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.
Elife. 2020 Apr 30;9:e53686. doi: 10.7554/eLife.53686.
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 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.
网格蛋白介导的内吞作用（CME）在哺乳动物细胞中由包括>70 种内吞辅助蛋白（EAP）的弹性机制驱动。因此，单个 EAP 的扰动通常会对 CME 的生化测量产生较小的影响，从而提供关于 EAP 功能的不确定/误导性信息。活细胞成像可以检测到货物内化之前 EAP 的早期作用；然而，由于需要但尚未完成明确区分无功能包被（ACs）和网格蛋白包被凹陷（CCPs），因此该方法受到限制。在这里，我们开发了一种受热力学启发的方法，“解体不对称评分分类（DASC）”，该方法基于单通道荧光电影从 CCPs 中区分 ACs。经过广泛验证，我们使用 DASC 区分的 ACs 和 CCPs 来量化 11 种 EAP 敲低条件下的 CME 进展。我们表明，DASC 是 CCP 动力学表型变化的敏感探测器，与 CME 的生化测量变化无关。因此，DASC 是揭示 EAP 功能的重要工具。
