Mechanisms of actin force production in clathrin-mediated endocytosis revealed by integrating computational modeling with in situ cryo-electron tomography

During clathrin-mediated endoytosis, the plasma membrane is deformed, forming clathrin-coated vesicles (CCVs) containing cargo. Membrane remodeling is supported by actin filament assembly, but its mode of function remains elusive. We previously used an experimentally constrained mathematical model to find that a minimal endocytic actin network can self-organize, bend, and produce sufficient force at sites of CME for pit internalization (Akamatsu et al., eLife 2020). Here we used cryo-electron tomography (cryo-ET) of intact mammalian cells to directly visualize networks of individual actin filaments at CME sites and CCVs, and used mathematical modeling to identify their mechanistic functions. Surprisingly, actin networks at CME sites consisted of both branched and unbranched filaments. Finally, we identified long proteins ~60 nm in length resembling the actin-CME linker Hip1R, both in the clathrin-coated area and in the neck of the pit. Mathematical modeling showed that this Hip1R neck localization not only directs filament growth toward the neck of CME sites, it also results in increased internalization efficiency. By combining mathematical modeling and in situ cryo-ET, deeper insights into actin mechanism during CME were achieved than either approach alone.