Forces generated during actin-based propulsion: A direct measurement by micromanipulation

Abstract

Dynamic actin networks generate forces for numerous types of movements such as lamellipodia protrusion or the motion of endocytic vesicles. The actin-based propulsive movement of Listeria monocytogenes or of functionalized microspheres have been extensively used as model systems to identify the biochemical components that are necessary for actin-based motility. However, quantitative force measurements are required to elucidate the mechanism of force generation, which is still under debate. To directly probe the forces generated in the process of actin-based propulsion, we developed a micromanipulation experiment. A comet growing from a coated polystyrene bead is held by a micropipette while the bead is attached to a force probe, by using a specially designed ''flexible handle.'' This system allows us to apply both pulling and pushing external forces up to a few nanonewtons. By pulling the actin tail away from the bead at high speed, we estimate the elastic modulus of the gel and measure the force necessary to detach the tail from the bead. By applying a constant force in the range of ؊1.7 to 4.3 nN, the force-velocity relation is established. We find that the relation is linear for pulling forces and decays more weakly for pushing forces. This behavior is explained by using a dimensional elastic analysis.T he growth of a polymer against a barrier is a general mechanism for production of force in cell biology (1). Spatially controlled polymerization of actin is responsible for a large variety of changes in cell shape leading to cell movement, like the protrusion of the lamellipodium (2), or the intracellular propulsion of vesicles (3, 4) and pathogens (5, 6). Actin assembly in these processes is controlled by various proteins present in the cytoplasm, which have been identified over the last 10 years (see refs. 7 and 8 for review). It is now well established that the Arp2͞3 complex associates with Wiskott-Aldrich Syndrome protein (WASp) family proteins to create new filaments locally by branching them (9), thus generating the dendritic organization of the actin array (10, 11). Because pure actin treadmilling is too slow to account for polymerization speed observed in these systems, this process has to be speeded up by regulatory proteins (8). One experimental model that has been used widely for the identification of these proteins is the bacteria Listeria monocytogenes, which are propelled inside cells by actin polymerization. The minimal subset of proteins necessary and sufficient to reconstitute the Listeria movement has been identified (12). In addition to Arp2͞3 and actin, the regulatory proteins ADF͞ cofilin, profilin, and a capping protein are necessary to enhance the efficiency of treadmilling (13) and control the life time and average length of filaments (14, 15). The essential role of these proteins has been confirmed recently (16) in vivo by using a RNA interference strategy.Biomimetic systems are useful for analyzing the physical aspects of actin-based motility. Listeria movement can be mimicked by fun...

Direct measurement of force generation by actin filament polymerization using an optical trap

“…However, it is also possible that parallel filament bundles are simply relatively inefficient at transducing the energy of actin polymerization into mechanical work against a rigid barrier, compared with other forms of actin filament organization such as the branched dendritic network that is found in lamellipodia (39) and actin ''comet tails'' associated with intracellular bacterial pathogens (40). In a dendritic network, actin filaments contact the load at a variety of angles, and constant branching nucleation of new filaments near the leading edge ensures that a large number of actively growing filament ends are in direct contact (7,22), enabling cooperation in generating force against the surface of a load that can result in efficient transduction of several nN/ m 2 (14,15). Biologically, the role of filopodia in exploring the environment and forming nascent adhesive structures in response to external signaling cues is well established (41,42).…”

“…Although the theoretical basis for understanding the origin of forces generated by actin polymerization is well developed, complementary experimental progress has been relatively slow. Recently, direct measurement of forces generated by the growth of densely branched networks comprising thousands of actin filaments has been achieved by using deflection of glass microneedles (14) and silicon cantilevers (15), and network forces have been estimated by using a variety of less direct experimental techniques (16)(17)(18)(19)(20)(21), giving values for the force of actin network growth in the range of several nN/ m 2 . It is not possible to extract information from these bulk experiments about the force-velocity relationship for single actin filaments, both because of the difficulty of accurately measuring the number of actin filaments in the networks and because the history of force loading on the network affects the network density (15,16,22).…”

A kinematic description of the trajectories of Listeria monocytogenes propelled by actin comet tails

“…In general, the propulsive force generated by the filaments is balanced either by the viscous drag from the surrounding fluid or by the frictional/adhesive force due to breaking of the bonds that tether some of the actin filaments to the surface of the bacterium (8,10,16). However, for bacteria moving in the cytoplasmic extract with a viscosity of Ϸ0.01 Pa⅐s, the drag force is Ϸ10 fN, which is much smaller than the forces that tether the filament network to the bacterial surface, which are in the range of few hundred piconewtons to a few nanonewtons (8,(11)(12)(13). A very recent experiment (16) that considers the temperature dependence of the speed of motile bacteria finds an activated Arrhenius behavior consistent with the breaking of tethered bonds.…”

Impact of branching on the elasticity of actin networks

“…Previous experiments based on these techniques have used purified actin filaments either entangled or crosslinked by various proteins, forming loose networks (16). Measurements have been performed on branched actin networks grown form a surface via the Arp2/3 complex, as in our experiments (17,18). These experiments are delicate, as they use macroscopic force sensor such as AFM and micropipette, which prevents the authors from studying a large range of parameters.…”