Kim Tae-Jin, Zheng Shuai, Sun Jie, Muhamed Ismaeel, Wu Jun, Lei Lei, Kong Xinyu, Leckband Deborah E, Wang Yingxiao
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Curr Biol. 2015 Jan 19;25(2):218-224. doi: 10.1016/j.cub.2014.11.017. Epub 2014 Dec 24.
The cytosolic protein α-catenin is a postulated force transducer at cadherin complexes. The demonstration of force activation, identification of consequent downstream events in live cells, and development of tools to study these dynamic processes in living cells are central to elucidating the role of α-catenin in cellular mechanics and tissue function. Here we demonstrate that α-catenin is a force-activatable mechanotransducer at cell-cell junctions by using an engineered α-catenin conformation sensor based on fluorescence resonance energy transfer (FRET). This sensor reconstitutes α-catenin-dependent functions in α-catenin-depleted cells and recapitulates the behavior of the endogenous protein. Dynamic imaging of cells expressing the sensor demonstrated that α-catenin undergoes immediate, reversible conformation switching in direct response to different mechanical perturbations of cadherin adhesions. Combined magnetic twisting cytometry with dynamic FRET imaging revealed rapid, local conformation switching upon the mechanical stimulation of specific cadherin bonds. At acutely stretched cell-cell junctions, the immediate, reversible conformation change further reveals that α-catenin behaves like an elastic spring in series with cadherin and actin. The force-dependent recruitment of vinculin—a principal α-catenin effector—to junctions requires the vinculin binding site of the α-catenin sensor. In cells, the relative rates of force-dependent α-catenin conformation switching and vinculin recruitment reveal that α-catenin activation and vinculin recruitment occur sequentially, rather than in a concerted process, with vinculin accumulation being significantly slower. This engineered α-catenin sensor revealed that α-catenin is a reversible, stretch-activatable sensor that mechanically links cadherin complexes and actin and is an indispensable player in cadherin-specific mechanotransduction at intercellular junctions.
胞质蛋白α-连环蛋白被认为是钙黏蛋白复合物中的一种力传感器。力激活的证明、活细胞中后续下游事件的鉴定以及研究活细胞中这些动态过程的工具的开发，对于阐明α-连环蛋白在细胞力学和组织功能中的作用至关重要。在这里，我们通过使用基于荧光共振能量转移（FRET）的工程化α-连环蛋白构象传感器，证明α-连环蛋白是细胞间连接处的一种力可激活的机械转导器。该传感器在α-连环蛋白缺失的细胞中重建了α-连环蛋白依赖性功能，并概括了内源性蛋白的行为。对表达该传感器的细胞进行动态成像表明，α-连环蛋白会直接响应钙黏蛋白黏附的不同机械扰动，立即发生可逆的构象转换。结合磁扭细胞术与动态FRET成像显示，在对特定钙黏蛋白键进行机械刺激时，会迅速发生局部构象转换。在急性拉伸的细胞间连接处，这种立即、可逆的构象变化进一步表明，α-连环蛋白的行为类似于与钙黏蛋白和肌动蛋白串联的弹性弹簧。α-连环蛋白的主要效应器纽蛋白向连接处的力依赖性募集需要α-连环蛋白传感器的纽蛋白结合位点。在细胞中，力依赖性α-连环蛋白构象转换和纽蛋白募集的相对速率表明，α-连环蛋白激活和纽蛋白募集是顺序发生的，而不是协同过程，纽蛋白的积累明显较慢。这种工程化的α-连环蛋白传感器表明，α-连环蛋白是一种可逆的、拉伸可激活的传感器，它在机械上连接钙黏蛋白复合物和肌动蛋白，并且是细胞间连接处钙黏蛋白特异性机械转导中不可或缺的参与者。
