a high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis分子动力学的高精度调查哺乳动物clathrin-mediated内吞作用.pdfVIP

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a high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis分子动力学的高精度调查哺乳动物clathrin-mediated内吞作用.pdf

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a high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis分子动力学的高精度调查哺乳动物clathrin-mediated内吞作用

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis 1 2,3 1 Marcus J. Taylor , David Perrais , Christien J. Merrifield * ´ 1 Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, 2 Universite de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France, 3 CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France 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, synaptojanin2b1, and