Membrane Fusion and Dynamics Using Supported Bilayers

说明(申请人提供)：脂双层是生物膜的基本结构。所有蛋白质中有很大一部分与膜有关，作为一类，这些蛋白质构成了药物开发的一个巨大而多样的靶点。流动性对于依赖于多个组分的侧向结合或聚集的生物功能以及改变膜拓扑结构的过程，如胞内和胞外以及融合，都是至关重要的。这项建议概述了探索膜动力学的这两个基本方面的新型实验：囊泡融合机制(目标1)和某些脂类和膜锚定蛋白进入筏子的横向结合(目标2)。这两个目标都依赖于新的支撑脂质双层结构和分析方法的发展，这些结构和分析方法可以对生物膜的研究产生广泛的影响。 目的1-利用可移动的栓系囊泡的囊泡融合机制：囊泡可以被拴在流体支持的双层上，并且可以平行于支持的双层平面自由扩散。单个栓系囊泡的轨迹和碰撞可以通过视频显微镜可视化。显示不同蛋白质的囊泡，如SNARE蛋白，将被拴在支撑的双层上的不同位置，然后可以在单个囊泡的水平上实时监测它们随后的扩散、对接、半融合和融合。这一新的检测系统非常适合对SNARE蛋白是融合所必需且足够的这一假说进行关键测试。提出了一种新的方法来制备非常明确的显示特定数量的v-和t-陷阱的系留小泡群体，从而可以建立每一步所需的同源蛋白质的阈值数量。然后将对其他蛋白质辅助因素进行研究。 目的2-脂筏相关运动-30 nm横向分辨率的成分分析：脂筏的概念对于解释膜内特定结合的起源是一个有用的主题，尽管膜的总体流动性。木筏已被证明是很难研究的，因为组件间的关联可能是瞬时的和/或延伸到小于光学显微镜的衍射极限的距离。我们建议开发新的方法来表征具有很高空间分辨率的膜的组成，用多同位素成像质谱学(MIMS)。MIMS可以用来确定单层分子的组成，横向分辨率约为30 nm，具有极高的灵敏度，其组成成分的身份由同位素替代编码。使用这种方法，应该可以在不使用荧光标记的情况下，以前所未有的横向分辨率建立脂类、糖脂和膜锚定蛋白的邻近和横向组成变化。

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.