Membrane bending occurs at all stages of clathrin-coat assembly and defines endocytic dynamics

Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. The key mechanistic question of how coat assembly produces membrane curvature has been studied with molecular and cellular structural biology approaches, without direct visualization of the process in living cells; resulting in two competing models for membrane bending. Here we use polarized total internal reflection fluorescence microscopy (pol-TIRF) combined with electron, atomic force, and super-resolution optical microscopy to measure membrane curvature during CME. Surprisingly, coat assembly accommodates membrane bending concurrent with or after the assembly of the clathrin lattice. Once curvature began, CME proceeded to scission with robust timing. Four color pol-TIRF showed that CALM accumulated at high levels during membrane bending, implicating its auxiliary role in curvature generation. We conclude that clathrin-coat assembly is versatile and that multiple membrane-bending trajectories likely reflect the energetics of coat assembly relative to competing forces.