Power transduction of actin filaments ratcheting in vitro against a load

The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.The actin cytoskeleton forms a signal-responsive protein system made of filaments that undergo constant remodeling via directional assembly and disassembly processes. At the leading edge of a migrating cell, protrusive force results from insertional polymerization of actin filament barbed ends against the membrane, pushing it forward (1, 2). A wealth of regulatory proteins adapts the organization of the filaments and their mechanical properties to the movement the cell needs to make. At the single filament level, Hill was the first to propose that the free energy of polymerization could be transduced into mechanical work against the membrane (3). Oster and colleagues transcribed Hill’s conceptual model into a mechanistic one coined Brownian ratchet (4), later refined in the tethered ratchet (5). They showed that a single filament can push against a load because thermal fluctuations of either the load (4) or the filament (5) allow for stochastic insertion of monomers at the polymerizing tip. The amplitude of the fluctuations are force dependent, making the elongation velocity of the filament force dependent as well. The whole force-velocity profile of a single actin filament was never measured experimentally. However, the stalling force of a filament, at which the elongation velocity drops to zero, could be estimated to a few piconewtons (6–8). Comparatively, forces of a few nanonewtons are required to stall the migration of cells (9, 10) or Listeria comet tails (11). This difference of three orders of magnitude points to the need for a large number of cooperating filaments to generate high forces in protrusive structures. Exploring the cooperation within an assembly of filaments polymerizing together against a load remains a hard task for experimentalists. Actin gels (12–14) or brushes (15) have been reconstituted in vitro to measure the amount of force they can generate. In these complex structures, regulatory proteins can cause tethering of the filaments to the load (14), rearrangements under force (16), or variations of filaments number in reaction to force (17). These phenomena shed light on the strong influence of regulatory proteins on force production but impede to draw information on the physical mechanism of force generation.Here we present an experimental setup that was designed to closely resemble the conceptual view of an array of about 100 independent filaments polymerizing perpendicularly to a load, their tip remaining nontethered to that load. Because the number of parameters affecting the force generation is minimal, we are able to concentrate on the physical interaction of filaments with the load and show that it is compatible with the Brownian ratchet. However, if the Brownian ratchet model is relevant to describe force generation at the single filament level, it gives no information about the collective behavior of the filaments population. This collective aspect of force production is further developed using an analytical model, revealing that filaments cooperation is optimal in a specific regime which ensures maximum transduction of mechanical power to the load.