginal zone cells involuting through the blastopore; somitogenic mesoderm during somite formation, and neural crest cells. XRnd1 also causes a severe loss of cell adhesion in overexpression experiments. These data and the expression pattern suggest that XRnd1 regulates morphogenetic movements by modulating cell adhesion in early embryos. Xenopus RhoA (XRhoA) is a potential XRnd1 antagonist, since overexpression of XRhoA increases cell adhesion in the embryo and reverses the disruption of cell adhesion caused by XRnd1. In addition to the potential roles of XRnd1 and XRhoA in the regulation of cell adhesion, a role for XRhoA has been found in axis formation. When coinjected with dominant-negative BMP receptor (tBR) in the ventral side of the embryo, XRhoA causes the formation of head structures resembling the phenotype seen after coinjection of wnt inhibitors with a dominant-negative BMP receptor. Since dominant-negative XRhoA is able to reduce the formation of head structures, it is proposed that XRhoA activity is essential for head formation. Thus, XRhoA may have a dual role in the embryo by regulating cell adhesion properties and pattern formation (Wunnenberg-Stapleton, 1999).

The differentiation of neural crest cells from progenitors located in the dorsal neural tube appears to involve three sequential steps: the specification of premigratory neural crest cell fate; the delamination of these cells from the neural epithelium, and the migration of neural crest cells in the periphery. BMP signaling has been implicated in the specification of neural crest cell fate but the mechanisms that control the emergence of neural crest cells from the neural tube remain poorly understood. To identify molecules that might function at early steps of neural crest differentiation, a PCR-based screen was performed to discover genes induced by BMPs in chick neural plate cells. One gene obtained from this screen, rhoB, is a member of the rho family of GTP-binding proteins. rhoB is expressed in the dorsal neural tube and its expression persists transiently in migrating neural crest cells. BMPs induce the neural expression of rhoB but not the more widely expressed rho family member, rhoA. Inhibition of rho activity by C3 exotoxin prevents the delamination of neural crest cells from neural tube explants but has little effect on the initial specification of premigratory neural crest cell fate or on the later migration of neural crest cells. These results suggest that rhoB has a role in the delamination of neural crest cells from the dorsal neural tube. Rho activity might contribute to the changes in cell shape and adhesion that occur during the delamination of neural crest cells by regulating actin polymerization and the formation of focal adhesions and stress fibers (Liu, 1998).

Rho family proteins play a critical role in muscle differentiation. The Rho family of GTP-binding proteins consists of the Rho, Rac, and Cdc42 subfamilies and has been demonstrated to regulate numerous aspects of cytoskeleton function. Rho family proteins also play a critical role in transcriptional regulation of the c-fos gene by modulating the transcription factor SRF. Since SRF also binds to the CArG box, which is a critical cis element in the promoters of many muscle-specific genes, an examination was made to determine whether the Rho family plays an important role in the expression of muscle-specific genes. To test whether Rho family G proteins are involved in transcription of muscle-specific genes, the role of Rho family proteins was examined in the transcription of the skeletal alpha-actin gene, which depends on the CArG box. A luciferase reporter plasmid containing the skeletal alpha-actin promoter (bp -394 to +24) was transiently transfected into C2C12 myoblasts together with dominant interfering mutants of Rac1 (Rac1N17), RhoA (RhoAN17), and Cdc42 (Cdc42N17) or the GDP dissociation inhibitor RhoGDI. Luciferase activities were measured at 36 h after induction of C2C12 cell differentiation (by changing the culture medium from GM to DM). The transcriptional activity of the skeletal alpha-actin gene is much higher in differentiated myotubes than in undifferentiated myoblasts and is reduced by cotransfection of either dominant interfering plasmid and RhoGDI. RhoGDI, which inhibits the functions of all Rho family members, most strongly inhibits the transcriptional activity of the skeletal alpha-actin gene during muscle differentiation; among the three dominant interfering mutants the inhibitory activity is strongest in Rac1N17. Transfection of these interfering mutants of the Rho family proteins and RhoGDI do not show such a strong inhibitory effect on transcription of nonmuscle gene promoters such as the SV40-derived promoter. The effects of wild-type and mutationally activated forms of Rho family G proteins (wild-type Rac1, Rac1V12, RhoAV12, or Cdc42V12) were tested on the activity of the skeletal alpha-actin promoter. Although overexpression of wild-type Rac1 has no significant effects on the activity of the skeletal alpha-actin promoter, all constitutively active mutants of Rho family proteins activate the promoter to various degrees. Rac1V12 most strongly activates the transcription, by more than 10-fold. It is concluded that dominant negative forms of Rho family proteins and RhoGDI, a GDP dissociation inhibitor, suppress transcription of muscle-specific genes, while mutationally activated forms of Rho family proteins strongly activate their transcription (Takano, 1998).

