成像“热连接”网格蛋白介导的内吞作用。

Imaging "Hot-Wired" Clathrin-Mediated Endocytosis.

作者信息

Wood Laura A, Royle Stephen J

机构信息

Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry, UK.

Department of Biology and Biochemistry, University of Bath, Bath, UK.

出版信息

Methods Mol Biol. 2018;1847:83-94. doi: 10.1007/978-1-4939-8719-1_7.

DOI:10.1007/978-1-4939-8719-1_7

PMID:30129011

Abstract

Clathrin-mediated endocytosis (CME) occurs continuously at the plasma membrane of eukaryotic cells. However, when a vesicle forms and what cargo it contains are unpredictable. We recently developed a system to trigger CME on-demand. This means that we can control when endocytosis is triggered and the design means that the cargo that is internalized is predetermined. The method is called hot-wired CME because several steps and proteins are bypassed in our system. In this chapter, we describe in detail how to use the hot-wiring system to trigger endocytosis in human cell lines and how to image the vesicles that form using microscopy and finally, how to analyze those images.

摘要

网格蛋白介导的内吞作用（CME）在真核细胞的质膜上持续发生。然而，囊泡何时形成以及其中包含何种货物是不可预测的。我们最近开发了一种按需触发CME的系统。这意味着我们可以控制内吞作用何时被触发，并且该设计意味着内化的货物是预先确定的。该方法被称为热连线CME，因为在我们的系统中绕过了几个步骤和蛋白质。在本章中，我们将详细描述如何使用热连线系统在人类细胞系中触发内吞作用，以及如何使用显微镜对形成的囊泡进行成像，最后，如何分析这些图像。