Stress-induced alignment of actin filaments and the mechanics of cytogel

Experimental evidence suggests that anisotropic stress induces alignment of intracellular actin filaments. We develop a model for this phenomenon, which includes a parameter reflecting the sensitivity of the microfilament network to changes in the stress field. When applied to a uniform cell sheet at rest, the model predicts that for sufficiently large values of the sensitivity parameter, all the actin filaments will spontaneously align in a single direction. Stress alignment can also be caused by a change in external conditions, and as an example of this we apply our model to the initial response of embryonic epidermis to wounding. Our solutions in this case are able to reflect the actin cable that has been found at the wound edge in recent experiments; the cable consists of microfilaments aligned with stress at the wound boundary of the epithelium. These applications suggest that stress-induced alignment of actin filaments could play a key role in some biological systems. This is the first attempt to include the alignment phenomenon in a mechanical model of cytogel.     

Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding

Epithelial wound healing relies on tissue movements and cell shape changes. Our work shows that, immediately after wounding, there was a dramatic cytoskeleton remodeling consisting of a pulse of actomyosin filaments that assembled in cells around the wound edge and flowed from cell to cell toward the margin of the wound. We show that this actomyosin flow was regulated by Diaphanous and ROCK and that it elicited a wave of apical cell constriction that culminated in the formation of the leading edge actomyosin cable, a structure that is essential for wound closure. Calcium signaling played an important role in this process, as its intracellular concentration increased dramatically immediately after wounding, and down-regulation of transient receptor potential channel M, a stress-activated calcium channel, also impaired the actomyosin flow. Lowering the activity of Gelsolin, a known calcium-activated actin filament–severing protein, also impaired the wound response, indicating that cleaving the existing actin filament network is an important part of the cytoskeleton remodeling process.     

Healing of incisional wounds in the embryonic chick wing bud: characterization of the actin purse-string and demonstration of a requirement for Rho activation

Small skin wounds in the chick embryo do not heal by lamellipodial crawling of cells at the wound edge as a skin wound does in the adult, but rather by contraction of an actin purse-string that rapidly assembles in the front row of epidermal cells (Martin, P., and J. Lewis. 1992. Nature (Lond.). 360:179-183). To observe the early time course of actin purse-string assembly and to characterize other cytoskeletal components of the contractile machinery, we have followed the healing of incisional or slash wounds on the dorsum of the chick wing; these wounds take only seconds to create and heal within approximately 6 h. Healing of the epithelium depends on a combination of purse-string contraction and zipper-like closure of the gap between the cut edges of the epithelium. Confocal laser scanning microscope studies show that actin initially aligns into a cable at the wound margin in the basal layer of the epidermis within approximately 2 min of wounding. Coincident with actin cable assembly, we see localization of cadherins into clusters at the wound margin, presumably marking the sites where segments of the cable in adjacent cells are linked via adherens junctions. A few minutes later we also see localization of myosin II at the wound margin, as expected if myosin is being recruited into the cable to generate a contractile force for wound healing. At the time of wounding, cells at the wound edge become transiently leaky, allowing us to load them with reagents that block the function of two small GTPases, Rho and Rac, which recently have been shown to play key roles in reorganiztion of the actin cytoskeleton in tissue-culture cells (Hall, A. 1994. Annu. Rev. Cell Biol. 10:31-54). Loading wound edge epidermal cells with C3 transferase, a bacterial exoenzyme that inactivates endogenous Rho, prevents assembly of an actin cable and causes a failure of healing. No such effects are seen with N17rac, a dominant inhibitory mutant Rac protein. These findings support the view that in this system the actin cable is required for healing-both the purse-string contraction and the zipping up-and that Rho is required for formation of the actin cable.     

Endocytosis-dependent coordination of multiple actin regulators is required for wound healing

The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form “signaling centers” along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.     

A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ~158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response.     

Model of For3p-Mediated Actin Cable Assembly in Fission Yeast

Formin For3p nucleates actin cables at the tips of fission yeast cells for polarized cell growth. The results of prior experiments have suggested a possible mechanism for actin cable assembly that involves association of For3p near cell tips, For3p-mediated actin polymerization, retrograde flow of actin cables toward the cell center, For3p dissociation from cell tips, and cable disassembly. We used analytical and computational modeling to test the validity and implications of the proposed coupled For3p/actin mechanism. We compared the model to prior experiments quantitatively and generated predictions for the expected behavior of the actin cable system upon changes of parameter values. We found that the model generates stable steady states with realistic values of rate constants and actin and For3p concentrations. Comparison of our results to previous experiments monitoring the FRAP of For3p-3GFP and the response of actin cables to treatments with actin depolymerizing drugs provided further support for the model. We identified the set of parameter values that produces results in agreement with experimental observations. We discuss future experiments that will help test the model''s predictions and eliminate other possible mechanisms. The results of the model suggest that flow of actin cables may establish actin and For3p concentration gradients in the cytoplasm that could be important in global cell patterning.     

Wound healing recapitulates morphogenesis in Drosophila embryos

The capacity to repair a wound is a fundamental survival mechanism that is activated at any site of damage throughout embryonic and adult life. To study the cell biology and genetics of this process, we have developed a wounding model in Drosophila melanogaster embryos that allows live imaging of rearrangements and changes in cell shape, and of the cytoskeletal machinery that draws closed an in vivo wound. Using embryos expressing green fluorescent protein (GFP) fusion proteins, we show that two cytoskeletal-dependent elements -- an actin cable and dynamic filopodial/lamellipodial protrusions -- are expressed by epithelial cells at the wound edge and are pivotal for repair. Modulating the activities of the small GTPases Rho and Cdc42 demonstrates that these actin-dependent elements have differing cellular functions, but that either alone can drive wound closure. The actin cable operates as a 'purse-string' to draw the hole closed, whereas filopodia are essential for the final 'knitting' together of epithelial cells at the end of repair. Our data suggest a more complex model for epithelial repair than previously envisaged and highlight remarkable similarities with the well-characterized morphogenetic movement of dorsal closure in Drosophila.     

Polarization and Movement of Keratocytes: A Multiscale Modelling Approach

Eukariotic cell motility is a complex phenomenon, in which the cytoskeleton and its major constituent, actin, play an essential role. Actin forms polymers of long, stiff filaments that are cross-linked into an anisotropic network inside a thin sheet-like cellular protrusion, the lamellipod. At the leading edge of this structure, polymerization of actin filaments creates the force that pushes out the membrane and leads to translocation of a motile cell. Dynamics of the actin network account for changes in cell shape, crawling motion and turning of the cell in response to external cues. Regulating the dynamics of the cytoskeleton, and playing a central role in signal transduction in the cell, are Cdc42, Rac and Rho (GTPases of the rho family, collectively known as the small G-proteins) and the actin nucleating complex, Arp2/3.In this paper, we use a multiscale modelling approach in a 2D model of a motile cell. We describe the mutual interactions of the small G-proteins, and their effects on capping and side-branching of actin filaments. We incorporate the pushing exerted by oriented actin filament ends on the cell edge, and a Rho-dependent contraction force. Combining these biochemical and mechanical aspects, we investigate the dynamics of a model epidermal fish keratocyte through in silico experiments. Our model gives insight into how, in response to some cue, a cell can polarize, form a leading edge, and move; concomitantly it explains how a keratocyte cell can maintain its shape and polarity, even after removal of the initial stimulus, and how it can change direction quickly in response to changes in its environment. We show that establishment of polarity stems from interactions of Cdc42, Rac and Rho, while maintenance and robustness of polarity is due to the rapid cytosolic diffusion of the inactive (GDI-bound) forms of the small G-proteins. Our model produces a cell shape that closely resembles the keratocytes and correct speeds for biologically reasonable parameter values. Movies of the simulations can be obtained from http://theory.bio.uu.nl/stan/keratocyte.     

Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure

During Drosophila melanogaster dorsal closure, lateral sheets of embryonic epidermis assemble an actomyosin cable at their leading edge and migrate dorsally over the amnioserosa, converging at the dorsal midline. We show that disappearance of the homophilic cell adhesion molecule Echinoid (Ed) from the amnioserosa just before dorsal closure eliminates homophilic interactions with the adjacent dorsal-most epidermal (DME) cells, which comprise the leading edge. The resulting planar polarized distribution of Ed in the DME cells is essential for the localized accumulation of actin regulators and for actomyosin cable formation at the leading edge and for the polarized localization of the scaffolding protein Bazooka/PAR-3. DME cells with uniform Ed fail to assemble a cable and protrude dorsally, suggesting that the cable restricts dorsal migration. The planar polarized distribution of Ed in the DME cells thus provides a spatial cue that polarizes the DME cell actin cytoskeleton, defining the epidermal leading edge and establishing its contractile properties.     

Dynamic analysis of actin cable function during Drosophila dorsal closure

Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. Here, we test the function of this cable by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. We show that the cable serves a dual role during dorsal closure. It is contractile and thus can operate as a "purse string," but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets.     

