Membrane Fusion, Organization, and Dynamics Using Supported Bilayers

使用受支持的双层的膜融合、组织和动力学

• 批准号: 8369852
• 负责人: STEVEN G. BOXER
• 金额: $ 30.98万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8369852
• 关键词: Address; Architecture; Area; Atomic Force Microscopy; Behavior; Biological; Biological Assay; Biological Process; Biophysics; Biotechnology; Calcium; Cell Communication; Cell membrane; Cells; Chimeric Proteins; Cholesterol; Collaborations; Complex; DNA; Dependence; Development; Devices; Goals; Grant; Image; Individual; Intercellular Junctions; Label; Lateral; Length; Lipid Bilayers; Lipids; Location; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Membrane Fluidity; Membrane Fusion; Membrane Proteins; Methods; Microfluidics; Microscopy; Monitor; Mono-S; Neurons; Oligonucleotides; Optics; Pattern; Pattern Formation; Phase; Population; Positioning Attribute; Process; Proteins; Resolution; Role; SNAP receptor; Scheme; Signal Transduction; Solid; Sphingomyelins; Structure; Synapses; System; Techniques; Testing; Vesicle; Work; analytical method; drug development; electric field; fluorescence imaging; imaging modality; immunological synapse; innovation; instrument; interest; interfacial; membrane model; molecular assembly/self assembly; novel; novel strategies; protein complex; receptor; research study; single molecule; synthetic construct

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. PUBLIC HEALTH RELEVANCE: A significant fraction of all proteins are associated with membranes, and, as a class, these constitute a huge and diverse target for drug development. This proposal outlines new methods for studying membranes and membrane-associated proteins that can impact our understanding of biological function and organization, as well as impact biotechnology.

描述（由申请人提供）：该项目的长期目标是开发探测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。脂质双层是生物膜共有的基本结构。膜流动性对于生物功能至关重要，生物功能取决于膜内的构象变化、脂质和蛋白质的横向缔合、细胞相互作用时的重组和模式形成，以及改变膜拓扑结构的过程（例如膜融合）。该提案描述了模型膜结构，以及探测膜动力学这些基本方面的成像和分析方法。已经开发了三种新颖的模型膜结构，这对于这项工作至关重要：（i）图案化支撑双层，用于组织和定位感兴趣的区域，以通过质谱、超分辨率光学显微镜和原子力显微镜进行并行成像； (ii) DN​​A-脂质束缚的移动囊泡，其个体碰撞和相互作用可以直接观察到； (iii) DN​​A-脂质束缚的双层斑块和巨型囊泡，它们作为膜融合和双层-双层相互作用与曲率控制的现实基质。已经选择了利用这些架构的两个具有当前生物学和生物医学意义的问题。 (i) 由新型 DNA-脂质缀合物或天然神经元蛋白质融合机制协调的囊泡融合机制。系留囊泡或双层膜片将用于精确探测单囊泡和单分子水平的融合步骤（目标 1）。我们最终计划组装一个人工突触，其中每个步骤以及单个蛋白质成分和钙的贡献都可以定量评估。 (ii) 将使用新型高空间分辨率和高灵敏度成像质谱技术来测量脂质和膜蛋白的组织（目标 2）。具体目标包括对单个小囊泡的组成进行定量分析， 被认为与膜筏相关的成分的集体行为，以及最终对免疫突触中的脂质和蛋白质聚集和重组的研究。每个目标都针对膜生物物理学的一个重要领域，整合创新的分子组装、界面制造和先进的成像方法，以解决广泛关注的复杂问题。 公共健康相关性：所有蛋白质中很大一部分与膜相关，作为一类，这些蛋白质构成了药物开发的巨大且多样化的目标。该提案概述了研究膜和膜相关蛋白的新方法，这些方法可以影响我们对生物功能和组织的理解，并影响生物技术。