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.

组织与器官时刻承受持续的物理扰动，单个细胞必须响应机械力以维持组织完整性。然而，细胞-细胞连接处介导机械转导的分子相互作用机制尚未完全阐明。这在一定程度上是因为力诱导的蛋白质复合物中普遍存在弱且瞬时的相互作用。我们采用融合标记于α-连环蛋白（α-catenin）的广谱生物素连接酶BirA进行原位近端生物素标记，并结合底物拉伸细胞腔室，旨在鉴定α-连环蛋白周围的力依赖性分子相互作用——α-连环蛋白是钙粘蛋白（cadherin）介导的细胞-细胞粘连位点处的肌动蛋白调控因子。尽管免疫荧光染色结果显示，E-钙粘蛋白（E-cadherin）、β-连环蛋白（β-catenin）、纽蛋白（vinculin）与肌动蛋白可在细胞-细胞连接处与α-连环蛋白共定位，但仅β-连环蛋白和盘状球蛋白（plakoglobin）被生物素标记，这表明该近端生物素标记仅局限于紧邻α-连环蛋白的分子。在机械拉伸的样本中，非肌肉肌球蛋白IIA（non-muscle myosin IIA）的生物素标记水平升高，而肌球蛋白IIB（myosin IIB）则无此变化，这提示在底物拉伸过程中，α-连环蛋白与肌球蛋白IIA存在紧密的空间邻近关系。当用blebbistatin抑制肌球蛋白II活性后，这种力诱导的生物素标记现象便会消失。综上，这项颇具前景的技术可帮助我们鉴定力敏感复合物，这类复合物可能在承受机械力的细胞粘连的机械响应中发挥关键作用。