The small GTPase Rac plays multiple roles in epithelial sheet fusion--dynamic studies of Drosophila dorsal closure

The coordinated migration and fusion of epithelial sheets is a crucial morphogenetic tool used on numerous occasions during the normal development of an embryo and re-activated as part of the wound healing response. Drosophila dorsal closure, whereby a hole in the embryonic epithelium is zipped closed late in embryogenesis, serves as an excellent, genetically tractable model for epithelial migration. Using live confocal imaging, we have dissected multiple roles for the small GTPase Rac in this process. We show that constitutive activation of Rac1 leads to excessive assembly of lamellipodia and precocious halting of epithelial sweeping, possibly through premature activation of contact-inhibition machinery. Conversely, blocking Rac activity, either by loss-of-function mutations or expression of dominant negative Rac1, disables the assembly of both actin cable and protrusions by epithelial cells. Movies of mutant embryos show that continued contraction of the amnioserosa is sufficient to draw the epithelial edges towards one another, allowing the zipper machinery to bypass non-functioning regions of leading edge. In addition to illustrating the key role of Rac in organization of leading edge actin, loss-of-function mutants also provide substantive proof that Rac acts upstream in the Jun N-terminal kinase (JNK) cascade to direct epithelial cell shape changes during dorsal closure.     

Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the Arp2/3 complex

The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.     

Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.     

Propagation of mechanical stress through the actin cytoskeleton toward focal adhesions: model and experiment

We investigate both theoretically and experimentally how stress is propagated through the actin cytoskeleton of adherent cells and consequentially distributed at sites of focal adhesions (FAs). The actin cytoskeleton is modeled as a two-dimensional cable network with different lattice geometries. Both prestrain, resulting from actomyosin contractility, and central application of external force, lead to finite forces at the FAs that are largely independent of the lattice geometry, but strongly depend on the exact spatial distribution of the FAs. The simulation results compare favorably with experiments with adherent fibroblasts onto which lateral force is exerted using a microfabricated pillar. For elliptical cells, central application of external force along the long axis leads to two large stress regions located obliquely opposite to the pulling direction. For elliptical cells pulled along the short axis as well as for circular cells, there is only one region of large stress opposite to the direction of pull. If in the computer simulations FAs are allowed to rupture under force for elliptically elongated and circular cell shapes, then morphologies arise which are typical for migrating fibroblasts and keratocytes, respectively. The same effect can be obtained also by internally generated force, suggesting a mechanism by which cells can control their migration morphologies.     

Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo

Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles.     

Actomyosin transports microtubules and microtubules control actomyosin recruitment during Xenopus oocyte wound healing

BACKGROUND: Interactions between microtubules and actin filaments (F-actin) are critical for cellular motility processes ranging from directed cell locomotion to cytokinesis. However, the cellular bases of these interactions remain poorly understood. We have analyzed the role of microtubules in generation of a contractile array comprised of F-actin and myosin-2 that forms around wounds made in Xenopus oocytes. RESULTS: After wounding, microtubules are transported to the wound edge in association with F-actin that is itself recruited to wound borders via actomyosin-powered cortical flow. This transport generates sufficient force to buckle and break microtubules at the wound edge. Transport is complemented by local microtubule assembly around wound borders. The region of microtubule breakage and assembly coincides with a zone of actin assembly, and perturbation of the microtubule cytoskeleton disrupts this zone as well as local recruitment of the Arp2/3 complex and myosin-2. CONCLUSIONS: The results reveal transport of microtubules in association with F-actin that is pulled to wound borders via actomyosin-based contraction. Microtubules, in turn, focus zones of actin assembly and myosin-2 recruitment at the wound border. Thus, wounding triggers the formation of a spatially coordinated feedback loop in which transport and assembly of microtubules maintains actin and myosin-2 in close proximity to the closing contractile array. These results are surprisingly reminiscent of recent findings in locomoting cells, suggesting that similar feedback interactions may be generally employed in a variety of fundamental cell motility processes.     

A Highlights from MBoC Selection: A central role for vimentin in regulating repair function during healing of the lens epithelium

Mock cataract surgery provides a unique ex vivo model for studying wound repair in a clinically relevant setting. Here wound healing involves a classical collective migration of the lens epithelium, directed at the leading edge by an innate mesenchymal subpopulation of vimentin-rich repair cells. We report that vimentin is essential to the function of repair cells as the directors of the wound-healing process. Vimentin and not actin filaments are the predominant cytoskeletal elements in the lamellipodial extensions of the repair cells at the wound edge. These vimentin filaments link to paxillin-containing focal adhesions at the lamellipodial tips. Microtubules are involved in the extension of vimentin filaments in repair cells, the elaboration of vimentin-rich protrusions, and wound closure. The requirement for vimentin in repair cell function is revealed by both small interfering RNA vimentin knockdown and exposure to the vimentin-targeted drug withaferin A. Perturbation of vimentin impairs repair cell function and wound closure. Coimmunoprecipitation analysis reveals for the first time that myosin IIB is associated with vimentin, linking vimentin function in cell migration to myosin II motor proteins. These studies reveal a critical role for vimentin in repair cell function in regulating the collective movement of the epithelium in response to wounding.     

An Atomic Model of the Tropomyosin Cable on F-actin

Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin’s coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations.