Probing protein interactions in living mammalian cells on a microtubule bench
| Autor: | Boca, Mirela, Kretov, Dmitry, Desforges, Bénédicte, Méphon-Gaspard, Alix, Curmi, Patrick, Pastre, David |
|---|---|
| Přispěvatelé: | Structure et activité des biomolécules normales et pathologiques (SABNP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE), Institute of Protein Research, Russian Academy of Sciences [Moscow] (RAS), Maciejak, Olek, Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM) |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Recombinant Fusion Proteins
Green Fluorescent Proteins DNA Helicases Gene Expression RNA-Binding Proteins tau Proteins Microtubules Article Rats RNA Recognition Motif Proteins Microscopy Fluorescence Connexin 43 Protein Interaction Mapping [SDV.BBM] Life Sciences [q-bio]/Biochemistry Molecular Biology Animals Humans [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Y-Box-Binding Protein 1 Carrier Proteins Poly A Poly-ADP-Ribose Binding Proteins RNA Helicases HeLa Cells Protein Binding |
| Zdroj: | Scientific Reports Scientific Reports, Nature Publishing Group, 2015, 5, pp.17304. ⟨10.1038/srep17304⟩ Scientific Reports, 2015, 5, pp.17304. ⟨10.1038/srep17304⟩ |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/srep17304 |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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