Membrane Fusion, Organization, and Dynamics Using Supported Bilayers

描述(由申请人提供)：该项目的长期目标是开发探索生物膜中脂类和蛋白质的组织和动态重组的方法，并将这些方法应用于具有广泛生物学重要性的问题。脂双层是生物膜共有的基本结构。膜流动性对生物功能至关重要，这些功能依赖于膜内构象的变化、脂类和蛋白质的侧向结合、细胞相互作用时它们的重组和图案形成，以及改变膜拓扑结构的过程，如膜融合。该提案描述了模型膜结构，以及探测膜动力学的这些基本方面的成像和分析方法。已经开发了三种新的模型膜结构，它们对这项工作至关重要：(I)图案化支撑双层，用于组织和定位感兴趣区域，用于通过质谱仪、超分辨率光学显微镜和原子力显微镜进行平行成像；(Ii)DNA-脂质拴系移动小泡，其单个碰撞和相互作用可以直接观察到；以及(Iii)DNA-脂系系双层贴片和巨大囊泡，可作为膜融合和双层-双层相互作用的真实底物，并控制曲率。选择了两个具有当前生物学和生物医学意义的问题来开发这些体系结构。(I)由新型DNA-脂质结合物或天然神经元蛋白质融合机制协调的囊泡融合机制。系留小泡或双层贴片将用于在单个小泡和单分子水平上精确探测融合步骤(目标1)。我们最终计划组装一种人工突触，其中的每一步及其对单个蛋白质成分和钙的贡献都可以得到定量评估。(2)将使用一种新型的高空间分辨率和高灵敏度的成像质谱学技术(目标2)来测量脂类和膜蛋白的组织。具体目标包括对单个小泡的成分进行定量分析， 被认为与膜筏相关的组件的集体行为，并最终研究免疫突触中的脂质和蛋白质聚集和重组。每个目标都以膜生物物理学的一个重要领域为目标，该领域整合了创新的分子组装、界面制造和先进的成像方法，以解决人们广泛感兴趣的复杂问题。

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop methods to probe the organization and dynamic reorganization of lipids and proteins in biological membranes and to apply these methods to problems of broad biological importance. The lipid bilayer is the basic structure common to biological membranes. Membrane fluidity is critical for biological functions that depend upon conformational changes within membranes, the lateral association of lipids and proteins, their reorganization and pattern formation when cells interact, and processes that change membrane topology such as membrane fusion. This proposal describes model membrane architectures, along with imaging and analytical methods that probe these basic aspects of membrane dynamics. Three novel model membrane architectures have been developed and are essential for this work: (i) patterned supported bilayers, used for organizing and locating regions of interest for parallel imaging by mass spectrometry, super-resolution optical microscopy and atomic force microscopy; (ii) DNA-lipid tethered mobile vesicles whose individual collisions and interactions can be observed directly; and (iii) DNA-lipid tethered bilayer patches and giant vesicles, which serve as realistic substrates for membrane fusion and bilayer-bilayer interactions with control of curvature. Two problems of current biological and biomedical significance have been selected that exploit these architectures. (i) The mechanism of vesicle fusion orchestrated either by novel DNA-lipid conjugates or the natural neuronal protein fusion machinery. Tethered vesicles or bilayer patches will be used to precisely probe the steps of fusion at the single vesicle and single molecule level (Aim 1). We ultimately plan to assemble an artificial synapse in which each step and contribution of individual protein components and calcium can be assessed quantitatively. (ii) The organization of lipids and membrane proteins will be measured by using a new type of high spatial resolution and high sensitivity imaging mass spectrometry technique (Aim 2). Specific targets include the quantitative analysis of the composition of individual small vesicles, collective behavior of components believed to be associated in membrane rafts, and ultimately studies of lipid and protein clustering and reorganization in the immunological synapse. Each Aim targets a significant area of membrane biophysics that integrates innovative molecular assemblies, interfacial fabrication, and advanced imaging methods to address a complex problem of wide interest.