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

Brandon L Scott,Adam D Hoppe,Shalini T Low-Nam,Steve Smith,Jason G Kerkvliet,Andrea M Dickey,Elizabeth M Bailey,Kem A Sochacki,Quocahn Luu,Justin W Taraska,Amy Hor

doi:10.1038/s41467-018-02818-8

Abstract

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.

Highlights

Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles
This work demonstrates that the clathrin coat is flexible and that the rate-limiting step for vesicle formation is the induction of membrane bending, which may be regulated by axillary proteins such as CALM
Measuring the dynamics of membrane bending during clathrin assembly at single endocytic sites in living cells is necessary to distinguish the possible modes of membrane bending

Summary

Introduction

Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. Energetic arguments suggest that this structural conversion would be energetically costly, requiring large protein contacts or ostensibly rigid structures to be broken and reformed during the hexagon to pentagon transition This leads to a model in which membrane bending occurs progressively with the assembly of clathrin[7,8,11] (Fig. 1a). We demonstrate that in cells genome edited to express fluorescent protein-tagged clathrin and dynamin, clathrin-coated structures can form as curved membranes that accumulate clathrin or as pre-accumulated flat clathrin sites that bend membrane to form pits Both behaviors were observed within single living cells, suggesting that local biochemical and biophysical factors can mediate a switch between these two modes. This work demonstrates that the clathrin coat is flexible and that the rate-limiting step for vesicle formation is the induction of membrane bending, which may be regulated by axillary proteins such as CALM

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Conclusion