While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.

We propose a model in which lamellar cytoplasm is considered a dynamically bistable system capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive-feedback loop intrinsic to the actin-myosin II machinery: anisotropic organization of the machinery drives translocation, which then reinforces the asymmetry of the machinery, favoring further translocation.

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base.

Abstract. The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo flbroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells.At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, o~-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.

Abstract. By immunogold labeling, we demonstrate that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actinbased cytoskeleton and possibly membrane components. Plectin binding to microtubules was significantly increased in cells from transgenic mice lacking IFs and was reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules did not show a preference for Glutubulin. Nor did it depend upon the presence of MAP4 since plectin links were retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survived actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. Our results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm.

The lamellipod, the locomotory region of migratory cells, is shaped by the balance of protrusion and contraction. The latter is the result of myosin-generated centripetal flow of the viscoelastic actin network. Recently, quantitative flow data was obtained, yet there is no detailed theory explaining the flow in a realistic geometry. We introduce models of viscoelastic actin mechanics and myosin transport and solve the model equations numerically for the flat, fan-shaped lamellipodial domain of keratocytes. The solutions demonstrate that in the rapidly crawling cell, myosin concentrates at the rear boundary and pulls the actin network inward, so the centripetal actin flow is very slow at the front, and faster at the rear and at the sides. The computed flow and respective traction forces compare well with the experimental data. We also calculate the graded protrusion at the cell boundary necessary to maintain the cell shape and make a number of other testable predictions. We discuss model implications for the cell shape, speed, and bi-stability.

Analysis of the relationship between actin network velocity and traction forces at the substrate shows that force transmission mechanisms vary with distinct regions of the cell.

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