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A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells
Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secre...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190671/ https://www.ncbi.nlm.nih.gov/pubmed/32350252 http://dx.doi.org/10.1038/s41467-020-15747-2 |
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author | Sung, Bong Hwan von Lersner, Ariana Guerrero, Jorge Krystofiak, Evan S. Inman, David Pelletier, Roxanne Zijlstra, Andries Ponik, Suzanne M. Weaver, Alissa M. |
author_facet | Sung, Bong Hwan von Lersner, Ariana Guerrero, Jorge Krystofiak, Evan S. Inman, David Pelletier, Roxanne Zijlstra, Andries Ponik, Suzanne M. Weaver, Alissa M. |
author_sort | Sung, Bong Hwan |
collection | PubMed |
description | Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes. |
format | Online Article Text |
id | pubmed-7190671 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-71906712020-05-01 A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells Sung, Bong Hwan von Lersner, Ariana Guerrero, Jorge Krystofiak, Evan S. Inman, David Pelletier, Roxanne Zijlstra, Andries Ponik, Suzanne M. Weaver, Alissa M. Nat Commun Article Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes. Nature Publishing Group UK 2020-04-29 /pmc/articles/PMC7190671/ /pubmed/32350252 http://dx.doi.org/10.1038/s41467-020-15747-2 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Sung, Bong Hwan von Lersner, Ariana Guerrero, Jorge Krystofiak, Evan S. Inman, David Pelletier, Roxanne Zijlstra, Andries Ponik, Suzanne M. Weaver, Alissa M. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
title | A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
title_full | A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
title_fullStr | A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
title_full_unstemmed | A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
title_short | A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
title_sort | live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190671/ https://www.ncbi.nlm.nih.gov/pubmed/32350252 http://dx.doi.org/10.1038/s41467-020-15747-2 |
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