A thorough understanding of actin polymerization-based crawling at the whole-cell scale has long been a challenge in the field of cell biology. A combination of biochemical and cell-biological experiments has led to a consensus model for the mechanism by which steady-state actin network treadmilling in the lamellipodia of motile cells contributes to protrusion of the leading edge: new filaments are nucleated near the leading edge, resulting in the assembly of a branched actin network, which is eventually disassembled by ADF/cofilin proteins, replenishing the pool of polymerizable actin monomers3
However, extending this model from the micrometer scale of the leading edge lamellipodium to the tens-of-micrometers scale of an entire cell requires a mechanism for longer-range coordination: a cell-scale spatial organization of network assembly and disassembly processes, giving rise to sustained whole-cell motility. The molecular mechanisms of actin network disassembly and its spatial regulation in motile cells are not completely understood (Supplementary Note 1
Here we use fish epidermal keratocytes () as a model system to investigate the spatial regulation of actin network turnover in motile cells. These cells are fast-moving with persistent velocity and shape4
and maintain a continuous actin network throughout the lamellipodium5,6
(), implying that the net rates of assembly at the front and disassembly at the rear must be closely and constantly coordinated. This property allows us to analyze network turnover based on steady-state measurements.
Myosin II in keratocytes colocalizes with the primary sites of actin network disassembly
To investigate the spatial organization of actin network movement and dynamics, we performed fluorescence speckle microscopy (FSM) on cells moving at steady state (). The direction and speed of actin network movement was determined by speckle flow tracking7
as a function of position within the lamellipodium (). Consistent with photoactivation experiments2
, we found that the actin network in the lamellipodium remains nearly stationary with respect to the substrate, with minimal retrograde flow (). At the cell rear, the actin network moved forward and rapidly inward from the sides.
To analyze the movement of the actin network relative to the boundaries of these fast-moving cells, we represented the FSM flow field in the cell’s moving frame of reference8
(, Supplementary Movie 1
). In the cell frame of reference, movement of the network appeared uniform and rearward in the front of the cell, and almost completely perpendicular to the direction of motion at the rear sides. Under the cell body, network flow ceased without significantly changing its direction.
The pattern of fluorescent speckle motion suggests that net actin network assembly occurred in the front of the cell and net disassembly in the rear. This was confirmed by calculating the spatial distribution of net filamentous actin (F-actin) assembly and disassembly, using actin speckle density and the divergence of the FSM flow field9
(; Supplementary Fig. 2a
). Intriguingly, we found that the rear-localized pattern of disassembly, with two foci flanking the cell body, was strongly reminiscent of the distribution of myosin II, as visualized by YFP-tagged myosin II regulatory light chain ().
While the role of myosin II in the rear of motile cells is conventionally associated with mechanical force generation and contraction (Supplementary Note 2
), several lines of evidence raise the possibility that myosin II may also play a specific role in driving actin network disassembly. Spatially correlated contraction and actin depolymerization in motile cells has been shown to be promoted by a drug that stimulates myosin II activity9
. In cytokinesis, where contraction of the cleavage furrow is driven in part by myosin II motor activity, inhibition of myosin II leads to increased accumulation of F-actin in the cleavage furrow, consistent with a role for myosin II in regulating or catalyzing F-actin disassembly in the contractile ring10,11
. During the early steps of inner ear morphogenesis, myosin II activity is required for depletion of F-actin from the basal cortex of an ectodermal sheet12
. More directly, myosin II has been shown to potentiate severing and disassembly of actin filament bundles at the base of neuronal growth cones13
. Finally, in a recent in vitro experiment, a loss of actin filaments in solution has been observed in the presence of purified myosin II14
Our observation of disassembly colocalized with myosin II density suggested that such a link between myosin II activity and actin network disassembly might be at work in keratocyte motility. To investigate this possibility, we treated keratocytes with the myosin II ATPase inhibitor, blebbistatin15
. Although blebbistatin-treated cells continued to move with little shape change at the leading edge (, Supplementary Movie 1
) and with only a moderate decrease in speed (), the pattern of disassembly was markedly altered: the two foci of disassembly adjacent to the cell body disappeared, and disassembly was distributed along the trailing edge (, Supplementary Note 3b
Inhibition of myosin II blocks inward flow and alters the pattern of disassembly of the actin network
Jasplakinolide specifically halts actin dynamics of cells in which myosin II is inhibited
Continued motility of keratocytes under blebbistatin treatment indicates that at least some actin network disassembly activity was retained. We judged that inhibition of myosin II was essentially complete under the conditions of our blebbistatin treatment (50 μM active enantiomer, 10 min) based on two criteria. First, two aspects of motility that are normally attributed to myosin II activity (Supplementary Note 2
) were reduced below the detection limit: inward flow in the cell rear (; Supplementary Movie 1
) and inward traction force (Supplementary Fig. 3, Supplementary Movie 2
). Second, increasing the blebbistatin concentration by two-fold did not have a further effect on cell speed (data not shown). These observations led us to hypothesize that there may be a parallel pathway that disassembles the actin network independent of myosin II.
