INTRACELLULAR TRANSLOCATION AND METABOLISM OF LIPIDS

本提案中概述的研究涉及以下广泛主题： 哺乳动物细胞内脂质分子的合成和转运， 重要的是正确理解膜生物发生。 以下 地区将进行调查。 我们将继续研究荧光 允许传统脂质生化分析的脂质类似物， 进行并与细胞内位置相关的信息 脂质代谢物的荧光显微镜测定在生活中 细胞 这一方法的普遍适用性将在 使用同位素标记的脂质和EM放射自显影进行平行研究。 我们将研究两种放射性标记的 脂质和荧光脂质类似物插入血浆后 通过脂质体的脂质转移来转移细胞膜。 这些数据将 与细胞中新合成脂质的出现率相比， 表面，以确定是否发生质膜脂质再循环。 我们 将研究荧光脂质、脂质体和其他试剂的行为 在显微注射到单个细胞后。 这些实验旨在 开发一种原位方法来研究脂质的区室化 在它们的生物合成过程中，并用于探测脂质转运的机制 在单个细胞内。 最后，使用基于 荧光光漂白，我们将继续分离突变细胞， 有缺陷的能力，纳入，代谢和/或 在细胞内膜之间转移荧光脂质类似物。 上面列出的研究是理解膜组装的基础 在真核生物中，并且是理性发展的基础。 膜或细胞表面相关疾病状态的治疗。

The research outlined in this proposal deals with the broad topics of synthesis and transport of lipid molecules within mammalian cells, and is important for a proper understanding of membrane biogenesis. The following areas will be investigated. We will continue our studies with fluorescent lipid analogues which permit traditional lipid biochemical analyses to be performed and correlated with information about the intracellular location of lipid metabolites determined by fluorescence microscopy in living cells. The general applicability of this approach will be examined in parallel studies using isotopically labeled lipids and EM autoradiography. We will study the internalization and degradation of both radiolabeled lipids and fluorescent lipid analogues after insertion into the plasma membrane of cells by lipid transfer from liposomes. These data will be compared to the rate of appearance of newly synthesized lipids at the cell surface to determine if recycling of plasma membrane lipids occurs. We will study the behavior of fluorescent lipids, liposomes, and other agents after microinjection into single cells. These experiments are designed to develop an in situ approach for studying the compartmentalization of lipids during their biosynthesis and for probing mechanisms of lipid transport within individual cells. Finally, using a selection procedure based on fluorescence photobleaching, we will attemtp to isolate mutant cells which are defective in their ability to incorporate, metabolize, and/or translocate the fluorescent lipid analogues among intracellular membranes. The studies listed above are basic to an understanding of membrane assembly in eukaryotes, and are fundamental to the development of rational treatments of membrane or cell surface-related disease states.