Abstract
During clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane, and their spatial and temporal coordination is crucial for efficient CME. Here, we show that the scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. Live-cell imaging of genome-edited cells revealed that endogenously labeled ITSN1 is recruited during CME site stabilization and growth and that ITSN1 knockdown impairs endocytic protein recruitment during this stage. Targeting ITSN1 to the mitochondrial surface was sufficient to assemble puncta consisting of the EPS15 and FCHO2 initiation proteins, the AP2 and epsin1 (EPN1) adaptor proteins, and the dynamin2 (DNM2) vesicle scission GTPase. ITSN1 can form puncta and recruit DNM2 independent of EPS15/FCHO2 or EPN1. Our findings redefine ITSN1's primary endocytic role as organizing and stabilizing CME protein interaction networks rather than initiation, providing deeper insights into the multi-step and multi-zone organization of CME site assembly.