Secretion Coupled Endocytosis in Neuroendocrine Cells

说明(申请人摘要)：单胺类递质的区域性缺陷 被认为在各种精神疾病的发病机制中起着关键作用 因此，有必要对其发病机制有详细的了解 这些毒剂的分泌物。神经元的分泌周期和 神经内分泌细胞通过以下途径释放神经递质和激素 胞吐作用，然后通过内吞作用恢复囊泡膜。我们 最近分析了一种新的内吞作用，称为快速内吞作用(RE)， 它存在于包括神经元在内的各种分泌细胞中，并紧密地 与分泌物相结合。RE不同于受体介导的内吞作用(RME) 因为这是一个更快的过程，而且不涉及氯氰菊酯。这种蛋白质 动力素被认为参与了囊泡裂变反应，即 对各种形式的内吞作用至关重要。存在多种形式的动力素，并且 在这项建议中，我们试图确定不同的亚型是否涉及 不同类型的内吞作用以及确定动力蛋白的哪些结构域是 对于蛋白质的功能来说是必不可少的。对于这些研究，我们建议使用 PC12细胞系，一种表现出双侧肾上腺特征的嗜铬细胞瘤 嗜铬(AC)细胞和交感神经元。首先，我们将演示 PC12细胞为细胞的分子分析提供了一个有用的模型系统 神经内分泌细胞的分泌周期。我们将描述它的动力学 并评估这些细胞的胞吐作用和内吞作用是否具有 这些过程类似于以前在AC细胞中看到的过程。第二，我们将 确定在这些细胞中表达的动力蛋白亚型及其在RE中的作用 还有RME。为此，我们将使用一组分子和 免疫程序，包括异构体特异性抗体。第三，我们 我将剖析哪些Dynamin结构域对于RE和RME是必不可少的，重点是 Pleckstrin同源(PH)结构域和富含Pro的C-末端。我们会 将各种Dynamin结构引入PC12细胞，随后 稀土元素和相对分子质量的测定。在这些研究中，我们将使用最先进的 电生理记录，包括膜电容的测量 直接监测胞吐和RE及安培检测 单细胞释放儿茶酚胺。这些研究将具有重要意义 对于一般的分泌生物学，应该进一步加深我们对 单胺能传递的细胞机制对于 开发新的治疗策略。

DESCRIPTION (Applicant's abstract): Regional deficits of monoamine transmitters are thought to play a critical role in the pathogenesis of various mental diseases, hence it is necessary to have detailed knowledge of the mechanisms underlying the secretion of these agents. The secretory cycle in neurons and neuroendocrine cells involves the release of neurotransmitters and hormones by exocytosis followed by recovery of vesicular membrane by endocytosis. We recently analyzed a novel form of endocytosis, called rapid endocytosis (RE), which occurs in a variety of secretory cells including neurons and is tightly coupled to secretion. RE is distinct from receptor-mediated endocytosis (RME) as it is a much faster process and does not involve clathrin. The protein dynamin is thought to participate in the vesicle fission reaction that is critical to various forms of endocytosis. Multiple forms of dynamin exist, and in this proposal we seek to establish if different isoforms are involved in distinct types of endocytosis as well as determine which domains of dynamin are essential to the function of the protein. For these studies we propose to use the PC12 cell line, a pheochromocytoma that exhibits properties of both adrenal chromaffin (AC) cells and sympathetic neurons. First, we will demonstrate that PC12 cells provide a useful model system for the molecular analysis of the secretory cycle in neuroendocrine cells. We will characterize the kinetics of exocytosis and endocytosis in these cells and assess whether the properties of these processes resemble those previously seen in AC cells. Second we will determine the dynamin isoforms expressed in these cells and their role in RE and RME. For this purpose we will employ a battery of molecular and immunological procedures, including isoform-specific antibodies. Thirdly, we will dissect which dynamin domains are essential for RE and RME, focussing on the pleckstrin-homology (PH) domain and the proline-rich C-terminal. We will introduce a variety of dynamin constructs into PC12 cells followed by measurement of RE and RME. In these studies we will use state-of-the-art electrophysiological recordings including measurements of membrane capacitance to directly monitor exocytosis and RE and amperometric detection of catecholamine release from single cells. These studies will have significance for secretory biology in general and should further our understanding of the cellular mechanisms of monoaminergic transmission so necessary for the development of novel therapeutic strategies.