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
The original version of this chapter was revised. A correction to this chapter can be found at https://doi.org/10.1007/978-1-4939-8719-1_19
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsChange history
24 October 2018
This book was inadvertently published with the incorrect title as Clathrin-Mediated Endoytosis: Methods and Protocols. This has now been corrected throughout the book to Clathrin-Mediated Endocytosis: Methods and Protocols.
References
Ehrlich M, Boll W, Van Oijen A, Hariharan R, Chandran K, Nibert ML, Kirchhausen T (2004) Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118(5):591–605. https://doi.org/10.1016/j.cell.2004.08.017
Taylor MJ, Perrais D, Merrifield CJ (2011) A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. PLoS Biol 9(3):e1000604. https://doi.org/10.1371/journal.pbio.1000604
Dannhauser PN, Ungewickell EJ (2012) Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat Cell Biol 14(6):634–639. https://doi.org/10.1038/ncb2478
Wood LA, Larocque G, Clarke NI, Sarkar S, Royle SJ (2017) New tools for “hot-wiring” clathrin-mediated endocytosis with temporal and spatial precision. J Cell Biol. https://doi.org/10.1083/jcb.201702188
Kelly BT, Graham SC, Liska N, Dannhauser PN, Honing S, Ungewickell EJ, Owen DJ (2014) Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch. Science 345(6195):459–463. https://doi.org/10.1126/science.1254836
Rivera VM, Clackson T, Natesan S, Pollock R, Amara JF, Keenan T, Magari SR, Phillips T, Courage NL, Cerasoli F Jr, Holt DA, Gilman M (1996) A humanized system for pharmacologic control of gene expression. Nat Med 2(9):1028–1032
Kozik P, Francis RW, Seaman MN, Robinson MS (2010) A screen for endocytic motifs. Traffic 11(6):843–855. https://doi.org/10.1111/j.1600-0854.2010.01056.x
Banaszynski LA, Liu CW, Wandless TJ (2005) Characterization of the FKBP.rapamycin.FRB ternary complex. J Am Chem Soc 127(13):4715–4721. https://doi.org/10.1021/ja043277y
Subach FV, Malashkevich VN, Zencheck WD, Xiao H, Filonov GS, Almo SC, Verkhusha VV (2009) Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc Natl Acad Sci U S A 106(50):21097–21102. https://doi.org/10.1073/pnas.0909204106
Owen DJ, Vallis Y, Pearse BM, McMahon HT, Evans PR (2000) The structure and function of the beta 2-adaptin appendage domain. EMBO J 19(16):4216–4227. https://doi.org/10.1093/emboj/19.16.4216
Acknowledgments
L.A. Wood was supported by the Medical Research Council Doctoral Training Partnership grant (MR/J003964/1).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wood, L.A., Royle, S.J. (2018). Imaging “Hot-Wired” Clathrin-Mediated Endocytosis. In: Swan, L. (eds) Clathrin-Mediated Endocytosis. Methods in Molecular Biology, vol 1847. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8719-1_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8719-1_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8717-7
Online ISBN: 978-1-4939-8719-1
eBook Packages: Springer Protocols