title: Force dependent biotinylation of myosin IIA by α-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 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.
authors: Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
authors: Shuji
authors: S
authors: Ueda
authors: Department of Biomedical Engineering, University of California Davis, Davis, CA, 95616, United States of America.
authors: Alexandra M
authors: AM
authors: Blee
authors: Department of Biomedical Engineering, University of California Davis, Davis, CA, 95616, United States of America.
authors: Katherine G
authors: KG
authors: Macway
authors: Department of Biomedical Engineering, University of California Davis, Davis, CA, 95616, United States of America.
authors: Derrick J
authors: DJ
authors: Renner
authors: Department of Biomedical Engineering, University of California Davis, Davis, CA, 95616, United States of America.
authors: Soichiro
authors: S
authors: Yamada
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013314:Stress, Mechanical; D013379:Substrate Specificity; D051178:alpha Catenin / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism; D051185:gamma Catenin / Q000378:metabolism
chemical_list: D000199:Actins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D051178:alpha Catenin; D051176:beta Catenin; D051185:gamma Catenin; D001710:Biotin; D024507:Nonmuscle Myosin Type IIA; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli