Affordable Access

Publisher Website

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Authors
  • Bhave, Madhura1
  • Mino, Rosa E1
  • Wang, Xinxin1, 2
  • Lee, Jeon2
  • Grossman, Heather M1, 2
  • Lakoduk, Ashley M1
  • Danuser, Gaudenz1, 2
  • Schmid, Sandra L3
  • Mettlen, Marcel3
  • 1 Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
  • 2 Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75390.
  • 3 Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; [email protected] [email protected].
Type
Published Article
Journal
Proceedings of the National Academy of Sciences
Publisher
Proceedings of the National Academy of Sciences
Publication Date
Dec 15, 2020
Volume
117
Issue
50
Pages
31591–31602
Identifiers
DOI: 10.1073/pnas.2020346117
PMID: 33257546
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME. Copyright © 2020 the Author(s). Published by PNAS.

Report this publication

Statistics

Seen <100 times