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PLoS One. 2015 Mar 25;10(3):e0122886. doi: 10.1371/journal.pone.0122886. eCollection 2015.

Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

Author information

1
Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
2
Department of Biomedical Engineering, University of California Davis, Davis, CA, 95616, United States of America.

Abstract

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.

PMID:
25806963
PMCID:
PMC4373798
DOI:
10.1371/journal.pone.0122886
[Indexed for MEDLINE]
Free PMC Article

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