Role of Phospholipid Remodeling in Secretion and Golgi Function in Mammalian Cell

描述(申请者提供)：这项资助侧重于哺乳动物细胞中通过分泌途径进行膜运输的基本细胞生物学过程。分泌物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，包括激素和消化酶的释放、神经传递和对外来病原体的防御。分泌涉及细胞内细胞器之间的货物运输，例如高尔基复合体和质膜之间的运输。通过这些途径的蛋白质运输缺陷与许多疾病有关，包括癌症、囊性纤维化、神经退行性变和动脉粥样硬化。在哺乳动物细胞中，膜小管(直径60-80 nm，长达许多微米)来自分泌途径的各种细胞器，这些小管参与了许多细胞内的运输步骤，例如从高尔基复合体到内质网的逆行运输和通过高尔基体的顺行运动。本实验室最近的研究表明，细胞质磷脂酶A(PLA)和膜溶血磷脂酰基转移酶(LPAT)具有相反的酶活性，它们通过直接改变膜磷脂的组成从而改变膜的形状来共同作用于介导膜小管的形成。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，并确定特定的解放军和LPAT酶在哺乳动物细胞分泌方面的生物学功能。这项工作的目标包括三个具体目标，重点是哺乳动物高尔基体复合体：1)鉴定和表征参与细胞内膜运输的PLA2和LPAT酶；2)确定PLA2和LPAT酶对高尔基体包被的囊泡和膜管的产生的贡献；以及3)确定PLA2和LPAT促进膜管形成的分子机制。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LA和LPAT酶在体内的作用将通过进行过表达和siRNA敲除实验来研究。这些实验将侧重于膜向高尔基复合体的运输、穿过高尔基复合体和从高尔基复合体传出，以及膜结合细胞器的囊泡分裂。此外，还将进行重建小管或囊泡形成的体外实验，以阐明聚乳酸和LPAT活性对膜形状的影响机制。这些研究将揭示PLA酶和LPAT酶在调节分泌途径的细胞内转运事件中的新的生物学作用，并促进我们对膜小管介导的转运的理解。

DESCRIPTION (provided by applicant): This grant focuses on the basic cell biological process of membrane trafficking through the secretory pathway in mammalian cells. Secretion plays fundamental biological roles at the level of both single cells and multi-cellular organisms t control a wide array of physiological processes including release of hormones and digestive enzymes, neurotransmission, and defense against foreign pathogens. Secretion involves the transport of cargo between intracellular organelles, e.g., between the Golgi complex and plasma membrane. Defects in the transport of proteins through these pathways are associated with a host of diseases including cancer, cystic fibrosis, neurodegeneration, and atherosclerosis. In mammalian cells, membrane tubules (60-80 nm in diameter and up to many microns in length) emanate from various organelles of the secretory pathway, and these tubules have been implicated in many intracellular trafficking steps, e.g., retrograde trafficking from the Golgi complex to the ER and anterograde movement through the Golgi stack. Recent evidence from my laboratory indicates that cytoplasmic phospholipase A (PLA) and integral membrane lysophospholipid acyltransferase (LPAT) enzymes, with opposing enzymatic activities, work in concert to mediate membrane 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 PLA and LPAT enzymes with respect to secretion in mammalian cells. The goals of this work are encompassed in three Specific Aims, with a focus on the mammalian Golgi complex: 1) identify and characterize PLA and LPAT enzymes involved in intracellular membrane trafficking; 2) determine the contribution of PLA2 and LPAT enzymes to the production of Golgi coated vesicles and membrane tubules; and 3) determine the molecular mechanisms by which PLA2 and LPATs contribute to membrane tubule formation. These goals will be achieved through a combination of molecular, cellular, and genetic approaches. The in vivo roles of candidate PLA and LPAT enzymes in secretory trafficking will be investigated by conducting over expression and siRNA knockdown experiments. These experiments will focus on membrane trafficking to, through, and from the Golgi complex, 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 PLA and LPAT activity on membrane shape. These studies will reveal novel biological roles for PLA and LPAT enzymes in mediating intracellular trafficking events in the secretory pathway and advance our understanding of membrane tubule-mediated transport.