Imaging “Hot-Wired” Clathrin-Mediated Endocytosis
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.
Key wordsClathrin Endocytosis Chemically induced dimerization Live cell imaging Immunofluorescence Image analysis
L.A. Wood was supported by the Medical Research Council Doctoral Training Partnership grant (MR/J003964/1).
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