RhoA-induced cytoskeletal tension controls adaptive cellular remodeling to mechanical signaling.

IF 1.4
Soon-Mi Lim, Jerome P Trzeciakowski, Harini Sreenivasappa, Lawrence J Dangott, Andreea Trache
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引用次数: 45

Abstract

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.

rhoa诱导的细胞骨架张力控制机械信号的适应性细胞重塑。
在亚细胞水平上测量对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。血管平滑肌细胞(VSMC)转染RhoA构建体(野生型,显性阴性或组成活性)或ML-7处理,以在机械刺激之前诱导特定的细胞骨架张力特征。使用细胞外基质(ECM)蛋白功能化的原子力显微镜探针对活VSMC施加拉伸应力。ECM诱导选择性整合素激活和局部黏附形成,从而通过ECM-整合素-肌动蛋白的活性连接直接操纵皮质肌动蛋白。因此,局部诱导的机械敏感事件触发了细胞内信号通路的下游激活,这些信号通路负责整个细胞的肌动蛋白和局灶性粘连重塑。机械刺激与旋转盘共聚焦和全内反射荧光显微镜同步荧光成像的结合,实现了亚细胞水平分子动力学事件的实时可视化和定量。结果提供证据表明,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适应外部施加压力的能力。RhoA激活诱导高细胞骨架张力,与应力纤维形成、细胞刚度、整合素激活和肌球蛋白磷酸化增加相关。相反,阻断rho激酶或肌球蛋白功能的特点是细胞骨架张力降低,应力纤维形成水平降低,细胞刚度降低,整合素激活降低。我们的研究结果表明,VSMC通过肌动球蛋白装置的协调响应来感知和适应物理微环境的变化,这是建立新的稳态所必需的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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