C2C12 cells overexpressing RhoGDI (C2C12RhoGDI cells) do not differentiate into myotubes, and expression levels of myogenin, MRF4, and contractile protein genes (but not MyoD and myf5 genes) are markedly reduced in C2C12RhoGDI cells. The promoter activity of the myogenin gene is suppressed by dominant negative mutants of Rho family proteins and is reduced in C2C12RhoGDI cells. Expression of myocyte enhancer binding factor 2 (MEF2), which has been reported to be required for the expression of the myogenin gene, is reduced at the mRNA and protein levels in C2C12RhoGDI cells. These results suggest that the Rho family proteins play a critical role in muscle differentiation, possibly by regulating the expression of the myogenin and MEF2 genes (Takano, 1998).

The first detectable expression of smooth muscle cell-specific proteins during coronary smooth muscle cell (CoSMC) differentiation from isolated proepicardial cells is restricted to cells undergoing epithelial-to-mesenchymal transformation (EMT). This study examines the relation between actin cytoskeletal rearrangements and serum response factor (SRF)-dependent transcription, and specifically tests whether rhoA-GTPase signaling is required for CoSMC differentiation. PDGF B chain homodimer (PDGF-BB) stimulates EMT and promotes SRF-dependent expression of SMC marker genes calponin, SM22alpha, and SMgammaactin (SMgammaA) in proepicardial cells. C3 exoenzyme or rhoGDI, inhibitors of rhoA signaling, blocks PDGF-BB-induced EMT, prevents actin reorganization into stress fibers, and inhibits CoSMC differentiation. Incubation with the selective p160 rho-kinase (p160RhoK) inhibitor Y27632 (RKI) blocks EMT, prevents the appearance of calponin and SMgammaA-positive cells, and abolishes expression and nuclear localization of SRF. To test the role of RhoK signaling for CoSMC differentiation in vivo, quail proepicardial organs (PEOs) were pretreated with RKI and then grafted into age-matched host chick embryos to produce a chimeric epicardium. The ability of grafted cells to participate in coronary vessel formation was monitored by staining with antibodies for quail cell nuclear antigen and SMC marker proteins. Proepicardial cells pretreated with RKI fail to form CoSMCs in vivo. Time course studies trace this deficiency to a failure of epicardial-derived mesenchymal cells to migrate into or survive within the myocardium. In summary, these data point to important roles for rhoA-RhoK signaling in molecular pathways controlling cytoskeletal reorganization, SRF-dependent transcription, and cell survival that are required to produce CoSMCs from proepicardial cells (Lu, 2001).

