Abstract
The bulk alignment of actin filament sliding movement, powered by randomly oriented myosin molecules, has been observed and studied using an in vitro motility assay. The well established, actin filament gliding assay is a minimal experimental system for studying actomyosin motility. Here, we show that when the assay is performed at densities of actin filaments approaching those found in living cells, filament gliding takes up a preferred orientation. The oriented patterns of movement that we have observed extend over a length scale of 10–100 μm, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. We developed a simple quantitative measure of filament sliding orientation and this enabled us to follow the time course of alignment and the formation and disappearance of oriented domains. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. Filament alignment due to near-neighbor mechanical interactions can propagate over a length scale of several microns; much greater than the size of individual filaments and analogous to a log drive. Self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which cell migration direction responds to chemical cues.
Highlights
In vitro motility assays (1, 2) provide a defined biochemical system to characterize the mechanochemistry of motor proteins and cytoskeletal polymer dynamics (3)
It is known that actin filaments become aligned by flow and the shear modulus is strongly dependent on filament length (20). It is of interest whether filament alignment might arise spontaneously within the cell cortex due to shear forces and filament movement generated by actomyosin interactions. We have addressed this issue using an in vitro actin filament gliding assay in which actin filament density approaches that found in the cell cortex
In addition to mechanical shearing, the filaments were further shortened in the flow cell due to surface interactions with heavy meromyosin (HMM) molecules
Summary
In vitro motility assays (1, 2) provide a defined biochemical system to characterize the mechanochemistry of motor proteins and cytoskeletal polymer dynamics (3). Under standard in vitro motility assay conditions (i.e. with no additional plain actin) the labeled filaments moved randomly over the surface (Fig. 4).
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