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Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions

The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retrovi...

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Autores principales: Hoffman, Huxley K., Fernandez, Melissa V., Groves, Nicholas S., Freed, Eric O., van Engelenburg, Schuyler B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827313/
https://www.ncbi.nlm.nih.gov/pubmed/31519756
http://dx.doi.org/10.1074/jbc.RA119.009372
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author Hoffman, Huxley K.
Fernandez, Melissa V.
Groves, Nicholas S.
Freed, Eric O.
van Engelenburg, Schuyler B.
author_facet Hoffman, Huxley K.
Fernandez, Melissa V.
Groves, Nicholas S.
Freed, Eric O.
van Engelenburg, Schuyler B.
author_sort Hoffman, Huxley K.
collection PubMed
description The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retroviruses, such as HIV-1, to release virions from infected cells. The crucial roles of the ESCRTs in cellular physiology and viral disease make it imperative to understand the membrane scission mechanism. Current methodological limitations, namely artifacts caused by overexpression of ESCRT subunits, obstruct our understanding of the spatiotemporal organization of the endogenous human ESCRT machinery. Here, we used CRISPR/Cas9-mediated knock-in to tag the critical ESCRT-I component tumor susceptibility 101 (Tsg101) with GFP at its native locus in two widely used human cell types, HeLa epithelial cells and Jurkat T cells. We validated this approach by assessing the function of these knock-in cell lines in cytokinesis, receptor degradation, and virus budding. Using this probe, we measured the incorporation of endogenous Tsg101 in released HIV-1 particles, supporting the notion that the ESCRT machinery initiates virus abscission by scaffolding early-acting ESCRT-I within the head of the budding virus. We anticipate that these validated cell lines will be a valuable tool for interrogating dynamics of the native human ESCRT machinery.
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spelling pubmed-68273132019-11-05 Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions Hoffman, Huxley K. Fernandez, Melissa V. Groves, Nicholas S. Freed, Eric O. van Engelenburg, Schuyler B. J Biol Chem Methods and Resources The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retroviruses, such as HIV-1, to release virions from infected cells. The crucial roles of the ESCRTs in cellular physiology and viral disease make it imperative to understand the membrane scission mechanism. Current methodological limitations, namely artifacts caused by overexpression of ESCRT subunits, obstruct our understanding of the spatiotemporal organization of the endogenous human ESCRT machinery. Here, we used CRISPR/Cas9-mediated knock-in to tag the critical ESCRT-I component tumor susceptibility 101 (Tsg101) with GFP at its native locus in two widely used human cell types, HeLa epithelial cells and Jurkat T cells. We validated this approach by assessing the function of these knock-in cell lines in cytokinesis, receptor degradation, and virus budding. Using this probe, we measured the incorporation of endogenous Tsg101 in released HIV-1 particles, supporting the notion that the ESCRT machinery initiates virus abscission by scaffolding early-acting ESCRT-I within the head of the budding virus. We anticipate that these validated cell lines will be a valuable tool for interrogating dynamics of the native human ESCRT machinery. American Society for Biochemistry and Molecular Biology 2019-11-01 2019-09-13 /pmc/articles/PMC6827313/ /pubmed/31519756 http://dx.doi.org/10.1074/jbc.RA119.009372 Text en © 2019 Hoffman et al. Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle Methods and Resources
Hoffman, Huxley K.
Fernandez, Melissa V.
Groves, Nicholas S.
Freed, Eric O.
van Engelenburg, Schuyler B.
Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions
title Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions
title_full Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions
title_fullStr Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions
title_full_unstemmed Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions
title_short Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions
title_sort genomic tagging of endogenous human escrt-i complex preserves escrt-mediated membrane-remodeling functions
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827313/
https://www.ncbi.nlm.nih.gov/pubmed/31519756
http://dx.doi.org/10.1074/jbc.RA119.009372
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