Role of Focal Adhesions and Mechanical Stresses in the Formation and Progression of the Lamellum Interface

Actin network in the front part of a moving cell is organized into a lamellipodium and a lamellum. A distinct lamellipodium-lamellum interface is associated with focal adhesions and consists of a series of arclike segments linking neighboring focal adhesions in the front row. The interface advances by leaping onto new rows of focal adhesions maturating underneath the lamellipodium. We propose a mechanism of the lamellipodium-lamellum boundary generation, shape formation, and progression based on the elastic stresses generated in the lamellipodial actin gel by its friction against the focal adhesions. The crucial assumption of the model is that stretching stresses trigger actin gel disintegration. We compute the stress distribution throughout the actin gel and show that the gel-disintegrating stresses drive formation of a gel boundary passing through the row of focal adhesions. Our computations recover the lamellipodium-lamellum boundary shapes detected in cells and predict the mode of the boundary transition to the row of the newly maturing focal adhesions in agreement with the experimental observations. The model fully accounts for the current phenomenology of the lamellipodium-lamellum interface formation and advancing, and makes experimentally testable predictions on the dependence of these phenomena on the sizes of the focal adhesions, the character of the focal adhesion distribution on the substrate, and the velocity of the actin retrograde flow with respect to the focal adhesions. The phase diagram resulting from the model provides a background for quantitative classification of different cell types with respect to their ability to form a lamellipodium-lamellum interface. In addition, the model suggests a mechanism of nucleation of the dorsal and arclike actin bundles found in the lamellum.     

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.     

New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.     

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.     

Dynamics of focal adhesions and reorganization of F‐actin in VEGF‐stimulated NSCs under varying differentiation states

Precise migration of neural stem/progenitor cells (NSCs) is crucially important for neurogenesis and repair in the nervous system. However, the detailed mechanisms are not clear. Our previous results showed that NSCs in varying differentiation states possess different migratory ability to vascular endothelial growth factor (VEGF). In this study, we demonstrate the different dynamics of focal adhesions (FAs) and reorganization of F‐actin in NSCs during spreading and migration stimulated by VEGF. We found that the migrating NSCs of 0.5 and 1 day differentiation possess more FAs at leading edge than cells of other states. Moreover, the phosphorylation of focal adhesion kinase (FAK) and paxillin in NSCs correlates closely with their differentiation states. VEGF promotes FA formation with broad lamellipodium generation at the leading edge in chemotaxing cells of 0, 0.5, and 1 day differentiation, but not in cells of 3 days differentiation. Furthermore, cells of 1 day differentiation show a maximal asymmetry of FAs between lamella and cell rear, orchestrating cell polarization and directional migration. Time‐lapse video analysis shows that the disassembly of FAs and the cell tail detachment in NSCs of 1 day differentiation are more rapid, along with the concurrent enlarged size of FAs at the leading edge, leading to the most effective chemotactic response to VEGF. Collectively, these results indicate that the dynamics of FAs and reorganization of F‐actin in NSCs that undergo directional migration correlate closely with their differentiation states, contributing to the different chemotactic responses of these cells to VEGF. J. Cell. Biochem. 114: 1744–1759, 2013. © 2013 Wiley Periodicals, Inc.     

Physical model for self-organization of actin cytoskeleton and adhesion complexes at the cell front

Cell motion is driven by interplay between the actin cytoskeleton and the cell adhesions in the front part of the cell. The actin network segregates into lamellipodium and lamellum, whereas the adhesion complexes are characteristically distributed underneath the actin system. Here, we suggest a computational model for this characteristic organization of the actin-adhesion system. The model is based on the ability of the adhesion complexes to sense mechanical forces, the stick-slip character of the interaction between the adhesions and the moving actin network, and a hypothetical propensity of the actin network to disintegrate upon sufficiently strong stretching stresses. We identify numerically three possible types of system organization, all observed in living cells: two states in which the actin network exhibits segregation into lamellipodium and lamellum, whereas the cell edge either remains stationary or moves, and a state where the actin network does not undergo segregation. The model recovers the asynchronous fluctuations and outward bulging of the cell edge, and the dependence of the edge protrusion velocity on the rate of the nascent adhesion generation, the membrane tension, and the substrate rigidity.     

