Intracellular Translocation and Metabolism of Lipids

描述（由申请人提供）：鞘脂（SLs）在多种细胞功能中发挥作用，包括细胞间相互作用，细胞生长和分化以及信号转导。它们可以与胆固醇相互作用形成膜结构域，许多研究数据表明，质膜（PM） SL和胆固醇组成受到严格调节。这项更新应用的研究将集中在SLs如何调节某些脂质和蛋白质进出细胞表面的运输。使用人类皮肤成纤维细胞，我们将追求三个特定目标来验证我们的中心假设，即细胞SLs对调节这些运输途径至关重要。在Aim 1中，我们将测试SLs通过促进PM结构域形成和组织内吞货物以进行后续内化的能力来调节网格蛋白非依赖性内吞作用的不同机制的假设。一种新型SL类似物对PM结构域形成和内吞货物组织的影响也将根据我们最近发现的这种SL选择性地抑制腔泡内吞作用而进行研究。在Aim 2中，我们将验证鞘糖脂通过诱导整合素和其他跨膜蛋白聚集到PM结构域，从而导致它们的激活和跨膜信号传导来调节空泡内吞作用的假设。抑制SL类似物对这些过程的影响也将被研究，我们将研究抑制整合素功能可能对细胞功能产生深远影响的其他细胞系统。我们还将研究SLs对RhoA-和cdc42依赖的内吞作用的调节，并测试我们的工作模型，即这种调节是由鞘磷脂调节这些gtpase（可能还有其他分子）向PM的易位的能力引起的。在Aim 3中，我们将研究沿分泌途径的选定脂质和蛋白质的运输可能与通过小泡的内吞作用特异性相关的可能性。这些研究还将测试抑制SL和类异戊二烯合成阻断选定蛋白质的分泌运输的潜在机制，以及脂质在调节这一分泌途径中涉及的一些机制中的潜在作用。拟议研究的数据将导致对内吞作用和分泌调节的更好理解，并可能导致通过不依赖网格蛋白的内化机制阻止某些毒素或病原体进入细胞的吸收的技术。抑制脂质的研究也可能在其他细胞系统中有重要的未来应用，其中抑制整合素功能可以对炎症反应或细胞迁移和侵袭性产生重大影响。

DESCRIPTION (provided by applicant): Sphingolipids (SLs) play roles in a wide variety of cell functions, including cell-cell interactions, cell growth and differentiation, and signal transduction. They can interact with cholesterol to form membrane domains, and data from many studies suggest that the plasma membrane (PM) SL and cholesterol composition is tightly regulated. Studies in this renewal application will focus on how SLs regulate transport of certain lipids and proteins to and from the cell surface. Using human skin fibroblasts, we will pursue three Specific Aims to test our central hypothesis that cellular SLs are critical for regulating these transport pathways. Under Aim 1, we will test the hypothesis that SLs regulate different mechanisms of clathrin-independent endocytosis by their ability to promote PM domain formation and to organize endocytic cargo for subsequent internalization. The effect of a novel SL analog on PM domain formation and organization of endocytic cargo will also be examined in light of our recent discovery that this SL selectively inhibits caveolar endocytosis. Under Aim 2, we will test the hypothesis that glycosphingolipids regulate caveolar endocytosis by inducing the clustering of integrins and other transmembrane proteins into PM domains, resulting in their activation and transmembrane signaling. The effects of the inhibitory SL analog on these processes will also be studied, and we will examine other cell systems in which inhibition of integrin function can have a profound effect on cell function. We will also study the regulation of RhoA- and Cdc42-dependent endocytosis by SLs and test our working model that this regulation results from sphingomyelin's ability to modulate the translocation of these GTPases, and perhaps other molecules, to the PM. Under Aim 3, we will investigate the possibility that transport of selected lipids and proteins along the secretory pathway may be specifically linked to endocytosis via caveolae. These studies will also test potential mechanisms by which inhibition of SL and isoprenoid synthesis block secretory transport of selected proteins, and the potential role for lipids in regulating some of the machinery involved in this secretory pathway. Data resulting from the proposed studies will lead to a greater understanding of the regulation of endocytosis and secretion and may lead to techniques for blocking uptake of certain toxins or pathogens that enter cells by clathrin-independent internalization mechanisms. Studies with the inhibitory lipid may also have important future applications in other cell systems where inhibition of integrin function can have significant effects on inflammatory responses, or cell migration and invasiveness.