Böcking Till, Upadhyayula Srigokul, Rapoport Iris, Capraro Benjamin R, Kirchhausen Tom
ARC Centre of Excellence in Advanced Molecular Imaging and EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.
Department of Cell Biology, Harvard Medical School; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
Methods Mol Biol. 2018;1847:121-146. doi: 10.1007/978-1-4939-8719-1_10.
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.
围绕被膜小泡的网格蛋白晶格的拆解是其生命周期中必不可少的最后一步。这一过程由辅助蛋白和Hsc70（一种由ATP驱动的分子钳）的协同募集介导。在此，我们描述了试剂的制备以及单颗粒荧光显微镜成像分析方法，通过该方法我们可以直接观察到Hsc70驱动的合成网格蛋白衣被或网格蛋白包被小泡的脱衣被过程。
