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Hot-wiring clathrin-mediated endocytosis in human cells

Laura A. Wood, Nicholas I. Clarke, Sourav Sarkar, View ORCID ProfileStephen J. Royle
doi: https://doi.org/10.1101/061986
Laura A. Wood
Centre for Mechanochemical Cell Biology Warwick Medical School, Gibbet Hill Road, Aerodyne Research, Inc. University of Warwick, Coventry, CV4 7AL, UK
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Nicholas I. Clarke
Centre for Mechanochemical Cell Biology Warwick Medical School, Gibbet Hill Road, Aerodyne Research, Inc. University of Warwick, Coventry, CV4 7AL, UK
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Sourav Sarkar
Centre for Mechanochemical Cell Biology Warwick Medical School, Gibbet Hill Road, Aerodyne Research, Inc. University of Warwick, Coventry, CV4 7AL, UK
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Stephen J. Royle
Centre for Mechanochemical Cell Biology Warwick Medical School, Gibbet Hill Road, Aerodyne Research, Inc. University of Warwick, Coventry, CV4 7AL, UK
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Abstract

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is complicated by the fact that initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. This is achieved by inducibly tethering a clathrin “hook” to a plasma membrane “anchor”. Our design incorporates temporal and spatial control of initiation using chemical and optical tools, and the cargo is defined. Since this system bypasses multiple steps in vesicle creation, we term it “hot-wiring”. In this paper, we use hot-wired endocytosis to define the functional interactions between clathrin and the β2 subunit of the AP2 complex. However, there are numerous applications for this new technology, which we hope will be broadly useful to the field.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted December 14, 2016.
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Hot-wiring clathrin-mediated endocytosis in human cells
Laura A. Wood, Nicholas I. Clarke, Sourav Sarkar, Stephen J. Royle
bioRxiv 061986; doi: https://doi.org/10.1101/061986
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Hot-wiring clathrin-mediated endocytosis in human cells
Laura A. Wood, Nicholas I. Clarke, Sourav Sarkar, Stephen J. Royle
bioRxiv 061986; doi: https://doi.org/10.1101/061986

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