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.     

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.     

Architectural dynamics of F-actin in eupodia suggests their role in invasive locomotion in Dictyostelium.

Eupodia are F-actin-containing cortical structures similar to vertebrate podosomes or invadopodia found in metastatic cells. Eupodia are rich in alpha-actinin and myosin IB/D, but not a Dictyostelium homologue of talin. In the present study, we localized other actin-binding proteins, ABP120, cofilin, coronin, and fimbrin, in the eupodia and examined the three-dimensional organization of their F-actin system by confocal microscopy and transmission electron microscopy. To examine their function, we analyzed the assembly and disassembly dynamics of the F-actin system in eupodia and its relation to lamellipodial protrusion. Actin dynamics was examined by monitoring S65T-GFP-coronin and rhodamine-actin using a real-time confocal unit and a digital microscope system. Fluorescence morphometric analysis demonstrates the presence of a precise spatiotemporal coupling between F-actin assembly in eupodia and lamellipodial protrusion. When a lamellipodium advances to invade a tight space, additional rows of eupodia are sequentially formed at the base of that lamellipodium. These results indicate that mechanical stress at the leading edge modulates the structural integrity of actin and its binding proteins, such that eupodia are formed when anchorage is needed to boost for invasive protrusion of the leading edge.     

Regulation of cofilin phosphorylation and asymmetry in collective cell migration during morphogenesis

During Drosophila oogenesis, two actin dynamics regulators, cofilin and Rac, are required for the collective migration of a coherent cluster of cells called border cells. Cell culture data have shown that Rac and cofilin are both essential for lamellipodium formation, but Rac signaling results in phosphorylation and hence inactivation of cofilin. So it remains unclear whether cofilin phosphorylation plays a promoting or inhibitory role during cell migration. We show here that cofilin is required for F-actin turnover and lamellipodial protrusion in the border cells. Interestingly, reducing the dosage of cofilin by half or expressing a phospho-mimetic mutant form, S3E, partially rescues the migration and protrusion defects of Rac-deficient border cells. Moreover, cofilin exhibits moderate accumulation in border cells at the migratory front of the cluster, whereas phospho-cofilin has a robust and uniform distribution pattern in all the outer border cells. Blocking or overactivating Rac signaling in border cells greatly reduces or increases cofilin phosphorylation, respectively, and each abolishes cell migration. Furthermore, Rac may signal through Pak and LIMK to result in uniform phosphorylation of cofilin in all the outer border cells, whereas the guidance receptor Pvr (PDGF/VEGF receptor) mediates the asymmetric localization of cofilin in the cluster but does not affect its phosphorylation. Our study provides one of the first models of how cofilin functions and is regulated in the collective migration of a group of cells in vivo.     

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.     

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.     

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.     

Coactosin-Like 1 Antagonizes Cofilin to Promote Lamellipodial Protrusion at the Immune Synapse

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell – B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.     

Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.     

Cofilin 1-mediated biphasic F-actin dynamics of neuronal cells affect herpes simplex virus 1 infection and replication

Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis.     

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.     

Reorganization of the actin cytoskeleton in the protruding lamellae of human fibroblasts.

To investigate the mechanisms of protrusion in vertebrate cells, the primary event in cell motility, human fibroblasts were treated with neomycin, an inhibitor of the phosphatidylinositol cycle, to induce protrusion. Changes in cell motility and the cytoskeleton were examined by video, fluorescence, scanning electron, and confocal microscopy and by cytofluorometry. Protrusion in neomycin-treated human fibroblasts is correlated with a transient overall decrease in F-actin followed by an increase in F-actin at the leading edge of the protruding lamella. In growing lamellae, F-actin is organized in a marginal band at the leading edge. Although actin is present in the lamella behind the leading edge, very little of it is F-actin. Scanning electron microscopy of detergent-extracted cells reveals a band of dense filaments at the leading edge, corresponding to the marginal band of F-actin seen in fluorescently labeled cells, and a sparse population of short, fragmented filaments, in the rest of the lamella. Gelsolin is colocalized with F-actin in the marginal band and is also present in the lamella where F-actin is largely absent. The data support the hypothesis that the protrusion is initiated by the breakdown of cortical actin filaments, possibly mediated by gelsolin, whereas expansion of the protrusion requires de novo polymerization of actin filaments at the leading edge.     

A role for actin arcs in the leading-edge advance of migrating cells

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion.     

Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion

Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulated in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin.     

Periodic lamellipodial contractions correlate with rearward actin waves

Cellular lamellipodia bind to the matrix and probe its rigidity through forces generated by rearward F-actin transport. Cells respond to matrix rigidity by moving toward more rigid matrices using an unknown mechanism. In spreading and migrating cells we find local periodic contractions of lamellipodia that depend on matrix rigidity, fibronectin binding and myosin light chain kinase (MLCK). These contractions leave periodic rows of matrix bound beta3-integrin and paxillin while generating waves of rearward moving actin bound alpha-actinin and MLCK. The period between contractions corresponds to the time for F-actin to move across the lamellipodia. Shortening lamellipodial width by activating cofilin decreased this period proportionally. Increasing lamellipodial width by Rac signaling activation increased this period. We propose that an actin bound, contraction-activated signaling complex is transported locally from the tip to the base of the lamellipodium, activating the next contraction/extension cycle.     

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.     

Adaptor Protein LRAP25 Mediates Myotonic Dystrophy Kinase-related Cdc42-binding Kinase (MRCK) Regulation of LIMK1 Protein in Lamellipodial F-actin Dynamics

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.     

Molecular requirements for actin-based lamella formation in Drosophila S2 cells

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.     

v-Crk regulates membrane dynamics and Rac activation

Cell migration is an integrated process that involves cell adhesion, protrusion and contraction. We recently used CAS (Crk-associated substrate, 130CAS)-deficient mouse embryo fibroblasts (MEFs) to examined contribution made to v-Crk to that process via its interaction with Rac1. v-Crk, the oncogene product of avian sarcoma virus CT10, directly affects membrane ruffle formation and is associated with Rac1 activation, even in the absence of CAS, a major substrate for Crk. In CAS-deficient MEFs, cell spreading and lamellipodium dynamics are delayed; moreover, Rac activation is significantly reduced, and it is no longer targeted to the membrane. However, expression of v-Crk by CAS-deficient MEFs increased cell spreading and active lamellipodium protrusion and retraction. v-Crk expression appears to induce Rac1 activation and its targeting to the membrane, which directly affects membrane dynamics and, in turn, cell migration. It thus appears that v-Crk/Rac1 signaling contributes to the regulation of membrane dynamics and cell migration, and that v-Crk is an effector molecule for Rac1 activation that regulates cell motility.