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

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 ?-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding ?-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, ?-catenin, vinculin and actin localize with ?-catenin at cell-cell contacts in immuno-fluorescent staining, only ?-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of ?-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between ?-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.

SUBMITTER: Ueda S 

PROVIDER: S-EPMC4373798 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature