Two distinct modes of myosin assembly and dynamics during epithelial wound closure

Actomyosin contraction powers the sealing of epithelial sheets during embryogenesis and wound closure; however, the mechanisms are poorly understood. After laser ablation wounding of Madin-Darby canine kidney cell monolayers, we observed distinct steps in wound closure from time-lapse images of myosin distribution during resealing. Immediately upon wounding, actin and myosin II regulatory light chain accumulated at two locations: (1) in a ring adjacent to the tight junction that circumscribed the wound and (2) in fibers at the base of the cell in membranes extending over the wound site. Rho-kinase activity was required for assembly of the myosin ring, and myosin II activity was required for contraction but not for basal membrane extension. As it contracted, the myosin ring moved toward the basal membrane with ZO-1 and Rho-kinase. Thus, we suggest that tight junctions serve as attachment points for the actomyosin ring during wound closure and that Rho-kinase is required for localization and activation of the contractile ring.     

A Rho-dependent signaling pathway operating through myosin localizes beta-actin mRNA in fibroblasts

BACKGROUND: The sorting of mRNA is a determinant of cell asymmetry. The cellular signals that direct specific RNA sequences to a particular cellular compartment are unknown. In fibroblasts, beta-actin mRNA has been shown to be localized toward the leading edge, where it plays a role in cell motility and asymmetry. RESULTS: We demonstrate that a signaling pathway initiated by extracellular receptors acting through Rho GTPase and Rho-kinase regulates this spatial aspect of gene expression in fibroblasts by localizing beta-actin mRNA via actomyosin interactions. Consistent with the role of Rho as an activator of myosin, we found that inhibition of myosin ATPase, myosin light chain kinase (MLCK), and the knockout of myosin II-B in mouse embryonic fibroblasts all inhibited beta-actin mRNA from localizing in response to growth factors. CONCLUSIONS: We therefore conclude that the sorting of beta-actin mRNA in fibroblasts requires a Rho mediated pathway operating through a myosin II-B-dependent step and postulate that polarized actin bundles direct the mRNA to the leading edge of the cell.     

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension

Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3–5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.     

Patterned gene expression directs bipolar planar polarity in Drosophila

During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. Here, we demonstrate that A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly

Laminin-5 is a major structural element of epithelial tissue basement membranes. In the matrix of cultured epithelial cells, laminin-5 is arranged into intricate patterns. Here we tested a hypothesis that myosin II-mediated actin contraction is necessary for the proper assembly of a laminin-5 matrix by cultured SCC12 epithelial cells. To do so, the cells were treated with ML-7, a myosin II light chain kinase inhibitor, or Y-27632, an inhibitor of Rho-kinase (ROCK), both of which block actomyosin contraction. Under these conditions, laminin-5 shows an aberrant localization in dense patches at the cell periphery. Since ROCK activity is regulated by the small GTPase Rho, this suggests that members of the Rho family of GTPases may also be important for laminin-5 matrix assembly by SCC12 cells. We confirmed this hypothesis since SCC12 cells expressing mutant proteins that inhibit RhoA, Rac, and Cdc42 assemble the same aberrant laminin-5 protein arrays as drug-treated cells. We have also evaluated the organization of the laminin-5 receptors alpha3beta1 and alpha6beta4 integrin and hemidesmosome proteins in ML-7- and Y-27632-treated cells or in cells in which RhoA, Rac, and Cdc42 activity were inhibited. In all instances, alpha3beta1 and alpha6beta4 integrin heterodimers, as well as hemidesmosome proteins, localize precisely with laminin-5 in the matrix of the cells. In summary, our results provide evidence that myosin II-mediated actin contraction and the activity of Rho GTPases are necessary for the proper organization of a laminin-5 matrix and localization of hemidesmosome protein arrays in epithelial cells.     

Actomyosin sliding is attenuated in contractile biomimetic cortices

Myosin II motors embedded within the actin cortex generate contractile forces to modulate cell shape in essential behaviors, including polarization, migration, and division. In sarcomeres, myosin II–mediated sliding of antiparallel F-actin is tightly coupled to myofibril contraction. By contrast, cortical F-actin is highly disordered in polarity, orientation, and length. How the disordered nature of the actin cortex affects actin and myosin movements and resultant contraction is unknown. Here we reconstitute a model cortex in vitro to monitor the relative movements of actin and myosin under conditions that promote or abrogate network contraction. In weakly contractile networks, myosin can translocate large distances across stationary F-actin. By contrast, the extent of relative actomyosin sliding is attenuated during contraction. Thus actomyosin sliding efficiently drives contraction in actomyosin networks despite the high degree of disorder. These results are consistent with the nominal degree of relative actomyosin movement observed in actomyosin assemblies in nonmuscle cells.     

