Endocytic Accessory Proteins Are Functionally Distinguished by Their Differential Effects on the Maturation of Clathrin-coated Pits

Abstract

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNAmediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation. INTRODUCTIONClathrin-mediated endocytosis (CME) proceeds through the sequential stages of 1) clathrin nucleation at the membrane, 2) clathrin-coated pit (CCP) maturation and invagination, 3) CCP scission, and 4) vesicle uncoating (Conner and Schmid, 2003). The core components of the CME machinery, namely clathrin, the tetrameric adaptor protein AP-2, and the large GTPase dynamin, are assisted by numerous endocytic accessory proteins throughout all stages of CCP maturation (Schmid and McMahon, 2007). Although the function of the core components of the CME machinery is increasingly well understood, the exact function of most other endocytic accessory proteins remains unknown. Among these factors, some such as CALM, epsin1, and SNX9 recognize specific subclasses of cargo molecules (Howard et al., 1999;Harel et al., 2008;Kazazic et al., 2009). Others, such as intersectin, Eps15, SNX9, and endophilin have multiple protein-interaction domains (e.g., Eps15-homolgy [EH] or Src-homology 3 [SH3] domains) and are thought to have scaffolding functions (Miliaras and Wendland, 2004;Ungewickell and Hinrichsen, 2007). Endophilin, SNX9, and epsin have lipid curvature sensing and/or generating domains (e.g., BAR or ENTH domains) and are thought to regulate CME by introducing local biochemical and physical changes into the lipid bilayer, i.e., membrane curvature (Ford et al., 2002;Itoh and De Camilli, 2006). Finally, Hip1R, SNX9, and intersectin are thought to link CME to the actin cytoskeleton and actin dynamics (McPherson, 2002;Le Clainche et al., 2007;Yarar et al., 2007). How these diverse and partially redundant components of the endocytic machinery are integrated and function to create a robust mech...

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…Previous studies defined different populations of CCSs on the basis of size (i.e., spot-like CCSs versus larger CCSs) and lifetime or whether CCSs disappeared following scission (terminal events) or persisted (non-terminal events) [10,23,28,61]. Detailed mechanistic inferences have been based on these types of dynamic classification [23].…”

Regulation of epidermal growth factor receptor signalling by inducible feedback inhibitors

“…The RED was shown to bind to AP-2, the major adaptor complex responsible for sorting cargo into clathrin-coated pits (Traub, 2009), and intersectins, which are SH3-domain-containing accessory proteins involved in cargo sorting and clathrin-coated pit maturation (Fig. 3) (O'Bryan, 2010;Mettlen et al, 2009). These molecular interactions allow coupling of EGFR-RALT complexes to clathrin-mediated endocytosis (Frosi et al, 2010).…”

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

“…Current models suggest that the FCHO proteins initiate membrane bending and cargo clustering via their FCH-BAR (F-BAR) and μ-homology domains, respectively (7,8,11). Simultaneously, Eps15 and intersectin recruit downstream adaptors and accessory proteins, which collectively drive maturation of the nascent endocytic pit (12). The early recruitment of FCHO1/2, Eps15, and intersectin to the plasma membrane suggest they play a role in the nucleation of clathrinmediated endocytosis (7,13).…”