Intracellular Translocation and Metabolism of Lipids

描述（由申请人提供）：鞘脂（SL）在多种细胞功能中发挥作用，包括细胞-细胞相互作用、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，许多研究的数据表明质膜（PM）SL和胆固醇组成受到严格调控。这项更新申请的研究将集中在SL如何调节某些脂质和蛋白质往返细胞表面的转运。使用人类皮肤成纤维细胞，我们将追求三个特定的目标来测试我们的中心假设，即细胞SL对调节这些转运途径至关重要。根据目标1，我们将测试的假设，SL调节网格蛋白独立的内吞作用的不同机制，他们的能力，以促进PM域的形成和组织内吞货物随后的内化。一种新的SL类似物PM结构域的形成和组织的内吞货物的影响也将根据我们最近的发现，这SL选择性地抑制小窝内吞检查。在目标2下，我们将测试鞘糖脂通过诱导整合素和其他跨膜蛋白聚集到PM结构域中，导致其激活和跨膜信号传导来调节小窝内吞作用的假设。抑制性SL类似物对这些过程的影响也将进行研究，我们将研究其他细胞系统，其中整合素功能的抑制可以对细胞功能产生深远的影响。我们还将研究调节RhoA和Cdc 42依赖的内吞SL和测试我们的工作模型，这种调节结果从鞘磷脂的能力，以调节这些GTP酶的易位，也许其他分子，PM。在目标3下，我们将研究选择的脂质和蛋白质的运输沿着分泌途径可能通过小窝特异性地与内吞作用相关的可能性。这些研究还将测试潜在的机制，SL和类异戊二烯合成的抑制块选定的蛋白质的分泌运输，以及脂质在调节参与这种分泌途径的一些机制的潜在作用。从拟议的研究中获得的数据将导致对内吞作用和分泌调节的更好理解，并可能导致用于阻断通过网格蛋白非依赖性内化机制进入细胞的某些毒素或病原体的摄取的技术。抑制性脂质的研究也可能在其他细胞系统中具有重要的未来应用，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显著影响。

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.