S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PIP3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PtdIns(3,4,5)P₃ and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia-inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.Key words: filopodia, integrins, migration, cancer     

Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow

The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.     

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.     

Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge.     

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 μM; IIB, K(d) = 8 μM; IIC, K(d) = 1.0 μM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.     

FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly

Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.     

RhoD activated by fibroblast growth factor induces cytoneme-like cellular protrusions through mDia3C

The small GTPase RhoD regulates actin cytoskeleton to collapse actin stress fibers and focal adhesions, resulting in suppression of cell migration and cytokinesis. It also induces alignment of early endosomes along actin filaments and reduces their motility. We show here that a constitutively activated RhoD generated two types of actin-containing thin peripheral cellular protrusions distinct from Cdc42-induced filopodia. One was longer, almost straight, immotile, and sensitive to fixation, whereas the other was shorter, undulating, motile, and resistant to fixation. Moreover, cells expressing wild-type RhoD extended protrusions toward fibroblast growth factor (FGF) 2/4/8–coated beads. Stimulation of wild-type RhoD-expressing cells with these FGFs also caused formation of cellular protrusions. Nodules moved through the RhoD-induced longer protrusions, mainly toward the cell body. Exogenously expressed FGF receptor was associated with these moving nodules containing endosome-like vesicles. These results suggest that the protrusions are responsible for intercellular communication mediated by FGF and its receptor. Accordingly, the protrusions are morphologically and functionally equivalent to cytonemes. RhoD was activated by FGF2/4/8. Knockdown of RhoD interfered with FGF-induced protrusion formation. Activated RhoD specifically bound to mDia3C and facilitated actin polymerization together with mDia3C. mDia3C was localized to the tips or stems of the protrusions. In addition, constitutively activated mDia3C formed protrusions without RhoD or FGF stimulation. Knockdown of mDia3 obstructed RhoD-induced protrusion formation. These results imply that RhoD activated by FGF signaling forms cytoneme-like protrusions through activation of mDia3C, which induces actin filament formation.     

The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts

The formation of protrusions at the leading edge of the cell is an essential step in fibroblast locomotion. Using fluorescent analogue cytochemistry, ratio imaging, multiple parameter analysis, and fluorescence photobleaching recovery, the distribution of actin and myosin was examined in the same protrusions at the leading edge of live, locomoting cells during wound-healing in vitro. We have previously defined two temporal stages of the formation of protrusions: (a) initial protrusion and (b) established protrusion (Fisher et al., 1988). Actin was slightly concentrated in initial protrusions, while myosin was either totally absent or present at extremely low levels at the base of the initial protrusions. In contrast, established protrusions contained diffuse actin and actin microspikes, as well as myosin in both diffuse and structured forms. Actin and myosin were also localized along concave transverse fibers near the base of initial and established protrusions. The dynamics of myosin penetration into a relatively stable, established protrusion was demonstrated by recording sequential images over time. Myosin was shown to be absent from an initial protrusion, but diffuse and punctate myosin was detected in the same protrusion within 1-2 min. Fluorescence photobleaching recovery indicated that myosin was 100% immobile in the region behind the leading edge containing transverse fibers, in comparison to the 21% immobile fraction detected in the perinuclear region. Possible explanations of the delayed penetration of myosin into established protrusions and the implications on the mechanism of protrusion are discussed.     

Maturation of Filopodia Shaft Adhesions Is Upregulated by Local Cycles of Lamellipodia Advancements and Retractions

While cell-substrate adhesions that form between the protruding edge of a spreading cell and flat surfaces have been studied extensively, processes that regulate the maturation of filopodia adhesions are far less characterized. Since little is known about how the kinetics of formation or disassembly of filopodia adhesions is regulated upon integration into the lamellum, a kinetic analysis of the formation and disassembly of filopodia adhesions was conducted at the leading edge of β3-integrin-EGFP-expressing rat embryonic fibroblasts spreading on fibronectin-coated glass or on soft polyacrylamide gels. Filopodia β3-integrin adhesions matured only if the lamellipodium in their immediate vicinity showed cyclic protrusions and retractions. Filopodia β3-integrin shaft adhesions elongated rapidly when they were overrun by the advancing lamellipodium. Subsequently and once the lamellipodium stopped its advancement at the distal end of the filopodia β3-integrin adhesion, these β3-integrin shaft adhesions started to grow sidewise and colocalize with the newly assembled circumferential actin stress fibers. In contrast, the suppression of the cyclic protrusions and retractions of the lamellipodium by blocking myosin light chain kinase suppressed the growth of filopodia adhesion and resulted in the premature disassembly of filopodia adhesions. The same failure to stabilize those adhesions was found for the advancing lamellipodium that rapidly overran filopodia shaft adhesions without pausing as seen often during fast cell spreading. In turn, plating cells on soft polyacrylamide gels resulted in a reduction of lamellipodia activity, which was partially restored locally by the presence of filopodia adhesions. Thus filopodia adhesions could also mature and be integrated into the lamellum for fibroblasts on soft polyacrylamide substrates.     

