INTRACELLULAR TRANSLOCATION AND METABOLISM OF LIPIDS

这项提案中概述的研究涉及以下广泛的主题 哺乳动物细胞内脂质分子的合成和运输，以及 对于正确理解膜的生物发生很重要。以下是 将对这些地区进行调查。我们将继续我们对荧光的研究 脂类类似物，允许传统的脂类生化分析 执行并与关于细胞内位置的信息相关联 荧光显微镜检测活体内脂代谢产物的含量 细胞。此方法的一般适用性将在 使用同位素标记的脂类和EM放射自显影的平行研究。 我们将研究两种放射性标记药物的内化和降解。 脂类和荧光脂类类似物进入血浆后 通过脂质体的脂类转移而形成细胞膜。这些数据将是 与细胞中新合成的脂质的出现率相比 以确定是否发生了质膜脂的循环。我们 将研究荧光脂类、脂质体和其他试剂的行为 在单个细胞内显微注射后。这些实验旨在 发展一种原位方法来研究脂类的房室划分 在它们的生物合成和脂类运输的探测机制中 在单个细胞内。最后，使用基于以下条件的选择程序 荧光光漂白，我们将尝试分离突变细胞 在合并、新陈代谢和/或 将荧光类脂类似物在细胞内膜之间转移。 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.