A reverse genetic approach has been employed to explore the role of Rho GTPases in murine cardiac development. Cardiac-specific inhibition of Rho family protein activities was achieved by expressing Rho GDIalpha, a specific GDP dissociation inhibitor for Rho family proteins, using the alpha-myosin heavy chain promoter, active at embryonic day (E) 8.0 during morphogenesis of the linear heart tube. RhoA, Rac1 and Cdc42 activities were significantly inhibited, as shown by decreased membrane translocation of these proteins in the transgenic hearts. Transgenic F1 mice for each of two independent lines expressing the highest levels of the transgene, died around E10.5. Homozygotes of the middle copy-number lines, in which Rho GDIalpha expression was increased four-fold over normal levels, were also embryonic lethal. Cardiac morphogenesis in these embryos was disrupted, with incomplete looping, lack of chamber demarcation, hypocellularity and lack of trabeculation. Cell proliferation was inhibited in the transgenic hearts, as shown by immunostaining with anti-phosphohistone H3, a marker of mitosis. In addition, ventricular hypoplasia was associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases, and with down-regulation of cyclin A, while cell survival was not affected. These results reveal new biological functions for Rho family proteins as essential determinants of cell proliferation signals at looping and chamber maturation stages in mammalian cardiac development (Wei, 2002).

A detailed analysis of Rho GTPase function during vertebrate development has been undertaken by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue are clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, are controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contribute separately to cell shape and filopodia formation. It is proposed that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension (Tahinci, 2003).

Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. The Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis 'decision.' Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision (Sordella, 2003).

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread undergo osteogenesis, while unspread, round cells became adipocytes. Cell shape regulates the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA commits hMSCs to become adipocytes, while constitutively active RhoA causes osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis is conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induces osteogenesis independent of cell shape. This RhoA-ROCK commitment signal requires actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate (McBeath, 2003).

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, it was found that ADF/cofilin is dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induces dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation is inhibited by Na3VO4 but not by other conventional phosphatase-inhibitors. A Xenopus homologue of Slingshot phosphatase (XSSH) was cloned, and antibody was raised specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppresses the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH binds to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity is increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and led to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation (Tanaka, 2005).

Rho-dependent amoeboid cell movement is a crucial mechanism in both tumor cell invasion and morphogenetic cell movements during fish gastrulation. Amoeboid movement is characterized by relatively non-polarized cells displaying a high level of bleb-like protrusions. During gastrulation, zebrafish mesodermal cells undergo a series of conversions from amoeboid cell behaviors to more mesenchymal and finally highly polarized and intercalative cell behaviors. This study demonstrates that Myosin phosphatase, a complex of Protein phosphatase 1 and the scaffolding protein Mypt1, functions to maintain the precise balance between amoeboid and mesenchymal cell behaviors required for cells to undergo convergence and extension. Importantly, Mypt1 has different cell-autonomous and non-cell-autonomous roles. Loss of Mypt1 throughout the embryo causes severe convergence defects, demonstrating that Mypt1 is required for the cell-cell interactions involved in dorsal convergence. By contrast, mesodermal Mypt1 morphant cells transplanted into wild-type hosts undergo dorsally directed cell migration, but they fail to shut down their protrusive behavior and undergo the normal intercalation required for extension. It was further shown that Mypt1 activity is regulated in embryos by Rho-mediated inhibitory phosphorylation, which is promoted by non-canonical Wnt signaling. It is proposed that Myosin phosphatase is a crucial and tightly controlled regulator of cell behaviors during gastrulation and that understanding its role in early development also provides insight into the mechanism of cancer cell invasion (Weiser, 2009).

Rho function during fertilization

Sperm-egg interaction was investigated in mouse eggs freed from the zona pellucida and injected with Clostridium difficile toxin B, the inhibitor of Rho family small G proteins. Toxin B reduces in a dose-dependent manner the percentage of eggs associated with sperm fusion on the surface or sperm nucleus decondensation in the ooplasm. The mean number of decondensed sperm nuclei per egg was remarkably decreased by ~1 micrograms/ml toxin B in the ooplasm. This was because spermatozoa were arrested at the fusion state without developing to sperm incorporation and tended to lose cytoplasmic continuity to the egg. The fusion-arrested spermatozoa caused transient small Ca2+ oscillations in most of eggs, while an injected spermatozoon produced repetitive large Ca2+ spikes unaffected by toxin B. A decrease in the rate of fused spermatozoa and decondensed sperm nuclei was also caused by 20-40 microM cytochalasin D, the inhibitor of actin polymerization. Immunostaining of Rho proteins shows that Rac1 and RhoB are present in the cortical ooplasm, but Cdc42 is absent. Actin filaments in the cortex appear to be reduced in toxin B-injected eggs. This study suggests that Rho protein(s) regulating actin-based cytoskeletal reorganization is involved in the process leading to sperm incorporation during fertilization of mouse eggs

