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Significance Clathrin-mediated endocytosis (CME), the major pathway for uptake into cells, is a multistep process involving a myriad of endocytic accessory proteins (EAPs). Although the biochemical activities of many EAPs have been extensively studied, their stage-specific role(s) during clathrin-coated pit (CCP) initiation, stabilization, and/or maturation are poorly defined. Here, using quantitative total internal reflection fluorescence microscopy and a rigorous experimental and multiparametric analytical pipeline, we study the effect of CRISPR interference (CRISPRi)- or small interfering RNA (siRNA)-mediated knockdown of 67 individual EAPs on CCP dynamics. Our comprehensive analyses combined with unsupervised phenotypic clustering reveal the complex and overlapping roles of EAPs during early, critical stages of CME, providing a valuable resource to spur further research into their function.

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