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia.     

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.     

E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens

In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell–cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

A Highlights from MBoC Selection: CGEF-1 and CHIN-1 Regulate CDC-42 Activity during Asymmetric Division in the Caenorhabditis elegans Embryo

The anterior–posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.     

Modulation of epithelial tubule formation by Rho kinase

We have developed a model system for studying integrin regulation of mammalian epithelial tubule formation. Application of collagen gel overlays to Madin-Darby canine kidney (MDCK) cells induced coordinated disassembly of junctional complexes that was accompanied by lamellipodia formation and cell rearrangement (termed epithelial remodeling). In this study, we present evidence that the Rho signal transduction pathway regulates epithelial remodeling and tubule formation. Incubation of MDCK cells with collagen gel overlays facilitated formation of migrating lamellipodia with membrane-associated actin. Inhibitors of myosin II and actin prevented lamellipodia formation, which suggests that actomyosin function was involved in regulation of epithelial remodeling. To determine this, changes in myosin II distribution, function, and phosphorylation were studied during epithelial tubule biogenesis. Myosin II colocalized with actin at the leading edge of lamellipodia thereby providing evidence that myosin is important in epithelial remodeling. This possibility is supported by observations that inhibition of Rho kinase, a regulator of myosin II function, alters formation of lamellipodia and results in attenuated epithelial tubule development. These data and those demonstrating myosin regulatory light-chain phosphorylation at the leading edge of lamellipodia strongly suggest that Rho kinase and myosin II are important modulators of epithelial remodeling. They support a hypothesis that the Rho signal transduction pathway plays a significant role in regulation of epithelial tubule formation. signaling pathway; polarity     

WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation

The Wnt-PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the re-organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide-exchange factors (GEFs), but the identity of the GEF involved in Wnt-PCP-mediated convergent extension is unknown. Here we report the identification of the weak-similarity GEF (WGEF) gene by a microarray-based screen for notochord enriched genes, and show that WGEF is involved in Wnt-regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant-negative Wnt-11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho-associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled-7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam-1, and deletion of the Dishevelled-binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt-PCP pathway that connects Dishevelled to Rho activation.     

Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell–cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.     

Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination

After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination.     

PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.     

The scaffold protein POSH regulates axon outgrowth

How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth. Complementation analysis reveals a requirement for the third Src homology (SH) 3 domain of POSH, and we find that the actomyosin regulatory protein Shroom3 interacts with this domain of POSH. Inhibition of Shroom3 expression by RNAi leads to increased process lengths, as observed for POSH RNAi, suggesting that POSH and Shroom function together to inhibit process outgrowth. Complementation analysis and interference of protein function by dominant-negative approaches suggest that Shroom3 recruits Rho kinase to inhibit process outgrowth. Furthermore, inhibition of myosin II function reverses the POSH or Shroom3 RNAi phenotype, indicating a role for myosin II regulation as a target of the POSH–Shroom complex. Collectively, these results suggest that the molecular scaffold protein POSH assembles an inhibitory complex that links to the actin–myosin network to regulate neuronal process outgrowth.     

Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis

Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.     

Myosin II and mechanotransduction: a balancing act

Adherent cells respond to mechanical properties of the surrounding extracellular matrix. Mechanical forces, sensed at specialized cell-matrix adhesion sites, promote actomyosin-based contraction within the cell. By manipulating matrix rigidity and adhesion strength, new roles for actomyosin contractility in the regulation of basic cellular functions, including cell proliferation, migration and stem cell differentiation, have recently been discovered. These investigations demonstrate that a balance of forces between cell adhesion on the outside and myosin II-based contractility on the inside of the cell controls many aspects of cell behavior. Disturbing this balance contributes to the pathogenesis of various human diseases. Therefore, elaborate signaling networks have evolved that modulate myosin II activity to maintain tensional homeostasis. These include signaling pathways that regulate myosin light chain phosphorylation as well as myosin II heavy chain interactions.