title: Endocytosis by random initiation and stabilization of clathrin-coated pits.
abstract: Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or 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 strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.
authors.affiliation: Department of Cell Biology, Harvard Medical School and The CBR Institute for Biomedical Research, 200 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Ehrlich
authors.forename: Werner
authors.initials: W
authors.lastname: Boll
authors.forename: Antoine
authors.initials: A
authors.lastname: Van Oijen
authors.forename: Ramesh
authors.initials: R
authors.lastname: Hariharan
authors.forename: Kartik
authors.initials: K
authors.lastname: Chandran
authors.forename: Max L
authors.initials: ML
authors.lastname: Nibert
authors.forename: Tomas
authors.initials: T
authors.lastname: Kirchhausen
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D000882:Haplorhini; D008077:Lipoproteins, LDL / Q000378:metabolism; D008164:Luminescent Proteins; D008954:Models, Biological; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012087:Reoviridae / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D033962:Adaptor Protein Complex 2; D049452:Green Fluorescent Proteins; D008077:Lipoproteins, LDL; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D000097573:Red Fluorescent Protein