INTRACELLULAR TRANSLOCATION AND METABOLISM OF LIPIDS

脂类分子的分子机制 合成、分类和运输到各种细胞质 我们将研究动物细胞内的隔间。一系列 荧光类脂将被用来关联 活细胞中脂代谢产物的细胞内分布， 通过荧光显微镜测定，用传统的脂类 生化分析。在可能的情况下，将进行类似的研究 使用放射性标记的脂类及其前体进行。这个 目标是：(I)研究囊泡和非囊泡的作用 脂类进出血浆的囊泡机制 膜；(Ii)研究新陈代谢、分选和转运 鞘磷脂通过内质网/高尔基复合体；(Iii)研究 一系列荧光物质的代谢和转运 正常人和突变人皮肤中的放射性神经节苷脂 成纤维细胞；(Iv)检查合成某些 亚细胞膜中的鞘磷脂及其前体 从大鼠肝脏中分离得到的部分；以及(V)研究它们之间的相互作用 脂质体胞内膜半完整 (“穿孔”)细胞。大豆油中荧光类物质的研究 活细胞将通过共振能量转移得到促进 显微镜，就像最近在这个实验室里开发的那样。并通过一个 新近获得的微光数字成像荧光 显微镜。一些研究将依赖于数字的综合 含荧光的类脂类似物(及其立体异构体)。 放射性或亲光性标签。这些将被用来 区分不同的传输机制，并 帮助确定与脂质合成有关的细胞蛋白质， 分拣和运输。本申请中提出的研究 是理解膜的组装和调节的基础 细胞中的膜脂成分，是细胞内 膜或细胞表面的合理处理的发展- 相关疾病状态。

The molecular mechanisms by which lipid molecules are synthesized, sorted, and transported to various cytoplasmic compartments within animal cells will be studied. A series of fluorescent lipid analogs will be used to correlate the intracellular distribution of lipid metabolites in living cells, as determined by fluorescence microscopy, with traditional lipid biochemical analyses. Where possible, similar studies will be performed using radiolabeled lipids and their precursors. The goals are to: (i) examine the role of vesicular and non- vesicular mechanisms of lipid transport to and from the plasma membrane; (ii) study the metabolism, sorting, and transport of sphingolipids through the ER/Golgi complex; (iii) study the metabolism and transport of a series of fluorescent and radioactive gangliosides in normal and mutant human skin fibroblasts; (iv) examine the sites of synthesis of some sphingolipids and their precursors in subcellular membrane fractions isolated from rat liver; and (v) study the interactions of liposomes with the intracellular membranes of semi-intact ("perforated") cells. Studies of the fluorescent lipids in living cells will be facilitated by resonance energy transfer microscopy, as recently developed in this laboratory. and by a recently acquired low-light-level digital imaging fluorescence microscope. Some studies will rely on the synthesis of a number of lipid analogs (and their stereoisomers) bearing fluorescent. radioactive, or photoaffinity labels. These will be used to distinguish various transport mechanisms from one another and to help identify the cellular proteins involved in lipid synthesis, sorting, and transport. The studies proposed in this application are basic to understanding membrane assembly and regulation of membrane lipid composition in cells and are fundamental to the development of rational treatments of membrane or cell surface- related disease states.