Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300–880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes. •FRET sensors in Sla2 report the actin-generated forces required for endocytosis•At least 8 pN force is transmitted by the Sla2-Ent1 membrane-actin protein linker•Large amount of force is needed for initial membrane bending and early invagination•Lowering cell turgor or membrane tension reduces force transmitted over the linker Abella et al., using FRET-based tension sensors inserted into the endocytic membrane-actin protein linker Sla2-Ent1 (Hip1R-epsin in human), measure the forces applied by the polymerizing actin cytoskeleton during endocytic membrane invagination under normal as well as genetically and environmentally perturbed conditions.