Mechanosensitivity of Cell Membranes: Role of Lipid-Protein Interactions

描述（由申请人提供）：血流动力学剪切应力刺激细胞内事件的数量，这些事件既调节血管结构，也影响血管病变的发展。内皮细胞将这种机械刺激转化为细胞内生化反应的确切分子机制尚未确定。中心假设是内皮细胞的质膜作为机械敏感元件；即，在机械应力作用下膜的物理性质的变化可以调节膜蛋白偶联到细胞内信号通路的活性。为了验证这一假设，我们将使用一种结合时间分辨荧光显微镜、生物化学、细胞生物学和膜微力学的综合方法。我们的初步实验首次表明：(1)当暴露于机械力时，膜横向流动性和水合水平发生变化；(2)膜张力的增加导致缓激肽G蛋白偶联受体（GPCR）的激活。拟议的研究涉及以下问题：(1)脂质双分子层的哪些物理性质在机械扰动下发生变化；(2)这些变化中哪些与膜相关蛋白（如GPCR、g蛋白和内皮型一氧化氮合酶（eNOS））的功能有明确的联系，并可以介导机械化学信号转导；(3)剪切应力导致GPCR受体、eNOS和g蛋白机械诱导激活的具体机制是什么。我们将使用最先进的皮秒时间分辨荧光、单分子和荧光相关光谱技术，详细研究在机械应力下脂质双分子层膜的物理性质在分子水平上发生了什么变化，以及这些变化是如何通过直接激活膜相关蛋白（如GPCR）和通过G-蛋白调节信号放大级联而耦合到机械化学信号转导的。具体来说，我们提出机械诱导的某些膜特性的变化，如厚度、横向流动性、极性、膜自由体积和/或跨膜横向力分布，能够启动和调节构象变化，这些构象变化负责实验观察到的GPCR和G蛋白信号转导途径的响应和eNOS激活。如果成功，它将为内皮细胞在正常生理和血管疾病中如何感知血流提供机制基础。

DESCRIPTION (provided by applicant): Hemodynamic shear stress stimulates number of intracellular events that both regulate vessel structure and also influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established yet. The central hypothesis is that the plasma membrane of endothelial cell acts as a mechanosensitive element; i.e. changes in physical properties of the membrane under mechanical stress can regulate activity of membrane proteins coupled to intracellular signaling pathways. To test this hypothesis, we will use an integrative approach that combines time-resolved fluorescence microscopy, biochemistry, cell biology, and membrane micromechanics. Our preliminary experiments show for the first time that (1) when exposed to mechanical forces, membrane lateral fluidity and hydration levels change and (2) that increases in membrane tension lead to activation of bradykinin G protein coupled receptor (GPCR). The proposed research addresses the following questions: (1) which physical properties of the lipid bilayer change in response to mechanical perturbation, (2) which of these changes has a clear link to function of membrane-associated proteins such as GPCRs, G-proteins and endothelial nitric oxide synthase (eNOS), and can mediate mechanochemical signal transduction, and (3) what are the specific mechanisms leading to mechanically induced activation of GPCR receptors, eNOS and G-proteins by shear stress. We will use state-of-the-art picosecond time-resolved fluorescence, single molecule and fluorescence correlation spectroscopy techniques to investigate in detail what happens to the physical properties of the lipid bilayer membrane at the molecular level under mechanical stress and how these changes are coupled to mechanochemical signal transduction via direct activation of the membrane associated proteins such as GPCR's and modulation of signal amplification cascades through G- proteins. Specifically we propose that mechanically-induced changes in certain membrane properties such as thickness, lateral fluidity, polarity, membrane free volume and/or trans-membrane lateral force profile are able to initiate and regulate conformational changes responsible for experimentally observed response of GPCR and G protein signal transduction pathways and eNOS activation. If successful it will provide the mechanistic basis on how endothelial cells sense flow in both normal physiology and in vascular disease.