Cells employ protrusive leading edges to navigate and promote their migration

Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. FLPs. We propose that actomyosin contraction acting against membrane tension advances the web of arcs between FLPs. Predictions of this model are verified experimentally. The dependence of myosin II in leading-edge advancement helps explain the previously reported defect in directional movement in the Arpc3-null fibroblasts. We provide further evidence that this defect is cell autonomous during chemotaxis. INTRODUCTION Actin polymerization drives protrusion of the leading edge in migrating cells through two types of structures lamellipodia and filopodia distinguished primarily by their morphological characteristics (Hall 1998 ; Pollard and Borisy 2003 ; Chhabra and Higgs 2007 ; Bugyi and Carlier 2010 ). Lamellipodia are dynamic veil-like edges made up of cross-linked orthogonal actin arrays and are typically observed in fibroblasts or keratocytes moving on two-dimensional (2D) surfaces. Enrichment of branched actin network and localization of the Arp2/3 complex an evolutionarily conserved actin-nucleating complex at the tip of lamellipodia led EW-7197 to the hypothesis that the Arp2/3 complex is the primary actin nucleator regulating the extension and organization of the lamellipodia actin network (Welch = 12). In mutant cells that had already spread blebbistatin treatment resulted in collapse of the arcs leaving behind long thin FLPs that often had branches (Figure 4D) in contrast to wt cells (Figure 4C). Soon after blebbistatin washout the membrane arcs between FLPs advanced promptly and recovered the same leading-edge morphology as untreated mutant cells (Figure 4 B and D and Supplemental Video 10). These results suggest that leading-edge advancement in ARPC3?/? cells is a product of both formin-mediated FLP extension and myosin II-dependent contractility of the regions between FLPs. FIGURE 4: Effects of the nonmuscle myosin II inhibitor blebbistatin on APRC3+/+ and ARPC3?/? fibroblast cells. (A and B) Montage of phase-contrast movies showing the morphology of representative ARPC3+/+ (A) and ARPC3?/? (B) fibroblast … Force-balance model of leading-edge protrusion in the absence of Arp2/3 complex On the basis of protein localization and functional data we propose a model for how fibroblast cells produce protrusive edges in the absence of the Arp2/3 complex. We assume that myosin II captures overlapping filaments at the base of adjacent FLPs and Rabbit Polyclonal to RPL30. produces the contractile force driving concerted advancement of the arc regions in between the FLPs (Figure 5A model 1 or 2 2). We hypothesize that this contractile force shortens the actomyosin assemblies in the arc regions between the bases of the FLPs in concert with filaments “peeling” from the FLP EW-7197 bases and being “reeled” into the contractile network. Together these processes lead to the advancement of the leading edge between FLPs. FIGURE 5: Force-balance model of leading-edge protrusion based on coordinated action of formin and myosin II. (A) Simple cartoon diagram depicting the key elements of the leading edge formed in the absence of the Arp2/3 complex. Small green circles: formin at actin … To evaluate whether this is mechanically plausible we considered the EW-7197 force balance between the effective pressure generated by actomyosin contraction and membrane tension (Figure 5A) which is described by Laplace’s law: = = (Bar-Ziv is the contractile force in the bundle and is the radius of the arc. At least two simple theories predict that the contractile force is a growing function of the actomyosin assembly length is force) (Rubinstein is viscosity is a coordinate along the fiber. We assume that at the ends of the actomyosin assembly where it is attached to the FLP base filaments are pulled into the arc with effective viscous friction against adhesions at the FLP base so the stress there is is the effective friction and 0 and are coordinates of the ends. The model excludes adhesion forces along the arc only taking into account adhesions at the end of the arc because the paxilin staining of mutant cells (Figure 1B) demonstrated the absence of adhesions along the arc. Integrating equation with unknown with boundary conditions = motors per unit length of the overlap the contractile force is equal to are positive constant parameters. EW-7197 Myosin is driven by this flow to the center of the arc while its detachment diffusion in the cytoplasm and reattachment redistributes it according to the equation.