RhoA-induced cytoskeletal tension controls adaptive cellular remodeling to mechanical signaling
RhoA-induced cytoskeletal tension controls adaptive cellular remodeling to mechanical signaling
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DOI：
10.1039/c2ib20008b
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发表时间：
2012-01-01
影响因子：
2.5
通讯作者：
Trache, Andreea
中科院分区：
文献类型：
--
作者：
Lim, Soon-Mi;Trzeciakowski, Jerome P.;Trache, Andreea
在亚细胞水平上测量响应于离散机械刺激的实时机械敏感事件的能力是理解机械诱导的细胞重塑的关键组成部分。用RhoA构建体（野生型、显性阴性或组成型活性）转染血管平滑肌细胞（VSMC）或用ML-7处理以在机械刺激之前诱导特异性细胞骨架张力特征。使用细胞外基质（ECM）蛋白功能化的原子力显微镜探针将拉伸应力施加到活VSMC。ECM诱导选择性整合素活化和粘着斑形成，使得能够通过活性ECM-整合素-肌动蛋白连接直接操纵皮质肌动蛋白。因此，局部诱导的机械敏感性事件触发了负责整个细胞内肌动蛋白和粘着斑重塑的细胞内信号通路的下游激活。整合机械刺激与同步荧光成像的转盘共聚焦和全内反射荧光显微镜，使可视化和定量的分子动态事件在亚细胞水平上的实时。结果提供的证据表明，预先存在的细胞骨架张力影响肌动球蛋白装置，这反过来又协调细胞的能力，以适应外部施加的压力。RhoA激活诱导高细胞骨架张力，其与增加的应力纤维形成、细胞硬度、整合素激活和肌球蛋白磷酸化相关。相反，阻断Rho激酶或肌球蛋白功能的特征在于细胞骨架张力低，应力纤维形成水平降低，细胞硬度降低和整合素活化。我们的研究结果表明，血管平滑肌细胞的感官和适应物理微环境的变化，通过协调反应的肌动球蛋白装置建立一个新的稳态。
The ability to measure real-time mechanosensitive events at the subcellular level in response to discrete mechanical stimulation is a critical component in understanding mechanically-induced cellular remodeling. Vascular smooth muscle cells (VSMC) were transfected with RhoA constructs (wild type, dominant negative or constitutively active) or treated with ML-7 to induce specific cytoskeletal tension characteristics prior to mechanical stimulation. Tensile stress was applied to live VSMC using an atomic force microscope probe functionalized with extracellular matrix (ECM) proteins. The ECM induces selective integrin activation and focal adhesion formation, enabling direct manipulation of cortical actin through an active ECM-integrin-actin linkage. Therefore, locally induced mechanosensitive events triggered downstream activation of intracellular signaling pathways responsible for actin and focal adhesion remodeling throughout the cell. Integration of mechanical stimulation with simultaneous fluorescence imaging by spinning-disk confocal and total internal reflection fluorescence microscopy enabled visualization and quantification of molecular dynamic events at the sub-cellular level in real-time. Results provide evidence that the pre-existing cytoskeletal tension affects the actomyosin apparatus which in turn coordinates the ability of the cell to adapt to the externally applied stress. RhoA activation induced high cytoskeletal tension that correlated with increased stress fiber formation, cell stiffness, integrin activation and myosin phosphorylation. In contrast, blocking Rho-kinase or myosin function was characterized by low cytoskeletal tension with a decreased level of stress fiber formation, lower cell stiffness and integrin activation. Our findings show that VSMC sense and adapt to physical microenvironmental changes by a coordinated response of the actomyosin apparatus necessary to establish a new homeostatic state.