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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2019
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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. |
format | Online Article Text |
id | pubmed-6827313 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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