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Vol. 20, Issue 14, 3251-3260, July 15, 2009
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Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
Submitted March 31, 2009;
Revised May 8, 2009;
Accepted May 12, 2009
Monitoring Editor: Sandra Lemmon
Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.
* These authors contributed equally to this work.
Author contributions: M.S., C.A., and M.M. performed all the cell biological, biochemical, and microscopic assays; D.L. designed and wrote the lifetime analysis software; M.M., M.S., and D.L. carried out data processing; S.L.S. and G.D. provided experimental design and assistance in interpretation; M.M. prepared the figures; M.M., M.S., D.L., G.D., and S.L.S. wrote the manuscript.
Present address: ETH, Institute for Biochemistry, Schafmattstraße, 18, 8093 Zurich, Switzerland.
Address correspondence to: Sandra L. Schmid (slschmid{at}scripps.edu)
Abbreviations used: CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; CME, clathrin-mediated endocytosis; TIR-FM, total internal reflection fluorescence microscopy.
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