Reconstitution of Clathrin Coat Disassembly for Fluorescence Microscopy and Single-Molecule Analysis

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2018 Aug 22

PMID 30129014

Citations 3

Authors

Till Bocking

Srigokul Upadhyayula

Iris Rapoport

Benjamin R Capraro

Tom Kirchhausen

Affiliations

Soon will be listed here.

Abstract

The disassembly of the clathrin lattice surrounding coated vesicles is the obligatory last step in their life cycle. It is mediated by the coordinated recruitment of auxilin and Hsc70, an ATP-driven molecular clamp. Here, we describe the preparation of reagents and the single-particle fluorescence microscopy imaging assay in which we visualize directly the Hsc70-driven uncoating of synthetic clathrin coats or clathrin-coated vesicles.

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References

Ehrlich M, Boll W, van Oijen A, Hariharan R, Chandran K, Nibert M . Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell. 2004; 118(5):591-605. DOI: 10.1016/j.cell.2004.08.017. View

Fotin A, Cheng Y, Sliz P, Grigorieff N, Harrison S, Kirchhausen T . Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature. 2004; 432(7017):573-9. DOI: 10.1038/nature03079. View

Fotin A, Cheng Y, Grigorieff N, Walz T, Harrison S, Kirchhausen T . Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating. Nature. 2004; 432(7017):649-53. DOI: 10.1038/nature03078. View

Merrifield C, Perrais D, Zenisek D . Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell. 2005; 121(4):593-606. DOI: 10.1016/j.cell.2005.03.015. View

Massol R, Boll W, Griffin A, Kirchhausen T . A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating. Proc Natl Acad Sci U S A. 2006; 103(27):10265-10270. PMC: 1502446. DOI: 10.1073/pnas.0603369103. View

Lee D, Wu X, Eisenberg E, Greene L . Recruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis. J Cell Sci. 2006; 119(Pt 17):3502-12. DOI: 10.1242/jcs.03092. View

Rapoport I, Boll W, Yu A, Bocking T, Kirchhausen T . A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction. Mol Biol Cell. 2007; 19(1):405-13. PMC: 2174180. DOI: 10.1091/mbc.e07-09-0870. View

Millare B, Thomas M, Ferreira A, Xu H, Holesinger M, Vullev V . Dependence of the quality of adhesion between poly(dimethylsiloxane) and glass surfaces on the conditions of treatment with oxygen plasma. Langmuir. 2008; 24(22):13218-24. DOI: 10.1021/la801965s. View

Bocking T, Aguet F, Harrison S, Kirchhausen T . Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol. 2011; 18(3):295-301. PMC: 3056279. DOI: 10.1038/nsmb.1985. View

10.

Aguet F, Antonescu C, Mettlen M, Schmid S, Danuser G . Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint. Dev Cell. 2013; 26(3):279-91. PMC: 3939604. DOI: 10.1016/j.devcel.2013.06.019. View

11.

Braell W, Schlossman D, Schmid S, Rothman J . Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase. J Cell Biol. 1984; 99(2):734-41. PMC: 2113264. DOI: 10.1083/jcb.99.2.734. View

12.

Ungewickell E, Ungewickell H, Holstein S, Lindner R, Prasad K, Barouch W . Role of auxilin in uncoating clathrin-coated vesicles. Nature. 1995; 378(6557):632-5. DOI: 10.1038/378632a0. View

13.

Barouch W, Prasad K, Greene L, Eisenberg E . Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets. Biochemistry. 1997; 36(14):4303-8. DOI: 10.1021/bi962727z. View