ABSTRACT Eukaryotic cells use clathrin-mediated endocytosis (CME) to internalize nutrients, receptors and recycle their plasma membrane. Defects in endocytosis are implicated in various diseases such as cancer, neuropathies, and metabolic syndromes, and the CME machinery can be hijacked by pathogens to infect cells. Deforming the membrane into ~50-nm endocytic vesicles requires the choregraphed assembly and disassembly of 60+ proteins. Because the CME machinery continuously exchanges and CME is diffraction-limited, the precise molecular mechanisms for force production have remained elusive. Models based on actin polymerization have been proposed but the amount of force they can realistically produce is 1-2 orders of magnitude too low. In this project, we will build on the discoveries we made during last funding period to study new mechanisms of force production at the CME site and determine mechanisms for the regulation of membrane tension, which is a key parameter for endocytosis. In aim 1, we hypothesize that crosslinking highly dynamic actin filaments can produce large amounts of force in a sustained way. We will characterize the biophysical properties of fimbrin in vitro, use mathematical modeling to uncover mechanisms of sustained force production by crosslinking of dynamic actin filaments, and test the model’s predictions by single-molecule tracking at the CME site. Aim 2 will focus on discovering the molecular mechanisms underlying the fast exchange of endocytic proteins we discovered during last funding period. We will test different hypotheses using mathematical modeling, measure the dependence of fimbrin’s detachment rate as a function of force, and determine whether the exchange of endocytic protein depends on the stage of endocytosis. In aim 3, we will characterize mechanisms for the regulation of plasma membrane tension and its influence on CME. We will use optical tweezers to determine whether membrane tension is locally and temporally regulated and use mathematical modeling to understand how a local reduction in membrane tension modulates the forces required for endocytosis. Altogether, our results will uncover new paradigms for dynamic force production and membrane tension control that will impact a variety of cellular processes.