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Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient inte...

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Autores principales: Ueda, Shuji, Blee, Alexandra M., Macway, Katherine G., Renner, Derrick J., Yamada, Soichiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373798/
https://www.ncbi.nlm.nih.gov/pubmed/25806963
http://dx.doi.org/10.1371/journal.pone.0122886
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author Ueda, Shuji
Blee, Alexandra M.
Macway, Katherine G.
Renner, Derrick J.
Yamada, Soichiro
author_facet Ueda, Shuji
Blee, Alexandra M.
Macway, Katherine G.
Renner, Derrick J.
Yamada, Soichiro
author_sort Ueda, Shuji
collection PubMed
description Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.
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spelling pubmed-43737982015-03-27 Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase Ueda, Shuji Blee, Alexandra M. Macway, Katherine G. Renner, Derrick J. Yamada, Soichiro PLoS One Research Article Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. Public Library of Science 2015-03-25 /pmc/articles/PMC4373798/ /pubmed/25806963 http://dx.doi.org/10.1371/journal.pone.0122886 Text en © 2015 Ueda et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ueda, Shuji
Blee, Alexandra M.
Macway, Katherine G.
Renner, Derrick J.
Yamada, Soichiro
Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase
title Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase
title_full Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase
title_fullStr Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase
title_full_unstemmed Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase
title_short Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase
title_sort force dependent biotinylation of myosin iia by α-catenin tagged with a promiscuous biotin ligase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373798/
https://www.ncbi.nlm.nih.gov/pubmed/25806963
http://dx.doi.org/10.1371/journal.pone.0122886
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