MEMBRANE TRAFFICKING IN MAMMALIAN CELLS

分泌和内吞作用在单个细胞和多细胞生物体的健康和功能中起着关键作用，以控制各种生理过程，包括营养摄取、激素和消化酶的分泌、神经细胞之间的突触传递以及对外来病原体的防御。 分泌和内吞都严重依赖于货物的运输，例如，多肽激素，这些途径的各种细胞内细胞器之间。 运输货物的一种机制涉及小的膜囊泡的形成，这些膜囊泡从一个隔室出芽并行进到靶隔室并与靶隔室融合。 然而，最近，另一种可能的手段，用于运输细胞内室之间的材料已成为越来越感兴趣的主题，即，从高尔基复合体，transGolgi网络（TGN），和内涵体形成大小均匀的膜小管。 关于这些小管的确切功能以及它们是如何形成的，我们知之甚少。在过去的两年里，我们一直在进行基础细胞生物学研究，以阐明这些小管的功能及其在哺乳动物细胞中形成的分子机制。 为此，我们已经开发并利用了体外和透化细胞系统，其从高尔基体和内体膜重建小管形成。 使用这些，和其他在体内测定，我们已经获得了补充线的证据表明，膜滋养层独特地需要一个细胞质钙离子依赖性磷脂酶A2（PLA2）酶。 我们建议测试的中心假设是，一个特定的细胞质PLA2是密切参与膜小管的形成，功能在高尔基体到ER逆行贩运，和组装一个完整的，完全互连的高尔基体复合体。 我们的计划是鉴定这种特殊的酶，分离cDNA，并制备针对这种蛋白质的抗体。 这些试剂，沿着PLA 2的药理学抑制剂，然后将用于通过过表达和消融以及体外重建测定来探索细胞中小管的特定功能。这些研究将有助于阐明新的PLA2依赖性，膜管介导的细胞内运输事件的分泌途径。

Secretion and endocytosis play critical roles in the health and function of both individual cells and multi-cellular organisms to control a wide variety of physiological processes including nutrient uptake, secretion of hormones and digestive enzymes, synaptic transmission between nerve cells, and defense against foreign pathogens. Both secretion and endocytosis critically depend on the transport of cargo, e.g., polypeptide hormones, between various intracellular organelles of these pathways. One mechanism for transporting cargo involves the formation of small membranous vesicles that bud from one compartment and travel to and fuse with a target compartment. More recently, however, another possible means for transporting material between intracellular compartments has become the subject of growing interest-namely, the formation of uniformly sized membrane tubules from the Golgi complex, trans Golgi network (TGN), and endosomes. Little is known about the exact function of these tubules, or how they are formed. Over the last two years of this grant, we have been performing basic cell biological research in order to elucidate the function of these tubules and their molecular mechanism of formation in mammalian cells. To do this, we have developed and utilized both in vitro and permeabilized cell systems which reconstitute tubule formation from both Golgi and endosome membranes. Using these, and other in vivo assays, we have obtained complementary lines of evidence indicating that membrane tubulation uniquely requires a cytoplasmic Ca2+- independent phospholipase A2 (PLA2) enzyme. The central hypothesis that we propose to test is that a specific cytoplasmic PLA2 is intimately involved in the formation of membrane tubules that function in Golgi-to-ER retrograde trafficking, and the assembly of an intact, fully interconnected Golgi complex. Our plan is to identify the specific enzyme, isolate a cDNA, and prepare antibodies against the protein. These reagents, along with pharmacological inhibitors of PLA2s, will then be used to explore the specific functions of tubules in cells through over- expression and ablation and in in vitro reconstitution assays. These studies will contribute to the elucidation of novel PLA2- dependent, membrane tubule-mediated intracellular trafficking events in the secretory pathway.