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

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nature Cell Biology (Impact Factor: 19.68). 03/2011; 13(4):371-81. DOI: 10.1038/ncb2205
Source: PubMed


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.

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    • "To facilitate comparison of frustrated phagocytosis with spreading and migratory processes we analyzed the dynamic spreading morphology using protrusion/retraction activity maps [44] [25] [34] [26] [13]. Similar to spreading fibroblasts [27], activity maps of FP spreading reveal distinct phases of behavior. "
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    DESCRIPTION: Conventional studies of dynamic phagocytic behavior have been limited in terms of spatial and temporal resolution due in large-part to the inherent 3-dimensionality and small feature sizes involved in phagocytosis. To overcome these issues, we developed a high-throughput dynamic characterization assay utilizing frustrated phagocytosis (FP). Using this assay, we quantitatively characterize phagocytic spreading dynamics. Our investigation reveals that FP spreading occurs in two phases and is punctuated by a distinct period of contraction. Characterization of hundreds of cells reveals that this behavior is highly reproducible. We also performed a comparison of FP dynamics at increasing levels of IgG opsonization. Spreading dynamics are independent of opsonin density, but the likelihood a cell exhibits an FP response increases logarithmically with IgG density. Based on this finding we propose that the dynamics of phagocytic spreading are determined primarily by cell material properties. To illustrate this idea, we develop a simple hydrodynamic model of cell spreading based only on measurable mechanical characteristics. The model reproduces initial cell-surface contact and rapid spreading behavior. We also characterize late-stage FP contraction. Using Traction Force Microscopy, we show that cells exert significant contractile forces following the FP spreading phase. These forces are reminiscent of contractile forces generated during phagosome closure. Blebbistatin treatment reveals that late-stage force generation is myosin II dependent. From this, we hypothesize that late-stage FP contraction and phagosome closure are signaled events. Although the origin of this signal is unclear, our results demonstrate that this signal is independent of particle geometry and therefore must depend either on some type of biochemical timer or a mechanosensing mechanism such as membrane tension sensors.
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    • "Thus the contact time and multiplicity of retraction cycles of the lamellipodium at the filopodia adhesions regulates the maturation or disassembly of filopodia adhesions. Cyclic protrusions and retractions of lamellipodia have also been associated with the deposition of circumferential actin filaments that run parallel to the cell edge in various cell types [35], [55], [63] which became efficiently coupled with focal adhesions at the onset of the lamellum right behind the transition zone [64]. Our data now suggest that the circumferential actin filaments become connected with filopodia adhesions behind the lamellipodium-lamellum transition (Fig. 7). "
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    ABSTRACT: 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.
    PLoS ONE 09/2014; 9(9):e107097. DOI:10.1371/journal.pone.0107097 · 3.23 Impact Factor
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    • "Although the spatiotemporal relationship between GFP-MLC2 punctae and actin filaments were not resolved in our experiments, we suggest that perturbations in nascent actomyosin arise in dynamin2-depleted cells from perturbations in lamellipodial actin filament organization. Recent findings that components of lamellar actomyosin arise, in part, from actin filaments of the lamellipod are consistent with this idea [2], [4]–[6], [54]. The mechanism by which dynamin2 controls actomyosin assembly remains to be determined but it is appealing to speculate that filament remodeling within the lamellipod creates bundled filament templates that are optimal for co-assembly with myosin II. "
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    ABSTRACT: Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.
    PLoS ONE 04/2014; 9(4):e94330. DOI:10.1371/journal.pone.0094330 · 3.23 Impact Factor
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