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 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

Cyclic stretch induces re-organization of actin fibers.

<p>(A) Schematic of the substrate stretch device and an image of the actual device used in this study. The adherent cells in the PDMS cell chamber (STREX) were stretched by a servo motor coupled to the chamber via mechanical linkages. (B) Immunofluorescence analysis of un-stretched or stretched BirA*-α-catenin expressing cells. The actin bundles of the stretched cells are oriented perpendicular to the stretch directions. Scale bar 20 μm. (C) The quantification of the actin bundle rearrangement. The angles are defined as shown in the diagram (left).</p

Characterization of promiscuous BirA (BirA*)-tagged α-catenin expressing cells.

<p>(A) Illustration of <i>in situ</i> proximal biotinylation and subsequent purification of biotinylated proteins. A stable cell line expressing BirA*-α-catenin grown in biotin containing media with subsequent biotinylated proteins purified with magnetic streptavidin-beads. Schematic of a BirA*-α-catenin construct. The BirA* is flanked by GFP and α-catenin. (B) Western blots of the wildtype (WT) and BirA*- α-catenin expressing MDCK stable cell lines. The cell lysates were analyzed using anti-α-catenin (top) and anti-tubulin antibodies (bottom). (C) The BirA*-α-catenin expressing cells were treated with biotin for 24 hours, then analyzed for the localization of BirA*-α-catenin and biotinylated proteins using AlexaFluor-568 labeled streptavidin. BirA*-α-catenin localized to cell-cell contacts, and in the presence of biotin in the media, streptavidin-specific labeling localized to cell-cell contacts, demonstrating the proximal biotinylation by BirA*-α-catenin. Scale bar 20 μm. (D) Detection of biotinylated proteins in BirA*-α-catenin expressing cells. The wildtype (WT) or BirA*-α-catenin expressing stable cell lines were cultured with or without biotin for 24 hours. The cell lysates were analyzed using western blots with streptavidin-HRP. (E) Most biotinylated proteins were in the soluble pool of cell lysates. Post-sonication cell lysates were centrifuged at 16,000g for 20 minutes, and the supernatant and pellet were analyzed using Western blot with streptavidin-HRP. (F) A streptavidin-bead purified sample from cells plated on a P150 dish was analyzed using Western blot with streptavidin-HRP. In adjacent lanes, cell lysate was analyzed using catenin antibodies. The molecular weights of major bands in the purified sample correspond to that of catenins. (G) Cell lysates and purified samples (bead) from cells plated on a P150 dish were analyzed with catenins and E-cadherin antibodies. The catenins were present in the bead fractions but E-cadherin was not.</p

Increased biotinylation of myosin IIA in stretched cells.

<p>(A) Western blot analysis of cell lysates and purified streptavidin-conjugated beads from control and stretched cells. With 0.1% SDS wash solution, the purified proteins included β-catenin and myosin IIA. The biotinlyation of myosin IIA increased in mechanically stretched samples. Myosin IIB (MyoIIB), vinculin (Vinc) and β-actin (β-act) were not purified with streptavidin-conjugated beads in unstretched or stretched samples. (B) Quantification of the relative band intensities of unstretched (control) and stretched (stretch) samples. βcat (n = 4), αcat (n = 4) and MyoIIA (n = 7). Results were analyzed using a one-way ANOVA; significance was determined using Dunnett’s post hoc test. Results were considered significant with P<0.05. (C) Immuno-precipitation of GFP-tagged BirA*-α-catenin using a GFP antibody. The immuno-precipitated samples were analyzed with α-catenin and myosin IIA antibodies. The blot of α-catenin is reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122886#pone.0122886.g002" target="_blank">Fig 2B</a>. (D) Immuno-precipitation of myosin IIA using a myosin IIA antibody. Immuno-precipitated samples were analyzed with a myosin IIA antibody and streptavidin. (E) Blebbistatin decreases biotinlyation of myosin IIA in both unstretched and stretched samples, suggesting that myosin IIA activity is required for the spatial proximity of α-catenin and myosin IIA. (F) Post-stretching, the cells were fixed and visualized with GFP (BirA*-α-catenin) and anti-myosin IIA antibodies. Yellow arrows point to the end of actin bundles localized at cell-cell contacts where myosin IIA also accumulated. The regions denoted by yellow rectangles (a, b) are magnified in the adjacent images. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122886#pone.0122886.s003" target="_blank">S1 Movie</a> for a 3D stack of stretched cells. Quantification of co-localization between BirA*-α-catenin and myosin IIA was analyzed by calculating overlapping coefficients. Scale bar 20 μm.</p

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 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