A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

TLDR

Dual‑color TIRF microscopy enables high‑precision tracking of clathrin‑mediated endocytosis, yet the reliability of using clathrin‑coated structure disappearance as a marker for canonical CME remains unclear. The authors employed a ~2‑s resolution TIRF assay to detect scission events and quantified the recruitment dynamics of 34 endocytic proteins, aligning ~1,000 profiles per protein to generate recruitment signatures that expose the modular organization of mammalian CME. Analysis uncovered unexpected recruitment patterns of SNX9, FBP17, and CIP4, showing that the same protein set is recruited in identical order to scission events across clathrin‑coated structures of varying size and lifetime, supporting a simplified canonical model in which actin‑driven CME operates uniformly.

Abstract

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.