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A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-c...

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Autores principales: Mageswaran, Shrawan Kumar, Yang, Wei Yuan, Chakrabarty, Yogaditya, Oikonomou, Catherine M., Jensen, Grant J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997517/
https://www.ncbi.nlm.nih.gov/pubmed/33771860
http://dx.doi.org/10.1126/sciadv.abc6345
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author Mageswaran, Shrawan Kumar
Yang, Wei Yuan
Chakrabarty, Yogaditya
Oikonomou, Catherine M.
Jensen, Grant J.
author_facet Mageswaran, Shrawan Kumar
Yang, Wei Yuan
Chakrabarty, Yogaditya
Oikonomou, Catherine M.
Jensen, Grant J.
author_sort Mageswaran, Shrawan Kumar
collection PubMed
description Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.
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spelling pubmed-79975172021-04-02 A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane Mageswaran, Shrawan Kumar Yang, Wei Yuan Chakrabarty, Yogaditya Oikonomou, Catherine M. Jensen, Grant J. Sci Adv Research Articles Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events. American Association for the Advancement of Science 2021-03-26 /pmc/articles/PMC7997517/ /pubmed/33771860 http://dx.doi.org/10.1126/sciadv.abc6345 Text en Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (https://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Research Articles
Mageswaran, Shrawan Kumar
Yang, Wei Yuan
Chakrabarty, Yogaditya
Oikonomou, Catherine M.
Jensen, Grant J.
A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
title A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
title_full A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
title_fullStr A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
title_full_unstemmed A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
title_short A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
title_sort cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997517/
https://www.ncbi.nlm.nih.gov/pubmed/33771860
http://dx.doi.org/10.1126/sciadv.abc6345
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