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.

网格蛋白介导的内吞作用（CME）始于网格蛋白在质膜上组装的成核，然后是网格蛋白包被的小坑（CCP）的稳定和生长/成熟，最终夹断并内化为网格蛋白包被的囊泡。这一高度调控的过程涉及大量的内吞辅助蛋白 (EAP)，其中许多是编码多种生化活性的多域蛋白。尽管 EAP 的特定领域活动已被广泛研究，但仅在少数情况下确定了其精确的特定阶段功能。使用单引导RNA (sgRNA)/dCas9和小干扰RNA (siRNA)介导的蛋白质敲除，结合基于图像的分析流程，我们确定了在CCP成熟过程中67个EAP的表型特征。基于这些数据，我们表明 EAP 可以分为表型簇，这对 CCP 的成熟和动态有不同的影响。重要的是，这些簇与基于生化活动的功能模块不相关。此外，我们发现 SNARE 蛋白及其接头在 CCP 成核和稳定的早期阶段发挥着关键作用，并强调了 GAK 在整个 CCP 成熟过程中的重要性，这与 GAK 的多功能域结构一致。总之，这些发现为 CME 的可塑性和稳健性提供了系统、机械的见解。