Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States
Marcel Mettlen
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States
Jungsik Noh
Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States
Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States
Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, “disassembly asymmetry score classification (DASC)”, that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.