Rho and axon extension and regeneration

Axons in the CNS of mammals do not regenerate after injury, and one barrier to regeneration is growth inhibition by CNS myelin. Myelin inhibits axon growth because it contains several different growth inhibitory proteins. Myelin-associated glycoprotein (MAG) inhibits axon growth both in vitro and in vivo. Also, a different high molecular weight inhibitory activity is present in myelin. To foster regeneration, a strategy to block the neuronal response to growth inhibitory signals has been investigated: injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme (C3 enzyme from Clostridium botulinum selectively ADP-ribosylates Rho in its effector domain without affecting Rac and Cdc42) to inactivate Rho and transfection with dominant negative Rho, allows neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extend neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, optic nerves of adult rat were crushed. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates (Lehmann, 1999).

Rac and Rho may have opposite effects on neurite growth: inactivation of Rho stimulates rapid neurite outgrowth, whereas activation of Rac stimulates neurite extension. Rho and Rac may have additive effects on growth cone morphology, with activated Rho and inactive Rac cooperating to give a spread out growth cone morphology, with lower rates of growth. Activation of Rho prevents growth cone collapse by myelin, but growth cone morphology is not always predictive of the growth state. Rapid neurite elongation in the presence of C3 occurs with a collapsed growth cone morphology, and in vivo, rapidly extending axons are bullet-shaped. Possibly, the prevention of myelin-derived growth cone collapse by activated Rho reflects the cooperative effects of Rac and Rho on growth cone morphology (Lehmann, 1999 and references).

Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. Multimode fluorescent speckle microscopy (FSM) was used to directly address this problem. Actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, Lysophosphatidic acid (LPA) mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration (Zhang, 2003).

Two actin-based structures, filopodia and actin arcs, had profound effects on MT organization and dynamics in the growth cone. Polarized actin bundles in filopodia guide MT growth into the peripheral (P) domain and simultaneously transport MTs rearward by retrograde actin flow. The second structure, actin arcs, appear in the T zone and move into the C domain, where they contribute to central actin bundle structure. Interestingly, MTs associated with actin arcs are less dynamic than those in the P domain, exhibiting prolonged periods of slow growth due to dramatically reduced catastrophe frequencies. Arcs have been identified as a novel motile actin structure in growth cones (Zhang, 2003).

FSM was used to investigate effects of altering Rho GTPase activity on cytoskeletal dynamics in growth cones. Actin arcs and central actin bundles derived from arcs are key cytoskeletal effectors of Rho and ROCK. Rho activity affects both the stability and contractility of actin arcs and strongly affects MT behavior as a consequence of arc-MT interactions. When cells were plated on laminin substrates, growth cone retractions were observed in response to Rho activation by LPA. Interestingly, these well-described chemorepellant responses do not involve changes in peripheral retrograde actin flow or filopodium number, but rather are driven by the contraction of more central actin structures (Zhang, 2003).

