Mechanics and Molecular Mobility of Endothelial Cells

描述（由申请人提供）：血流相关的剪切应力通过膜介导机制诱导血管内皮细胞（ECs）的生化和生理变化。为了了解质膜介导的机械转导的分子基础，我们提出了新的工程分析和单EC机械转导的实验研究。我们方法的核心是多模态显微镜的新使用，包括DIG， TIRFM，共聚焦荧光成像，时间分辨荧光和光子力显微镜，所有这些都在一个平台上。该基础设施为先进的3d图像处理算法、计算流体动力学求解器和有限元（FE）固体力学模型提供实验确定的输入，从而实现细胞膜应力与脂质介导的信号转导的时间和位置相关。为了验证我们的假设，即剪切应力引起膜应力，从而引起凝胶相脂质微域中g蛋白的激活，我们提出了3个特定目标（SAs）。在SA 1下，我们测量了三维膜拓扑结构、糖萼运输、各向异性膜和细胞质粘弹性，建立了一个完整的三维有限元力学模型，该模型预测了剪切诱导的膜应力在根尖表面、细胞连接处和局灶黏附处的分布。在SA 2下，我们使用膜相特异性脂质染料和BODIPY-GTP（一种新型的活化g蛋白荧光配体）的时间分辨荧光光谱，验证了膜应力浓度与EC膜中测量的剪切诱导的凝胶相脂质流动性和g蛋白活化的变化相关的假设。在sa3下，我们使用一种新的连续流波形发生器来测试普遍的剪切应力是否引起细胞质和膜微流变学和膜信号的适应性变化。研究结果将为血管功能障碍的分子干预提供新的思路，并为新型生物材料和组织工程血管的智能化发展提供依据。

DESCRIPTION (provided by applicant): Blood flow-related shear stress induces biochemical and physiological changes in vascular endothelial cells (ECs) through membrane-mediated mechanisms. To understand the molecular basis of plasma membrane-mediated mechanotransduction, we propose new engineering analyses and experimental studies of single EC mechanotransduction. Central to our approach is the novel use of multimodal microscopy including DIG, TIRFM, confocal fluorescence imaging, time-resolved fluorescence, and photonic-force microscopy, all on a single platform. This infrastructure provides experimentally-determined inputs to advanced 3-D image processing algorithms, computational fluid dynamics solvers, and finite element (FE) solid mechanics models enabling time-and position-dependent correlations of cell membrane stresses with lipid-mediated signal transduction. To test our hypothesis that shear stress causes membrane stresses which elicit G-protein activation in gel-phase lipid microdomains we propose 3 specific aims (SAs). Under SA 1 we measure 3-D membrane topology, glycocalyx transport, and anisotropic membrane and cytoplasmic viscoelasticity to develop a full 3-D finite element mechanical model of an EC which predicts the shear-induced membrane stress distribution in the apical surface, cell junctions and focal adhesions. Under SA 2 we test the hypothesis that membrane stress concentrations are correlated with measured shear-induced changes in gel-phase lipid mobility and G-protein activation in EC membranes using time-resolved fluorescence spectroscopy of membrane phase-specific lipoid dyes and BODIPY-GTP, a novel fluorescent ligand for activated-G-proteins. Under SA 3 we use a novel continuous flow waveform generator to test whether prevailing shear stress elicits adaptive changes in cytoplasmic and membrane microrheology and membrane signaling. Results will point to new molecular level interventions for vascular dysfunction and provide the basis for intelligent development of novel biomaterials and tissue engineered blood vessels.