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Imaging “Hot-Wired” Clathrin-Mediated Endocytosis

  • Laura A. Wood
  • Stephen J. Royle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1847)

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.

Key words

Clathrin Endocytosis Chemically induced dimerization Live cell imaging Immunofluorescence Image analysis 

Notes

Acknowledgments

L.A. Wood was supported by the Medical Research Council Doctoral Training Partnership grant (MR/J003964/1).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Centre for Mechanochemical Cell BiologyWarwick Medical SchoolCoventryUK
  2. 2.Department of Biology and BiochemistryUniversity of BathBathUK

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