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

Methods Mol Biol. 2018:1847:121-146. doi: 10.1007/978-1-4939-8719-1_10.

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.

Keywords: Auxilin; Clathrin uncoating; Clathrin-coated vesicle; Microfluidics; TIRF microscopy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Auxilins / metabolism
  • Biological Transport
  • Chromatography, Affinity
  • Clathrin / genetics
  • Clathrin / isolation & purification
  • Clathrin / metabolism*
  • Clathrin-Coated Vesicles / metabolism*
  • Clathrin-Coated Vesicles / ultrastructure
  • HSC70 Heat-Shock Proteins / isolation & purification
  • HSC70 Heat-Shock Proteins / metabolism
  • Liposomes / metabolism
  • Microfluidic Analytical Techniques
  • Microscopy, Fluorescence*
  • Molecular Imaging* / methods
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Staining and Labeling

Substances

  • Auxilins
  • Clathrin
  • HSC70 Heat-Shock Proteins
  • Liposomes
  • Recombinant Proteins