During embryonic development, tangentially migrating precerebellar neurons emit a leading process and then translocate their nuclei inside it (nucleokinesis). Netrin 1 (also known as netrin-1) acts as a chemoattractant factor for neurophilic migration of precerebellar neurons (PCN) both in vivo and in vitro. In the present work, Rho GTPases that could direct axon outgrowth and/or nuclear migration were analyzed. The expression pattern of Rho GTPases in developing PCN is consistent with their involvement in the migration of PCN from the rhombic lips. Pharmacological inhibition of Rho enhances axon outgrowth of PCN and prevents nuclei migration toward a netrin 1 source, whereas inhibition of Rac and Cdc42 sub-families impairs neurite outgrowth of PCN without affecting migration. Through pharmacological inhibition, it has been shown that Rho signaling directs neurophilic migration through Rock activation. Altogether, these results indicate that Rho/Rock acts on signaling pathways favoring nuclear translocation during tangential migration of PCN. Thus, axon extension and nuclear migration of PCN in response to netrin 1 are not strictly dependent processes because: (1) distinct small GTPases are involved; (2) axon extension can occur when migration is blocked, and (3) migration can occur when axon outgrowth is impaired (Causeret, 2004).

Rho and activity-dependent dendritic growth

Studies suggest that neuronal activity may guide the development of synaptic connections in the central nervous system through mechanisms involving glutamate receptors and GTPase-dependent modulation of the actin cytoskeleton. In vivo time-lapse imaging of optic tectal cells in Xenopus laevis tadpoles demonstrates that enhanced visual activity driven by a light stimulus promotes dendritic arbor growth. The stimulus-induced dendritic arbor growth requires glutamate-receptor-mediated synaptic transmission, decreased RhoA activity and increased Rac and Cdc42 activity. The results delineate a role for Rho GTPases in the structural plasticity driven by visual stimulation in vivo (Sin, 2002).

In vivo time-lapse imaging of single optic tectal neurons in Xenopus tadpoles was used to test the function of visual activity in neuronal development. The dendritic arbor growth rates of individual tectal neurons during a 4-h period with a visual stimulus were compared to a preceding 4-h period in the absence of light. This imaging protocol allows the comparison of dendritic arbor structures of the same neurons over time and therefore provides a sensitive measure of structural plasticity. Visual stimulation significantly enhances dendritic arbor elaboration compared with growth rates in the preceding 4-h period in the dark. Neurons from animals exposed to visual stimulus throughout the 8-h protocol maintain a constant rate of dendritic arbor elaboration. This indicates that growth rates do not change with longer periods of stimulation. Another set of animals was exposed to visual stimulus within the first 4-h period and then returned to the dark for the second 4-h period. Notably, the enhanced growth rates of these neurons in response to light stimulation were maintained during the 4-h dark period. This suggests that exposure to visual stimulation triggers mechanisms that enhance dendritic arbor growth over a period following termination of the stimulus (Sin, 2002).

Dendritic arbor development may proceed through repeated branching of dendritic growth cones or through a mechanism called 'interstitial/back branching' in which new branches emerge from more stable branches within the arbor. Tectal projection neurons imaged in this study have a single stable primary dendrite, an average of 12 secondary branches originating from the primary dendrite, and complex arborizations with up to fifth-order branches. Light stimulus significantly increases the number of secondary branches, indicating that interstitial branching is a prominent mechanism controlling dendritic arbor elaboration in response to visual stimulation in vivo. Expression of dominant-negative RacN17, Cdc42N17, RhoAN19 or constitutively active RhoAV14 prevents the light-induced increase in secondary branches. This indicates that the GTPases regulate the formation of interstitial branches in response to visual stimulation (Sin, 2002).

The data indicate that visual system activity affects dendritic arbor elaboration through a mechanism involving Rho GTPases. Expression of mutant GTPases that interfere with endogenous GTPases blocks light-induced dendritic arbor development. It is suggested that enhanced Rac and Cdc42 activity promotes branch dynamics. Decreased Rho activity promotes branch elongation mediated by several downstream effectors, including ROK. Rac- and Cdc42-mediated branch addition and stabilization and RhoA-mediated branch elongation cooperate to result in dendritic arbor growth (Sin, 2002 and references therein).

