Single-molecule live-cell imaging of clathrin-based endocytosis

Biochem Soc Symp. 2005:(72):71-6. doi: 10.1042/bss0720071.

Abstract

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.

Publication types

  • Review

MeSH terms

  • Adaptor Protein Complex 2 / chemistry
  • Adaptor Protein Complex 2 / metabolism
  • Animals
  • Clathrin / chemistry
  • Clathrin / metabolism*
  • Clathrin-Coated Vesicles / chemistry
  • Clathrin-Coated Vesicles / metabolism
  • Endocytosis / physiology*
  • Models, Biological
  • Molecular Structure

Substances

  • Adaptor Protein Complex 2
  • Clathrin