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: 8707472
• 负责人: STEVEN G. BOXER
• 金额: $ 33.63万
• 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/8707472
• 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.

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广泛的生物学重要性问题。脂质双层是生物膜共有的基本结构。膜的流动性对于依赖于膜内构象变化的生物学功能至关重要，脂质和蛋白质的横向缔合，细胞相互作用时它们的重组和模式形成以及改变膜拓扑的过程，例如膜融合。该建议描述了模型膜体系结构，以及探测膜动力学这些基本方面的成像和分析方法。已经开发了三个新型模型膜结构，对于这项工作至关重要：（i）由质谱法，超分辨率光学显微镜和原子力显微镜的图案化支持的双层层，用于组织和定位感兴趣的区域，用于平行成像； （ii）可以直接观察到的单个碰撞和相互作用的DNA纤维束缚的移动囊泡； （iii）DNA-脂连系双层斑块和巨囊泡，它们是膜融合的逼真的底物和双层双层与曲率控制的相互作用。已经选择了当前的生物学和生物医学意义的两个问题，这些问题利用了这些体系结构。 （i）新型DNA-脂质结合物或天然神经元蛋白融合机械策划的囊泡融合机制。束缚囊泡或双层斑块将用于精确探测单个囊泡和单分子水平的融合步骤（AIM 1）。我们最终计划组装人工突触，在该突触中，可以定量评估单个蛋白质成分和钙的每个步骤和贡献。 （ii）通过使用新型的高空间分辨率和高灵敏度成像质谱技术（AIM 2）来测量脂质和膜蛋白的组织。具体目标包括对单个小囊泡组成的定量分析， 被认为与膜筏相关的成分的集体行为，最终研究了免疫突触中脂质和蛋白质聚类和重组的研究。每个目标都靶向膜生物物理学的重要领域，这些膜生物物理学整合了创新的分子组件，界面制造和先进的成像方法，以解决一个广泛关注的复杂问题。