Membrane Fusion and Dynamics Using Supported Bilayers

使用受支持的双层的膜融合和动力学

• 批准号: 7163536
• 负责人: STEVEN G. BOXER
• 金额: $ 28万
• 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 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7163536
• 关键词: Address; Architecture; Biologic Characteristic; Biological; Biological Assay; Biological Process; Cholesterol; Class; Classification; Complementary DNA; Data; Deposition; Development; Diffuse; Diffusion; Docking; Elements; Fluorescence; Gangliosides; Glycolipids; Image; Individual; Isotopes; Label; Laboratories; Lateral; Lipid Bilayers; Lipids; Liquid substance; Location; Mass Spectrum Analysis; Measurement; Membrane; Membrane Fluidity; Membrane Fusion; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Methods; Microscopy; Monitor; Motion; Numbers; Oligonucleotides; Optics; Outcome; Pattern; Population; Process; Proteins; Range; Resolution; SNAP receptor; Solid; Sphingomyelins; Structure; Surface; System; Techniques; Testing; Time; Variant; Vesicle; Video Microscopy; Work; analytical method; concept; drug development; electric field; fluidity; fluorescence imaging; research study; size; target SNARE proteins; two-dimensional

DESCRIPTION (provided by applicant): The lipid bilayer is the basic structure common to biological membranes. A large fraction of all proteins are associated with membranes, and, as a class, these constitute a huge and diverse target for drug development. Fluidity is critical for biological functions that depend upon the lateral association or clustering of multiple components as well as for processes that change membrane topology such as endo- and exocytocis and fusion. This proposal outlines new types of experiments that probe these two basic aspects of membrane dynamics: the mechanism of vesicle fusion (Aim 1) and the lateral association of certain lipids and membrane anchored proteins into rafts (Aim 2). Both aims depend upon the development of new supported lipid bilayer architectures and analytical methods that can have a broad impact on studies of biological membranes. Aim 1 - Vesicle fusion mechanisms using mobile tethered vesicles: Vesicles can be tethered to fluid supported bilayers and are free to diffuse parallel to the plane of the supported bilayer. The trajectories and collisions of individual tethered vesicles can be visualized by video microscopy. Vesicles displaying different proteins, such as the SNARE proteins, will be tethered at different locations on the supported bilayer, and their subsequent diffusion, docking, hemifusion and fusion can then be monitored in real time at the level of individual vesicles. This new assay system is ideally suited for critical tests of the hypothesis that SNARE proteins are necessary and sufficient for fusion. New methods are proposed for preparing very well defined populations of tethered vesicles displaying specific numbers of v- and t-SNAREs, so that the threshold numbers of cognate proteins needed for each step can be established. Other protein co-factors will then be investigated. Aim 2 - Correlated motion in rafts - composition analysis with 30 nm lateral resolution: The concept of lipid rafts is a useful theme for explaining the origin of specific associations within membranes, despite the overall fluidity of the membrane. Rafts have proven to be quite difficult to study as inter-component correlations may be transient and/or extend over distances that are smaller than the diffraction limit of optical microscopy. We propose to develop new methods to characterize the composition of membranes with very high spatial resolution by multi-isotope imaging mass spectrometry (MIMS). MIMS can be used to determine the composition of a single layer of molecules with a lateral resolution of about 30 nm and extraordinary sensitivity, with the identities of the components encoded by isotopic substitution. Using this method it should be possible to establish the proximity and lateral composition variations of lipids, glycolipids and membrane anchored proteins with unprecedented lateral resolution and without the use of fluorescence labels.

描述（由申请人提供）：脂质双层是生物膜常见的基本结构。所有蛋白质中的很大一部分与膜相关，并且作为一类，这些构成了药物开发的巨大而多样的目标。流动性对于依赖于多个组分的横向缔合或聚集的生物功能以及改变膜拓扑结构的过程（例如胞内和胞外以及融合）是至关重要的。该提案概述了新类型的实验，探测这两个基本方面的膜动力学：囊泡融合的机制（目的1）和横向协会的某些脂质和膜锚定蛋白成筏（目的2）。这两个目标都依赖于新的支持脂质双层结构和分析方法的发展，可以对生物膜的研究产生广泛的影响。 目的1 -使用移动的栓系囊泡的囊泡融合机制：囊泡可以栓系到流体支撑的双层上，并且可以平行于支撑的双层的平面自由扩散。单个拴系囊泡的轨迹和碰撞可以通过视频显微镜可视化。展示不同蛋白质（例如SNARE蛋白质）的囊泡将被拴系在所支持的双层上的不同位置处，然后可以在单个囊泡的水平上真实的时间监测它们随后的扩散、对接、半融合和融合。这种新的检测系统非常适合于对SNARE蛋白是融合所必需和足够的假设进行关键性测试。提出了新的方法，用于制备非常明确的群体的拴系囊泡显示特定数量的v-和t-SNARE，这样就可以建立每个步骤所需的同源蛋白的阈值数量。然后将研究其他蛋白质辅因子。 目的2 -筏中的相关运动-具有30 nm横向分辨率的组成分析：脂筏的概念是解释膜内特定关联起源的有用主题，尽管膜的整体流动性。筏已被证明是相当困难的研究，因为组件间的相关性可能是短暂的和/或延伸的距离小于光学显微镜的衍射极限。我们建议开发新的方法来表征膜的组成具有非常高的空间分辨率的多同位素成像质谱（MIMS）。MIMS可用于确定单层分子的组成，其横向分辨率约为30 nm，具有非凡的灵敏度，组分的身份由同位素取代编码。使用这种方法，它应该是可能的，以前所未有的横向分辨率，而不使用荧光标记建立的接近和横向组成变化的脂质，糖脂和膜锚定蛋白。