The data demonstrating that NMDAR activity and GTPases are both required for dendritic arbor development suggest that calcium influx through NMDAR affects regulators of Rho GTPase activity. Such regulation may occur through GTPase regulators or effectors such as SynGAP, kalarin or trio. The Rho GTPases may also affect intracellular calcium levels by regulating calcium influx into cells or calcium-induced calcium release from intracellular stores. Calcium-dependent events, including calcium- and calmodulin-dependent kinase type II (CaMKII)-mediated branch stabilization, may cooperate with GTPase-mediated branch initiation and extension to control dendritic arbor development. These results indicate that visual stimulation affects structural plasticity in vivo, through NMDA receptor-induced intracellular signaling events mediated by the Rho GTPases (Sin, 2002 and references therein).

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. This study shows that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, it was further shown that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity (H. Zhang, 2008).

Rho and apoptosis

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Primary murine bone marrow-derived macrophages (using alphavß3 integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton. Inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process (Leverrier, 2001).

Rho and cancer

The most damaging change during cancer progression is the switch from a locally growing tumor to a metastatic killer. This switch is believed to involve numerous alterations that allow tumor cells to complete the complex series of events needed for metastasis1. Relatively few genes have been implicated in these events. An in vivo selection scheme has been used to select highly metastatic melanoma cells. By analyzing these cells on DNA arrays, a pattern of gene expression has been defined that correlates with progression to a metastatic phenotype. In particular, enhanced expression has been shown for several genes involved in extracellular matrix assembly and for a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumor cell invasion. The genomic approach allows the identification of families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis (Clark, 2000).

Transformation by oncogenic Ras requires the function of the Rho family GTPases. Ras-transformed cells have elevated levels of RhoA-GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho-GTP are not a direct consequence of Ras signaling but are selected for in response to sustained ERK-MAP kinase signaling. While the elevated levels of Rho-GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibers in transformed cells. The sustained ERK-MAP kinase signaling resulting from transformation by oncogenic Ras down-regulates ROCK1 and Rho-kinase, two Rho effectors required for actin stress fiber formation. The repression of Rho-dependent stress fiber formation by ERK-MAP kinase signaling contributes to the increased motility of Ras-transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibers and inhibits the motility of Ras-transformed fibroblasts. A model is proposed in which Ras and Rho signaling pathways cross-talk to promote signaling pathways favoring transformation (Sahai, 2001).

Expression of p21/Waf1 following mitogenic stimulation is dependent on the ERK-MAP kinase pathway. In the control of cell cycle progression, p21/Waf1 has a dual role: it serves as an assembly factor for active complexes of D-type cyclins and their cyclin-dependent kinases (CDKs) and is an inhibitor of CDK2. In studies employing transient assays of Ras-driven proliferation, the absence of signaling through RhoA results in the Ras-driven ERK-MAP kinase pathway, inducing levels of p21/Waf1 that are inhibitory to cell cycle progression. signaling through RhoA is required to suppress growth inhibitory levels of p21/Waf1 in Ras-transformed Swiss-3T3 cells and in two human colorectal cancer cell lines. In these human cell lines, inhibition of the Raf/MEK/ERK and PI-3-kinase Ras effector pathways does not affect Rho-GTP levels, thus supporting the model that Rho activity is determined by selection; however, the levels of Rho-GTP in these cells compared with untransformed cells could not be determined due to the lack of genotypically matched controls. It is proposed that cells with high levels of Rho activity are selected for because they counteract the high levels of p21/Waf1 induced by oncogenic Ras and proliferate, while cells with low Rho activity remain growth arrested by high p21/Waf1 levels. Interestingly, the Ras-transformed Swiss-3T3 cells have higher levels of p21/Waf1 than parental non-transformed cells. However, these cells proliferate presumably because the levels of p21/Waf1 are such that they enable the assembly of active cyclin D-CDK complexes rather than inhibit CDK2. It is proposed that the elevated levels of Rho-GTP in the transformed cells set a threshold level of p21/Waf1 that is compatible with proliferation (Sahai, 2001).

Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. This study shows that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell-cell contact. When actomyosin is not downregulated at cell-cell contacts, migrating cells lose cohesion. A molecular mechanism is provided for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1-Par3/Par6 complex controls the localization of RhoE to cell-cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell-cell contacts, a loss of cell-cell cohesion and defective collective cell invasion (Hidalgo-Carcedo, 2011).

Collective movement requires the coordination of actomyosin organization between cells. Actomyosin contractility is high around the edge of the cell cluster and low between cells. At the margin of the group, actomyosin is organized in a supracellular structure analogous to the 'purse-string' observed in epithelial wound closure. Both the elevated actomyosin levels observed around the edges of groups of invading cancer cells and 'purse-string' wound closure are dependent on Cdc42. Force is transmitted between cells through cell-cell contacts near the edge of the group. However, if force is applied uniformly around the cell margin, the cell junctions become compromised and the coordination of movement between neighbouring cells fails. Consistent with this, contact inhibition of locomotion and cell-cell repulsion are associated with increased Rho-driven actomyosin contraction function after cell-cell contact. Therefore a mechanism is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. The localization of DDR1 to cell-cell contacts requires E-cadherin. Once localized at cell-cell contacts, DDR1 helps to recruit Par3 and Par6; these molecules are required for efficient collective invasion. Cell polarity regulators are required for optimal migration in two-dimensional scratch/wound assays, which have some aspects of a collective nature. Moreover, Par3 and Par6 are required for collective migration of border cells in the Drosophila embryo41. The DDR1-Par3/Par6 complex then controls the localization of RhoE. RhoE may be localized through the intermediary p190ARhoGAP, which can bind both Par6 and RhoE. Consistent with this, depletion of p190ARhoGAP gave a similar phenotype to that after DDR1 or Par3/Par6 depletion. Both RhoE and p190ARhoGAP can antagonize Rho-ROCK-mediated regulation of actomyosin. The Par3-dependent suppression of actomyosin that was observe reciprocates the suppression of Par3 function by the actomyosin regulator ROCK. It is likely that the reciprocal nature of these negative interactions serves to segregate Par3 and actomyosin robustly. The DDR1-dependent mechanism that is describe most probably acts together with other proteins to decrease Rho-ROCK function at cell-cell contacts, such as p120 catenin-dependent mechanisms. This analysis has not yet allowed determination of all the components of DDR1 complexes at cell-cell contacts (Hidalgo-Carcedo, 2011).

Various regulators of cell polarity become misregulated in cancer; this has been linked to increased metastasis. It is believed that disruption of DDR1-dependent Par3 localization to cell-cell contacts might be expected to favour blood-borne metastasis. The data do not exclude a positive role for DDR1 in metastasis as a collagen receptor. Indeed, it was found that interference with DDR1 function in metastatic MTLn3 cells decreased their ability to colonize lung tissue. DDR1 expression may therefore not correlate simply with metastatic ability, but it is important to consider whether it is acting in a cell-matrix or cell-cell adhesion context: in the former it may promote single-cell cancer invasion and processes such as lung colonization; in the latter it may only promote more local and lymphatic invasion and hinder haematogenous metastasis. It is likely that DDR1 engages in different molecular complexes depending on whether it is involved in cell-cell interactions or cell-matrix interactions. For example, the data suggest that DDR1 does not associate with myosin IIa at cell-cell contacts but it has been reported to associate with myosin IIa in other contexts (Hidalgo-Carcedo, 2011).

This study described a mechanism that is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. DDR1 helps to recruit Par3 and Par6; this complex then controls the localization of RhoE, which can antagonize Rho-ROCK-mediated regulation of actomyosin. Thus, DDR1 functions at cell-cell contacts to keep actomyosin activity at low levels. Without this decrease in actomyosin activity, cell cohesion cannot be maintained during collective cell migration (Hidalgo-Carcedo, 2011).

Table of contents

Rho1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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