Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells

Abstract

Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, β2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones.

Molecular mechanisms of force production in clathrin‐mediated endocytosis

“…The push-pull model is supported by experimental observations that WASp and myosin-I are distributed in a ring-shaped region around the CCP base in budding yeast, while the HIP1R homologs (S. cerevisiae Sla2p and S. pombe End4p), which connect actin filaments with the membrane, are concentrated inside the ring [36,57]. Ongoing experimental efforts will help determine how the actin machinery generates forces in CME, with novel geometries or previously unobserved dynamics (such as enhanced rates of assembly or turnover).…”

Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

“…Filament growth decreases with load according to the Brownian ratchet mechanism (Mogilner and Oster, 1996;Peskin et al, 1993) . Growth of the actin network is coupled to internalization of the endocytic pit by an actin-linking protein (Hip1/Hip1R/Epsin, simplified here as Hip1R), which is embedded in the coated pit and binds to actin filaments (Clarke and Royle, 2018;Engqvist-Goldstein et al, 1999, 2001Sochacki et al, 2017) . Importantly, most of the parameters in this model have been determined with measurements in vitro or in vivo , including the dimensions of the endocytic pit, its resistance to internalization (modeled as a spring, Figure 1D), rates of association and dissociation of different proteins, branching angles, capping rates, filament persistence length, and stall force (Table S3 and Methods).…”

“…Our finding that self-organized endocytic actin networks grow toward the base of the pit prompted us to explore the molecular mechanism by which actin filaments self-organize. Actin dynamics in association with the endocytic machinery can be thought of as a polymerization engine constrained by two spatial boundary conditions --active Arp2/3 complex at the base of the pit (Almeida-Souza et al, 2018;Idrissi et al, 2008;Kaksonen et al, 2003;Mund et al, 2018;Picco et al, 2015 ;Kaplan et al, in preparation) and Hip1R/actin attachments on the curved pit surface (Clarke and Royle, 2018;Engqvist-Goldstein et al, 1999, 2001Sochacki et al, 2017) ( Figure 4A). Given that such spatial boundary conditions confer unique mechanical properties and adaptation to loads under flat geometries in vitro (Bieling et al, 2016) , we aimed to understand how the boundary conditions corresponding to the curved endocytic pit affect endocytic actin organization and internalization.…”

“…Clathrin-mediated endocytosis (CME) is an especially attractive process for studies of actin's role in membrane shape changes due to the relatively complete parts list and available quantitative information about the positions, recruitment timing and biochemical function of many of the participating proteins (Kaksonen et al, 2003;Sochacki et al, 2017;Taylor et al, 2011) . CME is a ubiquitous and essential cellular process by which cells take macromolecules from the extracellular space and the plasma membrane into the cell interior (Kaksonen and Roux, 2018) .…”

“…Using this framework, we sought to answer the following questions: How do branched actin networks assemble, organize, and produce force around an endocytic pit? How does the spatial segregation of Arp2/3 complex activators (Almeida-Souza et al, 2018;Mund et al, 2018) and actin-binding proteins associated with endocytic coats (Clarke and Royle, 2018;Engqvist-Goldstein et al, 2001;Sochacki et al, 2017) influence this organization? And finally, how do endocytic actin networks adapt to changing loads due to the stochastic environment and changes in membrane tension?…”