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Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis
How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966983/ https://www.ncbi.nlm.nih.gov/pubmed/27307587 http://dx.doi.org/10.1091/mbc.E16-01-0057 |
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author | Trexler, Adam J. Sochacki, Kem A. Taraska, Justin W. |
author_facet | Trexler, Adam J. Sochacki, Kem A. Taraska, Justin W. |
author_sort | Trexler, Adam J. |
collection | PubMed |
description | How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells. |
format | Online Article Text |
id | pubmed-4966983 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-49669832016-10-16 Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis Trexler, Adam J. Sochacki, Kem A. Taraska, Justin W. Mol Biol Cell Articles How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells. The American Society for Cell Biology 2016-08-01 /pmc/articles/PMC4966983/ /pubmed/27307587 http://dx.doi.org/10.1091/mbc.E16-01-0057 Text en © 2016 Trexler et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. |
spellingShingle | Articles Trexler, Adam J. Sochacki, Kem A. Taraska, Justin W. Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
title | Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
title_full | Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
title_fullStr | Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
title_full_unstemmed | Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
title_short | Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
title_sort | imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966983/ https://www.ncbi.nlm.nih.gov/pubmed/27307587 http://dx.doi.org/10.1091/mbc.E16-01-0057 |
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