Self-Organization and Force Production by the Branched Actin Cytoskeleton during Mammalian Clathrin-Mediated Endocytosis

Abstract

During clathrin-mediated endocytosis (CME), the cell's plasma membrane is deformed from a flat sheet into a round vesicle to internalize transmembrane proteins and extracellular cargo. Actin participates in mammalian CME, but the mechanism by which it ensures internalization of endocytic pits under variable physical constraints is not understood. To determine the molecular mechanism by which actin organizes to produce force in CME, we combined multi-scale mathematical modeling, cryo-electron tomography, and quantitative fluorescence imaging of diploid human cells endogenously expressing fluorescent fusion proteins. We developed a fluorescence microscopy-based calibration method that relates fluorescence intensity to numbers of molecules in live cells, to measure the number of Arp2/3 complexes at endocytic sites over time. These measurements constrained a multiscale mathematical model of actin polymerization coupled to the internalization of an endocytic pit. The model accounts for the mechanics of a coated plasma membrane, as well as individual actin filaments that diffuse, polymerize, and bend under force. Branched actin filaments are nucleated by active Arp2/3 complex at the base of the pit and attach to the coat via actin-binding proteins. Stochastic simulations of the model revealed that these branched actin networks self-organize at endocytic sites in a dendritic cone between the base and tip of the endocytic pit, which directs the growth of actin filaments toward the base of the pit. This self-organization was robust over a range of conditions, ensuring pit internalization against physiological membrane tension. Surprisingly, long actin filaments bent between their attachment sites at the coat and the base of the pit, which we confirmed with cryo-electron tomography of intact mammalian cells. We suggest that the self-organization and bending of actin filaments allows the actin network to robustly internalize cellular membranes across a range of physical constraints.