REGULATION OF ENDOCYTOSIS IN NEUROENDOCRINE CELLS

描述（申请人描述的逐字）：神经元和神经内分泌 细胞通过胞吐作用释放神经递质/激素，然后恢复 通过内吞作用形成囊泡膜。多年来，P1的重点是 实验室已经了解分泌周期的基本过程 肾上腺嗜铬细胞系中致密核心囊泡的形成。的 分泌的生理和分子基础已经被广泛研究， 但与胞吐作用相结合的内吞过程的本质是 只是部分理解。因此，当前非常感兴趣的一个领域是 分泌细胞内吞作用的机制。两个相互竞争的假设：完全 囊泡膜与质膜融合，然后在 网格蛋白包被的囊泡和“吻-跑”胞吐作用， 完全融合并从膜上完整地恢复一直存在争议 在过去的25年里。使用电容测量，我们表征了 一种新的内吞形式，称为“快速内吞”（RE; 秒），这发生在胞吐作用被短暂触发后， 小牛AC细胞这个过程是网格蛋白独立的，可能是一部分， “吻了就跑”的机制最近，新的初步数据表明， 另一种类型的膜回收，我们称之为“缓慢内吞作用”（SE;半 几分钟的时间）被更持续的生理激活 刺激，并可能参与恢复囊膜后充分 核聚变我们将在这里研究的假设是RE和SE是 在机械上和动力学上都是不同的，我们将探索关键 监管机构的组成部分，以证明这一点。有三 具体目标：（一）确定RE和SE之间的关系，并确定 两个过程中任一过程占主导地位的生理条件。我们将 分析SE的动力学和调节特性，并确定 由网格蛋白包被的囊泡介导。(2)确定不同的角色 RE和SE中的发动蛋白亚型。小牛AC细胞表达动力蛋白1和2， 我们的假设是发动蛋白-1特异性调节RE，而发动蛋白-2 规范SE。我们还将确定发动蛋白的哪些领域是关键的 其功能和动力蛋白结合的差异参与测试 参与两种不同形式的内吞作用。(3)确定的作用 RE和SE中的钙、钙调蛋白和钙调神经磷酸酶。我们知道所有这些 调节剂参与RE，但它们的作用方式以及它们是否也 参与SE是未知的。为了实现这些目标，我们将使用 多学科方法，包括生物化学，分子和单细胞 嗜铬细胞的电生理方法。该项目将提高我们的 理解囊泡回收过程中的囊泡回收 在分泌细胞中。

DESCRIPTION (Verbatim from Applicant's Description): Neurons and neuroendocrine cells release neurotransmitters/ hormones by exocytosis followed by recovery of vesicular membrane by endocytosis. For many years, the focus of the P1's laboratory has been to understand the processes underlying the secretory cycle of dense-core-vesicles (DCVs) in the adrenal chromaffin (AC) cell system. The physiological and molecular basis of secretion has been studied extensively, but the nature of the endocytotic processes that are coupled to exocytosis is only partly understood. As such, an area of intense current interest is the mechanism of endocytosis in secretory cells. Two competing hypotheses: complete fusion of vesicular membrane with plasma membrane followed by retrieval in clathrin-coated vesicles and "kiss-and-run" exocytosis where vesicles never completely fuse and are recovered intact from the membrane have been debated for the last 25 years. Using capacitance measurements we have characterized a novel form of endocytosis, termed "rapid endocytosis" (RE; half time of seconds), that occurs immediately after exocytosis is transiently triggered in bovine calf AC cells. This process is clathrin-independent and may be part of the "kiss-and-run" mechanism. Recently, new preliminary data suggest that another type of membrane retrieval which we call "slow endocytosis" (SE; half time of several minutes) is activated by more sustained physiological stimulation and may be involved in recovering vesicular membrane after full fusion. The hypothesis we will investigate here is that RE and SE are mechanistically as well as kinetically distinct and we will probe key components of the regulatory machinery to prove this point. There are three specific aims: (I) Determine the relationship between RE and SE and determine the physiological conditions under which either process is predominant. We will analyze the kinetic and regulatory properties of SE and determine if is mediated by clathrin coated vesicles. (2) Determine the role of different dynamin isoforms in RE and SE. Calf AC cells express both dynamin-l and 2 and our hypothesis is that dynamin-l specifically regulates RE while dynamin-2 regulates the SE. We will also identify which domains of dynamin are critical for its function and test for differential involvement of dynamin-binding partners in the two different forms of endocytosis. (3) Determine the role of calcium, calmodulin and calcineurin in RE and SE. We know that all these modulators are involved in RE but their mode of action and whether they also participate in SE is not known. To accomplish these aims we will use a multidisciplinary approach that includes biochemical, molecular and single-cell electrophysiological methods in chromaffin cells. This project will enhance our understanding of vesicle retrieval that underlies vesicle recvclin2 processes in secretory cells.