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

Journal Article


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. © 2005 The Biochemical Society.

Publication Date


  • 2005

Citation


  • Kirchhausen, T., Boll, W., Van Oijen, A., & Ehrlich, M. (2005). Single-molecule live-cell imaging of clathrin-based endocytosis. Biochemical Society Symposium, 72, 71-76. doi:10.1042/bss0720071

Scopus Eid


  • 2-s2.0-15544367894

Start Page


  • 71

End Page


  • 76

Volume


  • 72

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. © 2005 The Biochemical Society.

Publication Date


  • 2005

Citation


  • Kirchhausen, T., Boll, W., Van Oijen, A., & Ehrlich, M. (2005). Single-molecule live-cell imaging of clathrin-based endocytosis. Biochemical Society Symposium, 72, 71-76. doi:10.1042/bss0720071

Scopus Eid


  • 2-s2.0-15544367894

Start Page


  • 71

End Page


  • 76

Volume


  • 72