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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Association for the Advancement of Science
2021
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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. |
format | Online Article Text |
id | pubmed-7997517 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Association for the Advancement of Science |
record_format | MEDLINE/PubMed |
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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