Endocytosis by random initiation and stabilization of clathrin-coated pits.
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.
Ehrlich
Marcelo
M
Department of Cell Biology, Harvard Medical School and The CBR Institute for Biomedical Research, 200 Longwood Avenue, Boston, MA 02115, USA.
Boll
Werner
W
Van Oijen
Antoine
A
Hariharan
Ramesh
R
Chandran
Kartik
K
Nibert
Max L
ML
Kirchhausen
Tomas
T
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
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