We thus proceeded to examine the effect of myosin II inhibition in a sensitized background where actin depolymerization was partially inhibited. The F-actin stabilizing drug jasplakinolide11,16
, at 1 μM, decreased cell speed moderately, an effect comparable to that of 50 μM blebbistatin (). For cells pre-incubated with 50 μM blebbistatin, however, 1 μM jasplakinolide dramatically inhibited motility (; Supplementary Movie 3
), often causing a rapid and complete cessation of both movement and actin network flow (; Supplementary Movie 3
). To test the specificity of this synthetic effect, we examined the effect of latrunculin A, which slows actin network turnover by sequestering monomer (inhibiting polymerization) rather than by inhibiting depolymerization. While 50 nM latrunculin A caused keratocytes to lose polarity and stop moving (data not shown), 5 nM latrunculin A caused a speed decrease comparable to sole treatment with either 50 μM blebbistatin or 1 μM jasplakinolide (). Treatment with the combination of blebbistatin and latrunculin A, or jasplakinolide and latrunculin A, did not have a synthetic effect on cell speed (). This suggested that the synthetic effect was not simply a result of actin monomer depletion, but specific to disassembly inhibition. Both blebbistatin-treated cells and jasplakinolide-treated cells showed an unusual accumulation of F-actin in the cell rear, but the patterns were distinct: in blebbistatin-treated cells, F-actin accumulated primarily along the trailing edge and in tail-like structures (), whereas in jasplakinolide-treated cells F-actin accumulated under the cell body (). These observations are consistent with the existence of two complementary and partially redundant mechanisms for actin network disassembly, one dependent on myosin II activity and the other inhibited by jasplakinolide.
Our in vivo
experiments do not distinguish between a direct or indirect contribution of myosin II activity to actin network disassembly. To investigate the specific contribution of myosin II activity, it was essential to analyze disassembly of the rear network in isolation—in the absence of the continuous replenishment that occurs in a live forward-moving cell—and to exclude the action of cytosolic factors. Accordingly, we performed experiments using isolated cytoskeletons of Triton X-100–extracted keratocytes6
. Strikingly, addition of ATP to the extracted cytoskeletons caused not only a rapid inward contraction at the cell rear, but also a concomitant dissolution of the actin network in that region (, Supplementary Movie 4, Supplementary Fig. 6
). The leading edge of the cell was not significantly affected by this process. The rear-dominant disassembly of actin (as well as contraction) was significantly suppressed (sometimes to undetectable levels) when cells were incubated with blebbistatin prior to the detergent extraction (, Supplementary Movie 4, Supplementary Fig. 6
). The ATP dependence, blebbistatin sensitivity, rear localization, and synchrony with contraction of the network disassembly are consistent with myosin II as the active agent, and recapitulation of this myosin II–dependent disassembly in extracted cells strongly suggests that soluble proteins are not required for this process.
Actin network disassembly in the rear of detergent-extracted keratocyte cytoskeletons is ATP-dependent and blebbistatin-sensitive, consistent with a direct role for myosin II in this process
To test for the possibility that an unrelated mechanism was protecting the actin network in the front of the cell from disassembly, we treated extracted cytoskeletons with the gelsolin-family Ca2+
-dependent actin severing protein villin17,18
, in the absence of ATP. GST-villin solubilized F-actin in most regions of the cell, including the front (, Supplementary Movie 4
). Interestingly, however, a portion of the network in the rear was relatively insensitive to GST-villin. This region of the network, which lingered during prolonged villin treatment (Supplementary Movie 4
), was reminiscent of the pattern of myosin II localization () and net actin network disassembly in intact cells () and complementary
to the pattern of actin network stability after ATP treatment. Addition of both
ATP and GST-villin, in either order, gave nearly complete destruction of the actin cytoskeleton (; Supplementary Movie 4
). Thus the rear-dominant pattern of ATP-dependent disassembly () is not due to other regions being impervious to disassembly per se; it likely reflects regions where myosin II is abundant and integrated into the actin network in a configuration that brings about maximal disassembling activity.
Our combined experimental observations point to a direct role for myosin II in actin network disassembly in motile fish keratocytes. A second, unidentified disassembly mechanism appears to act in parallel with myosin II in unperturbed cells. We found this pathway to be sensitive to jasplakinolide, and we conjecture that it may involve cofilin or gelsolin activity (Supplementary Note 1
). Previous reports showing that F-actin disassembly increases upon stimulation of myosin II phosphorylation9
and that jasplakinolide selectively halts actin turnover in regions of cells lacking myosin II19
are consistent with these conclusions.
The observed spatial and temporal coincidence of contraction and disassembly (, ; ref. 9
) is most consistent with a simple mechanism for network disassembly by myosin II: mechanical breakage of actin filaments20
. Such an effect could be assisted by the ability of the nonmuscle myosin II motor domain to stably bind actin filaments when under tension21
, and may represent a common feature of myosin II function in all but the most specialized organization found in striated muscle.
The contribution of myosin II–driven network disassembly to whole-cell motility may vary in different cell types depending on the spatial arrangement of the cytoskeletal elements. In keratocytes, we find myosin II to be concentrated at the cell rear and to contribute to cell-scale network treadmilling; given the similar localization of myosin II to the rear in Dictyostelium22
, we suggest that its role there may be very similar. In contrast, myosin II in neurons appears to be involved in network disassembly at the base of growth cones13
. In fibroblasts and larger epithelial cells, the concentration of myosin II in the lamella (behind the lamellipodium) may primarily contribute to local network disassembly in the transition zone between the lamellipodial and lamellar networks19,24
The involvement of myosin II in actin network disassembly motivates an appealing model for the long-range spatial and temporal coordination of disassembly in motile cells (Supplementary Fig. 1
): slow incorporation of myosin II into the network and subsequent myosin II–mediated rearrangement of F-actin occur over the same time scale as whole cell translocation6
, serving as a timing mechanism. Progressively assembled myosin II bipolar filaments gradually coalesce the initially dendritic actin network into a more parallel organization6
that permits efficient action of the myosin II motor, eventually leading to disassembly. The spatiotemporal organization of myosin II incorporation and actin network rearrangement takes place at a large enough scale to serve as a basis for cell-scale treadmilling of the actin cytoskeleton during steady-state motility in these cells.