Mechanosensitivity of Cell Membranes: Role of Lipid-Protein Interactions

细胞膜的机械敏感性：脂质-蛋白质相互作用的作用

• 批准号: 7268267
• 负责人: MIRIANAS CHACHISVILIS
• 金额: $ 37.88万
• 依托单位: LA JOLLA BIOENGINEERING INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-04 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7268267
• 关键词: Address; Affect; Angiotensins; Arts; Biochemical; Biochemistry; Biomedical Engineering; Blood Vessels; Bradykinin; Cell membrane; Cellular biology; Chemicals; Complex; Coupled; Development; Diffusion; Elements; Endothelial Cells; Energy Transfer; Environment; Event; Fluorescence; Fluorescence Microscopy; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Heterotrimeric GTP-Binding Proteins; Hydration status; Label; Lateral; Lead; Ligands; Link; Lipid Bilayers; Lipids; Liposomes; Liquid substance; Measures; Mechanical Stress; Mechanics; Mediating; Membrane; Membrane Fluidity; Membrane Proteins; Molecular; Molecular Conformation; Monitor; Numbers; Pathology; Physiology; Property; Proteins; Regulation; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Spectrum Analysis; Stimulus; Stress; Structure; Supporting Cell; Techniques; Testing; Thick; Time; Vascular Diseases; Work; base; conformational conversion; fluidity; hemodynamics; human NOS3 protein; membrane polarity; physical property; pressure; programs; receptor; reconstitution; research study; response; shear stress; single molecule; time use

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.

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精确分子机制尚未建立。中心假设是内皮细胞的质膜充当机械敏感元件;即在机械应力下膜的物理性质的变化可以调节与细胞内信号传导途径偶联的膜蛋白的活性。为了验证这一假设，我们将使用结合时间分辨荧光显微镜、生物化学、细胞生物学和膜微观力学的综合方法。我们的初步实验首次表明：（1）当暴露于机械力时，膜的侧向流动性和水化水平发生变化;（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）脂质双层的哪些物理性质响应于机械扰动而改变，（2）这些改变中的哪些与膜相关蛋白如GPCR、G蛋白和内皮一氧化氮合酶（eNOS）的功能有明确的联系，并且可以介导机械化学信号转导，（3）切应力诱导GPCR受体、eNOS和G蛋白活化的具体机制是什么？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，以详细研究在机械应力下在分子水平上脂质双层膜的物理性质发生了什么，以及这些变化如何通过膜相关蛋白如GPCR的直接活化和信号放大级联的调节与机械化学信号转导偶联通过G蛋白。具体而言，我们建议，机械诱导的某些膜特性，如厚度，侧向流动性，极性，膜自由体积和/或跨膜侧向力分布的变化能够启动和调节构象变化负责实验观察到的GPCR和G蛋白信号转导途径和eNOS激活的响应。如果成功的话，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。