Imaging “Hot-Wired” clathrin-mediated endocytosis

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.