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

TLDR

Actin networks generate forces for cellular movements such as lamellipodia protrusion and vesicle transport, and actin‑based propulsion of Listeria or functionalized beads has been used to identify the biochemical components required for motility. The study aims to obtain quantitative force measurements to clarify the debated mechanism of actin‑generated force. A micromanipulation assay was developed in which a comet‑forming bead is held by a micropipette and a force probe via a flexible handle, allowing application of pulling or pushing forces up to a few nanonewtons to measure elastic modulus, detachment force, and to establish a force–velocity curve. The force–velocity relation is linear for pulling forces and decays more weakly for pushing forces, a behavior explained by dimensional elastic analysis.

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.