Single-molecule live-cell imaging of clathrin-based endocytosis.
Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.
Kirchhausen
Tomas
T
Department of Cell Biology, Harvard Medical School and Center for Blood Research, 200 Longwood Avenue, Boston, MA 02115, U.S.A. Kirchhausen@crystal.harvard.edu
Boll
Werner
W
van Oijen
Antoine
A
Ehrlich
Marcelo
M
D033962:Adaptor Protein Complex 2 / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000737:chemistry / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D008954:Models, Biological; D015394:Molecular Structure
D033962:Adaptor Protein Complex 2; D002966:Clathrin