BPS2026 – A FRET-based molecular tension sensor reveals actin-driven force dynamics in clathrin-mediated endocytosis

Cytoskeleton assembly generates forces that drive many biological processes; however, understanding how these forces change over time has remained challenging. In budding yeast, clathrin-mediated endocytosis (CME) is a conserved and regular process that proceeds against high turgor pressures comparable to a bike tire, making actin polymerization a requirement for membrane invagination. Complex machinery assembles to harness actin polymerization- and myosin motor-generated forces for membrane invagination. This machinery includes the coat protein Sla2 (Hip1R in humans)—the primary actin-membrane adaptor that transmits forces from the actin cytoskeleton to the plasma membrane. While the timing of recruitment for most CME proteins has been determined, how forces evolve during the generation of endocytic membrane curvature is poorly understood in vivo. Building on prior studies designed to detect forces transmitted through Sla2 and its homologs, we optimized a series of FRET-based molecular tension sensors, enabling simultaneous, quantitative, high-throughput measurements of the time evolution of force, protein recruitment, and internalization for hundreds of individual endocytic sites. We observe that single Sla2-actin interactions bear at least 10pN of mechanical force after the onset of actin polymerization. When inducing elevated membrane tension by hypotonic shock, we observe a large increase in actin-generated force. Our data support a multi-phase model of force generation, by which (1) before actin assembly, no detectable force is transmitted through Sla2; (2) force increases as actin polymerization begins and the membrane starts to invaginate; and (3) force decreases despite continued buildup of the actin network concomitant with vesicle internalization. The transition between the second and third phases is consistent with published mechanical simulations and time-resolved EM of membrane shapes. These tension sensors will enable further understanding of how actin networks generate force and adapt to changes in mechanical load.