Membrane Trafficking in Mammalian Cells

描述(由申请者提供)：这项资助侧重于哺乳动物细胞中通过分泌和内吞途径进行膜运输的基本细胞生物学过程。分泌和内吞作用在单细胞和多细胞生物水平上都发挥着基本的生物学作用，控制着一系列广泛的生理过程，包括营养吸收、激素和消化酶的分泌以及对外来病原体的防御。分泌和内吞作用涉及这些途径的细胞内细胞器之间的货物运输，例如在内质网(ER)和高尔基复合体之间。通过这些途径的蛋白质运输缺陷与包括癌症、囊性纤维化和动脉粥样硬化在内的一系列疾病有关。在哺乳动物细胞中，膜小管(直径60-80 nm，长达许多微米)从分泌和内吞途径的各种细胞器发出，这些小管参与了许多细胞内运输步骤，包括从高尔基复合体到内质网的逆行运输和从内体到细胞表面的受体循环。最近来自我的实验室的证据表明，细胞质中钙离子非依赖性磷脂酶A2(PLA2)和溶血磷脂酰基转移酶(LPAT)具有相反的酶活性，它们可能通过直接改变膜磷脂组成从而改变膜形状来协同作用，介导肾小管的形成。这项资助的长期目标是阐明膜小管在细胞内运输中的作用，并确定特定的PLA2和LPAT酶在哺乳动物细胞分泌和内吞方面的生物学功能。这项工作的目标包括两个大的具体目标：1)识别和表征参与刺激小管形成的PLA2酶和辅助蛋白；2)识别和表征参与介导小管形成和/或包被囊泡分裂的LPAT。这些目标将通过分子、细胞和遗传方法的组合来实现。候选的PLA2和LPAT酶在体内的作用将通过过表达和siRNA敲除实验来研究。这些实验将集中在高尔基体到内质网的逆行运输，内细胞室的输出，以及膜结合细胞器的囊泡分裂。也将进行重建小管或囊泡形成的体外试验，以阐明PLA2活性对膜形状的影响的机制。这些研究将揭示PLA2和LPAT酶在调节分泌和内吞途径中的细胞内转运事件中的新生物学作用。

DESCRIPTION (provided by applicant): This grant focuses on the basic cell biological process of membrane trafficking through the secretory and endocytic pathways in mammalian cells. Secretion and endocytosis play fundamental biological roles at the levels of both single cells and multi-cellular organisms to control a wide array of physiological processes including nutrient uptake, secretion of hormones and digestive enzymes, and defense against foreign pathogens. Secretion and endocytosis involve the transport of cargo between intracellular organelles of these pathways, e.g., between the endoplasmic reticulum (ER) and Golgi complex. Defects in the transport of proteins through these pathways are associated with a host of diseases including cancer, cystic fibrosis, and atherosclerosis. In mammalian cells, membrane tubules (60-80 nm in diameter and to many microns in length) emanate from various organelles of the secretory and endocytic pathways, and these tubules have been implicated in many intracellular trafficking steps, including retrograde trafficking from the Golgi complex to the ER and recycling from receptors from endosomes to the cell surface. Recent evidence from my laboratory shows that cytoplasmic Ca2+independent phospholipase A2 (PLA2) and lysophospholipid acyltransferase (LPAT) enzymes, with opposing enzymatic activities, may work in concert to mediate tubule formation by directly altering membrane phospholipid composition and consequently membrane shape. The long-term objectives of this grant are to elucidate the roles of membrane tubules in intracellular trafficking, and to determine the biological functions of specific PLA2 and LPAT enzymes with respect to secretion and endocytosis in mammalian cells. The goals of this work are encompassed in two large Specific Aims: 1) identify and characterize the PLA2 enzymes and accessory proteins involved in stimulating tubule formation; 2) identify and characterize the LPATs involved in mediating tubule formation and/or coated vesicle fission. These goals will be achieved through a combination of molecular, cellular, and genetic approaches. The in vivo roles of candidate PLA2 and LPAT enzymes in secretory and endocytic trafficking will be investigated by conducting over-expression and siRNA knock-down experiments. These experiments will focus on Golgi-to-ER retrograde trafficking, export from endocytic compartments, and vesicle fission from membrane-bound organelles. In vitro assays that reconstitute tubule or vesicle formation will also be conducted to elucidate the mechanisms underlying the effects of PLA2 activity on membrane shape. These studies will reveal novel biological roles for PLA2 and LPAT enzymes in mediating intracellular trafficking events in the secretory and endocytic pathways.