Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation

Markus Mund,Frannois Nnddlec,Marko Kaksonen,Jonas Ries,Serge Dmitrieff,Andrea Picco,Joran Deschamps,Jooske Louiser Monster,Johannes Albertus Van Der Beek

doi:10.2139/ssrn.3155759

Markus Mund, Frannois Nnddlec + Show 7 more

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https://doi.org/10.2139/ssrn.3155759

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Abstract

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

Highlights

Clathrin-mediated endocytosis (CME) is critical for many biological processes such as signaling, nutrient uptake, and pathogen entry and involves the internalization of cargo molecules from the cell surface into small membrane vesicles
Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies
Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis

Summary

Introduction

Clathrin-mediated endocytosis (CME) is critical for many biological processes such as signaling, nutrient uptake, and pathogen entry and involves the internalization of cargo molecules from the cell surface into small membrane vesicles. CME follows a stereotypic order of events: first, a protein coat assembles on the membrane, which invaginates to form a vesicle with cargo molecules inside. This vesicle is pinched off the plasma membrane and rapidly uncoats allowing fusion with endosomes. Live-cell imaging has revealed the order of assembly of components and categorized them into modules based on their dynamics (Kaksonen et al, 2003, 2005) This modular organization is remarkably conserved in metazoans (Boettner et al, 2011; Kaksonen and Roux, 2018; McMahon and Boucrot, 2011; Weinberg and Drubin, 2012)

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