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发展探索生物膜中脂质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。脂质双分子层是生物膜的基本结构。膜流动性对于依赖于膜内构象变化的生物功能、脂质和蛋白质的横向结合、细胞相互作用时它们的重组和模式形成以及改变膜拓扑结构的过程（如膜融合）至关重要。本提案描述了膜结构模型，以及探讨膜动力学这些基本方面的成像和分析方法。已经开发了三种新型膜结构模型，并且对这项工作至关重要：(i)有图案的支持双层，用于组织和定位感兴趣的区域，以便通过质谱、超分辨率光学显微镜和原子力显微镜进行平行成像；（ii） dna -脂质系留的可移动囊泡，其单个碰撞和相互作用可以直接观察到；（iii） dna -脂系双分子层斑块和巨囊泡，它们作为膜融合和双分子层-双分子层相互作用的现实底物，具有曲率控制。目前生物学和生物医学意义的两个问题已经选择利用这些架构。(1)由新型dna -脂质偶联物或天然神经元蛋白融合机制协调的囊泡融合机制。栓系囊泡或双层贴片将用于精确探测单囊泡和单分子水平的融合步骤（目的1）。我们最终计划组装一个人工突触，其中每个步骤和单个蛋白质成分和钙的贡献可以定量评估。（ii）脂质和膜蛋白的组织将通过使用一种新型的高空间分辨率和高灵敏度成像质谱技术来测量（目标2）。具体目标包括对单个小泡组成的定量分析，

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.