Mechanosensitivity of Cell Membranes: Role of Lipid-Protein Interactions

描述(申请人提供)：血流动力学剪切力刺激细胞内事件的数量，既调节血管结构，也影响血管病理的发展。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建立。核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与细胞内信号通路耦合的膜蛋白的活性。为了验证这一假设，我们将使用一种结合了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)当受到机械力作用时，膜的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。这项研究涉及以下问题：(1)机械扰动时，脂质双层的哪些物理性质会发生变化；(2)这些变化中的哪些与膜相关蛋白如GPCRs、G蛋白和内皮型一氧化氮合酶(ENOS)的功能有明显的联系，并可以介导机械力化学信号转导；(3)切应力作用下机械诱导GPCR型受体、eNOS和G蛋白激活的具体机制是什么。我们将利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，在分子水平上详细研究机械应力下脂质双层膜的物理性质发生了什么变化，以及这些变化是如何通过直接激活膜相关蛋白(如GPCRs)和通过G蛋白调节信号放大级联作用而与机械力化学信号转导相耦合的。具体地说，我们认为，机械诱导的某些膜性质的变化，如厚度、侧向流动性、极性、膜自由体积和/或跨膜侧向力分布，能够启动和调节构象变化，这些变化与实验观察到的GPCRs和G蛋白信号转导通路的反应以及eNOS的激活有关。如果成功，它将为内皮细胞如何在正常生理和血管疾病中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.