Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide.

BACKGROUND: In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer. RESULTS: In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide. CONCLUSIONS: Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.     

Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks

Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals.     

ADF/cofilin controls cell polarity during fibroblast migration

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. We have previously shown that actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, we assessed their role in polarized lamellipodium protrusion in migrating fibroblasts. We compared the spatial distribution of AC and the inactive, phosphorylated AC (pAC) in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, we increased the proportion of pAC in migrating fibroblasts by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. We conclude that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration.     

Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics

Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency.     

Adhesive F-actin waves: a novel integrin-mediated adhesion complex coupled to ventral actin polymerization

At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These "adhesive F-actin waves" require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Retrograde flow and myosin II activity within the leading cell edge deliver F-actin to the lamella to seed the formation of graded polarity actomyosin II filament bundles in migrating fibroblasts

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

Conformational dynamics of loop 262-274 in G- and F-actin

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.     

The actin-based nanomachine at the leading edge of migrating cells.

Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined-its thickness in living cells (176 +/- 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 +/- 613 microm of F-actin/microm(3)), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 +/- 100/microm) and the maximum force (1.86 +/- 0.83 nN/microm) and pressure (10.5 +/- 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (. J. Cell Sci. 44:187-200;. Proc. Natl. Acad. Sci. USA. 79:5327-5331) and an estimate of the force required to stall the polymerization of a single filament (. Proc. Natl. Acad. Sci. USA. 78:5613-5617;. Biophys. J. 65:316-324) to argue that actin assembly alone could drive lamellipodial extension directly.     

The quantitation of G- and F-actin in cultured cells

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.     

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.     

Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.     

Regulation of intestinal brush border microvillus length during development by the G- to F-actin ratio

We examined ultrastructural changes in developing chicken intestinal microvilli and correlated these with changes in the G- to F-actin ratio and the amount of actin per milligram cell protein. Three discrete morphological and temporal changes occur during late microvillus morphogenesis: an increase in microvillus number associated with microvilli becoming hexagonally packed on the cell surface; an increase in core actin filament number; and an increase in the length of microvilli. Dramatic rises in the amount of cell actin occur at the time of the first two morphological changes. Changes in the G- to F-actin ratio suggest that increases in the level of monomeric actin drive the elongation phase of microvillus growth since immediately prior to growth the G- to F-actin ratio shifts from its embryonic and adult 3:7 ratio to a 1:1. Our results also indicate, but do not prove, that an increase in the amount of G-actin precedes the rise in level of F-actin and growth of microvilli by 1 day, implying that an increase in the content of G-actin stimulates actin polymerization. Our findings also suggest that the G- to F-actin ratio and their absolute amounts, perhaps in combination with cytoskeletal protein turnover and/or the pool size of actin binding proteins, plays a role in restricting the mature constant length of microvilli.     

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.     

Ecdysterone induction of actin synthesis and polymerization in a Drosophila melanogaster cultured cell line

Actin pools have been evaluated in Drosophila melanogaster Kc 0% cells, through an actin assay based on differential inhibition of DNase I by globular (G) and filamentous (F) actin. Total actin represents about 4 % of total proteins and 54 % is G-actin. In ecdysterone treated cells (0.1 μM), the total actin content increases up to 9 % of total proteins after 3 days of treatment. Ecdysterone induces increase of G-actin as well as F-actin. Increase of both actins, detectable after only 24 hrs of treatment, is roughly parallel during the first two days of treatment. For longer hormonal treatment, actin polymerization is more important than accumulation of G-actin. Indirect immunofluorescence microscopy with antibodies to exogeneous DNase I suggests that actin is widely distributed in the whole cytoplasm before and after ecdysterone treatment. These results suggest that ecdysterone induces actin synthesis and polymerization in Drosophila melanogaster cells.     

Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.