Imaging supported lipid bilayers and actual cell membrane fragments by NanoSIMS

描述(由申请人提供)：支撑的脂类双分子层已被用作研究具有特定组成的模型膜的生物物理性质的工具。胆固醇在脂膜相行为中的作用，特别是对脂类“筏”或结构域和复合体的形成，已经得到了特别的关注。用来解释二元和三元类脂双分子膜相行为的常用技术(即荧光和原子力显微镜)由于无法提供关于膜的空间组成的直接信息而受到限制。二次离子质谱仪(NanoSIMS)是一种对脂质双层的侧向组织和组成进行成像的有力工具。由于脂质双层中的成分是由同位素标记唯一识别的，因此NanoSIMS可以明确地识别在小至50 nm的区域内共存的不同分子。在这些研究中，描述了一种在高13C浓缩水平下生产胆固醇同位素异构体的方法。此外，NanoSIMS将用于成像胆固醇和蛋白质在天然细胞膜碎片中的空间分布和共定位。 与公共卫生相关：就横向组成和组织而言，细胞膜的脂质双层是一个复杂而动态的系统。活细胞依赖于膜成分的协调重排，如胆固醇、脂类和蛋白质；然而，人们对这种组织在细胞-细胞相互作用、信号转导、膜运输以及膜结合蛋白的活性和可及性等基本过程中的变化知之甚少。在这些研究中，二次离子质谱仪(NanSIMS)成像将被用来获得模型和实际细胞膜的纳米级分辨率的定量组成信息。

DESCRIPTION (provided by applicant): Supported lipid bilayers have been used as a tool to study the biophysical properties of model membranes with defined compositions. Special attention has been given to the role of cholesterol on the phase behavior of lipid membranes, in particular, to the formation of lipid "rafts" or domains and complexes. Common techniques used to elucidate the phase behavior of binary and ternary lipid bilayers (i.e. fluorescence and atomic force microscopy) have been limited by their inability to provide direct information on the spatial composition of membranes. Secondary ion mass spectrometry (NanoSIMS) is a powerful tool for imaging the lateral organization and composition of lipid bilayers. Because the components in a lipid bilayer are uniquely identified by isotopic labels, NanoSIMS can unambiguously identify different molecules co-localized in a region as small as 50nm. In these studies, a method for producing cholesterol isotopomers at high levels of 13C enrichment is described. Furthermore, NanoSIMS will be used to image the spatial distribution and co-localization of cholesterol and proteins in native cell membrane fragments. PUBLIC HEALTH RELEVANCE: The lipid bilayer of a cell membrane is a complex and dynamic system in terms of lateral composition and organization. Living cells depend on the concerted rearrangement of membrane components, such as cholesterol, lipids, and proteins; yet very little is known about how this organization changes during essential processes, such as, cell-cell interactions, signal transduction, membrane trafficking, and membrane-bound protein activity and accessibility. In these studies, secondary ion mass spectrometry (nanoSIMS) imaging will be used to obtain quantitative compositional information with nanometer scale resolution of model and actual cell membranes.