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.     

Molecular Mechanism of Fascin Function in Filopodial Formation

Filopodia are cell surface protrusions that are essential for cell migration. This finger-like structure is supported by rigid tightly bundled actin filaments. The protein responsible for actin bundling in filopodia is fascin. However, the mechanism by which fascin functions in filopodial formation is not clear. Here we provide biochemical, cryo-electron tomographic, and x-ray crystal structural data demonstrating the unique structural characteristics of fascin. Systematic mutagenesis studies on 100 mutants of fascin indicate that there are two major actin-binding sites on fascin. Crystal structures of four fascin mutants reveal concerted conformational changes in fascin from inactive to active states in the process of actin bundling. Mutations in any one of the actin-binding sites impair the cellular function of fascin in filopodial formation. Altogether, our data reveal the molecular mechanism of fascin function in filopodial formation.     

C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia

We have identified a gene by microarray analysis that is located on chromosome 6 (c6orf32), whose expression is increased during human fetal myoblast differentiation. The protein encoded by c6orf32 is expressed both in myogenic and non-myogenic primary cells isolated from 18-week old human fetal skeletal muscle. Immunofluorescent staining indicated that C6ORF32 localizes to the cellular cytoskeleton and filopodia, and often displays polarized expression within the cell. mRNA knockdown experiments in the C2C12 murine myoblast cell line demonstrated that cells lacking c6orf32 exhibit a myogenic differentiation defect, characterized by a decrease in the expression of myogenin and myosin heavy chain (MHC) proteins, whereas MyoD1 was unaltered. In contrast, overexpression of c6orf32 in C2C12 or HEK293 cells (a non-muscle cell line) promoted formation of long membrane protrusions (filopodia). Analysis of serial deletion mutants demonstrated that amino acids 55-113 of C6ORF32 are likely involved in filopodia formation. These results indicate that C6ORF32 is a novel protein likely to play multiple functions, including promoting myogenic cell differentiation, cytoskeletal rearrangement and filopodia formation.     

Filopodia: molecular architecture and cellular functions

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

The key feature for early migratory processes: Dependence of adhesion,actin bundles,force generation and transmission on filopodia

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process, filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.Key words: filopodia, focal complexes, cell migration, focal adhesion, myosin II, force, actin flow, maturation     

Migration of periodontal ligament fibroblasts on nanometric topographical patterns: influence of filopodia and focal adhesions on contact guidance

Considered to be the "holy grail" of dentistry, regeneration of the periodontal ligament in humans remains a major clinical problem. Removal of bacterial biofilms is commonly achieved using EDTA gels or lasers. One side effect of these treatment regimens is the etching of nanotopographies on the surface of the tooth. However, the response of periodontal ligament fibroblasts to such features has received very little attention. Using laser interference lithography, we fabricated precisely defined topographies with continuous or discontinuous nanogrooves to assess the adhesion, spreading and migration of PDL fibroblasts. PDL fibroblasts adhered to and spread on all tested surfaces, with initial spreading and focal adhesion formation slower on discontinuous nanogrooves. Cells had a significantly smaller planar area on both continuous and discontinuous nanogrooves in comparison with cells on non-patterned controls. At 24 h post seeding, cells on both types of nanogrooves were highly elongated parallel to the groove long axis. Time-lapse video microscopy revealed that PDL fibroblast movement was guided on both types of grooves, but migration velocity was not significantly different from cells cultured on non-patterned controls. Analysis of filopodia formation using time-lapse video microscopy and labeling of vinculin and F-actin revealed that on nanogrooves, filopodia were highly aligned at both ends of the cell, but with increasing time filopodia and membrane protrusions developed at the side of the cell perpendicular to the cell long axis. We conclude that periodontal ligament fibroblasts are sensitive to nanotopographical depths of 85-100 μm, which could be utilized in regeneration of the periodontal ligament.     

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

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

Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.