Assembly and mechanosensory function of focal adhesions: experiments and models

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.     

The comings and goings of actin: coupling protrusion and retraction in cell motility

Cells utilize actin filaments to produce protrusive and contractile arrays that cooperate to drive cell motility. The generation of the two arrays and the coupling between them result from the unique properties of the lamellipodium, a protrusive leaflet of cytoplasm at the cell edge. From the lamellipodium into the lamella behind, there is a transition from a fast retrograde flow of actin polymer driven by polymerization to a slow flow driven by the interaction of anti-parallel arrays of actin with myosin. In addition to driving protrusion, the lamellipodium appears to play a role in supplying filaments to the lamella for the assembly of the contractile network required for traction.     

Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.     

Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.     

Discrete mechanical model of lamellipodial actin network implements molecular clutch mechanism and generates arcs and microspikes

Mechanical forces, actin filament turnover, and adhesion to the extracellular environment regulate lamellipodial protrusions. Computational and mathematical models at the continuum level have been used to investigate the molecular clutch mechanism, calculating the stress profile through the lamellipodium and around focal adhesions. However, the forces and deformations of individual actin filaments have not been considered while interactions between actin networks and actin bundles is not easily accounted with such methods. We develop a filament-level model of a lamellipodial actin network undergoing retrograde flow using 3D Brownian dynamics. Retrograde flow is promoted in simulations by pushing forces from the leading edge (due to actin polymerization), pulling forces (due to molecular motors), and opposed by viscous drag in cytoplasm and focal adhesions. Simulated networks have densities similar to measurements in prior electron micrographs. Connectivity between individual actin segments is maintained by permanent and dynamic crosslinkers. Remodeling of the network occurs via the addition of single actin filaments near the leading edge and via filament bond severing. We investigated how several parameters affect the stress distribution, network deformation and retrograde flow speed. The model captures the decrease in retrograde flow upon increase of focal adhesion strength. The stress profile changes from compression to extension across the leading edge, with regions of filament bending around focal adhesions. The model reproduces the observed reduction in retrograde flow speed upon exposure to cytochalasin D, which halts actin polymerization. Changes in crosslinker concentration and dynamics, as well as in the orientation pattern of newly added filaments demonstrate the model’s ability to generate bundles of filaments perpendicular (actin arcs) or parallel (microspikes) to the protruding direction.     

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.     

Slipping or gripping? Fluorescent speckle microscopy in fish keratocytes reveals two different mechanisms for generating a retrograde flow of actin

Fish keratocytes can generate rearward directed traction forces within front portions of the lamellipodium, suggesting that a retrograde flow of actin may also occur here but this was not detected by previous photoactivation experiments. To investigate the relationship between retrograde flow and traction force generation, we have transfected keratocytes with GFP-actin and used fluorescent speckle microscopy, to observe speckle flow. We detected a retrograde flow of actin within the leading lamellipodium that is inversely proportional to both protrusion rate and cell speed. To observe the effect of reducing contractility, we treated transfected cells with ML7, a potent inhibitor of myosin II. Surprisingly, ML7 treatment led to an increase in retrograde flow rate, together with a decrease in protrusion and cell speed, but only in rapidly moving cells. In slower moving cells, retrograde flow decreased, whereas protrusion rate and cell speed increased. These results suggest that there are two mechanisms for producing retrograde flow. One involves slippage between the cytoskeleton and adhesions, that decreases traction force production. The other involves slippage between adhesions and the substratum, which increases traction force production. We conclude that a biphasic relationship exists between retrograde actin flow and adhesiveness in moving keratocytes.     

Vinculin–actin interaction couples actin retrograde flow to focal adhesions,but is dispensable for focal adhesion growth

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium–lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin–F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.     

LIM kinase has a dual role in regulating lamellipodium extension by decelerating the rate of actin retrograde flow and the rate of actin polymerization

Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow, and lamellipodium extension using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin-stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization, and in MCF-7 cells endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.     

Tracking retrograde flow in keratocytes: news from the front

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1-3 microm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.     

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.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

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.     

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.