MEMBRANE TRAFFICKING IN MAMMALIAN CELLS

分泌和内吞作用在单个细胞和多细胞生物的健康和功能中起着至关重要的作用，控制着多种生理过程，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和对外来病原体的防御。分泌和内吞作用都严重依赖于这些途径的各种胞内细胞器之间的货物运输，例如多肽激素。运输货物的一种机制涉及小膜囊的形成，这些膜囊从一个隔室萌发，并移动到目标隔室并与之融合。然而，最近，另一种在细胞内区室之间运输物质的可能方法已成为人们日益关注的主题，即由高尔基复合体、反高尔基网络（TGN）和核内体形成均匀大小的膜小管。人们对这些小管的确切功能以及它们是如何形成的所知甚少。在过去的两年里，我们一直在进行基本的细胞生物学研究，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。为了做到这一点，我们已经开发并利用了体外和渗透细胞系统，这些系统可以从高尔基体膜和核内体膜中重建小管形成。通过这些和其他体内试验，我们已经获得了补充的证据，表明膜管化独特地需要细胞质Ca2+独立磷脂酶A2 （PLA2）酶。我们提出要验证的中心假设是，特定的细胞质PLA2密切参与膜小管的形成，膜小管在高尔基体到内质网逆行运输中起作用，以及完整的、完全相互连接的高尔基复合体的组装。我们的计划是鉴定特异性酶，分离cDNA，并制备针对该蛋白的抗体。这些试剂，连同PLA2s的药理学抑制剂，将通过过表达、消融和体外重构实验来探索细胞中小管的特定功能。这些研究将有助于阐明在分泌途径中新的依赖PLA2的膜小管介导的细胞内运输事件。

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.