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Probing protein interactions in living mammalian cells on a microtubule bench
Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661529/ https://www.ncbi.nlm.nih.gov/pubmed/26610591 http://dx.doi.org/10.1038/srep17304 |
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author | Boca, Mirela Kretov, Dmitry A. Desforges, Bénédicte Mephon-Gaspard, Alix Curmi, Patrick A. Pastré, David |
author_facet | Boca, Mirela Kretov, Dmitry A. Desforges, Bénédicte Mephon-Gaspard, Alix Curmi, Patrick A. Pastré, David |
author_sort | Boca, Mirela |
collection | PubMed |
description | Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases. |
format | Online Article Text |
id | pubmed-4661529 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-46615292015-12-02 Probing protein interactions in living mammalian cells on a microtubule bench Boca, Mirela Kretov, Dmitry A. Desforges, Bénédicte Mephon-Gaspard, Alix Curmi, Patrick A. Pastré, David Sci Rep Article Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases. Nature Publishing Group 2015-11-27 /pmc/articles/PMC4661529/ /pubmed/26610591 http://dx.doi.org/10.1038/srep17304 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Boca, Mirela Kretov, Dmitry A. Desforges, Bénédicte Mephon-Gaspard, Alix Curmi, Patrick A. Pastré, David Probing protein interactions in living mammalian cells on a microtubule bench |
title | Probing protein interactions in living mammalian cells on a microtubule bench |
title_full | Probing protein interactions in living mammalian cells on a microtubule bench |
title_fullStr | Probing protein interactions in living mammalian cells on a microtubule bench |
title_full_unstemmed | Probing protein interactions in living mammalian cells on a microtubule bench |
title_short | Probing protein interactions in living mammalian cells on a microtubule bench |
title_sort | probing protein interactions in living mammalian cells on a microtubule bench |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661529/ https://www.ncbi.nlm.nih.gov/pubmed/26610591 http://dx.doi.org/10.1038/srep17304 |
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