REGULATION OF ENDOCYTOSIS IN NEUROENDOCRINE CELLS

描述(逐字摘自申请者的描述)：神经元和神经内分泌 细胞通过胞吐释放神经递质/激素，随后恢复 通过内吞作用形成囊泡膜。多年来，关注的焦点是P1‘S 实验室一直在了解分泌周期背后的过程 肾上腺嗜铬细胞系统中的致密核小泡(DCV)。这个 分泌物的生理和分子基础已被广泛研究， 但与胞吐作用相结合的内吞过程的本质是 我只了解了一部分。因此，目前最受关注的领域是 分泌细胞的内吞作用机制。两个相互竞争的假设：完全 囊泡膜与质膜融合后取材 包被网状蛋白的小泡和“接吻就跑”的胞吐，其中小泡从不 关于完全融合和从膜上完好无损地恢复存在争议 在过去的25年里。使用电容测量，我们已经表征了一种 新形式的内吞作用，称为“快速内吞作用”(RE； 秒)，在瞬时触发胞吐作用后立即发生 牛犊AC细胞。这一过程是不依赖于笼状蛋白的，并且可能是 “接吻就跑”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“慢内吞”(SE；Half 几分钟的时间)由更持久的生理激活 刺激，并可能参与恢复水泡膜后完全 核聚变。我们在这里要研究的假设是RE和SE是 在机械和运动上都是不同的，我们将探索关键 监管机构的组成部分证明了这一点。一共有三个 具体目标：(一)确定RE和SE之间的关系并确定 这两个过程都占优势的生理条件。我们会 分析SE的动力学和调节特性，并确定是否 由包被网状蛋白的囊泡介导。(2)确定不同主体的作用 在RE和SE中，Dynamin亚型。小牛AC细胞同时表达Dynamin-L和2和 我们的假设是Dynamin-L特异性地调节RE，而Dynamin-2 监管SE。我们还将确定Dynamin的哪些区域是关键的 对其功能和动力蛋白结合差异牵连的检验 在两种不同形式的内吞作用中配对。(三)明确角色定位 钙、钙调素和钙调神经磷酸酶在RE和SE中的表达。我们知道这一切 调节剂参与了RE，但它们的作用模式以及它们是否也 是否参与SE尚不清楚。为了实现这些目标，我们将使用 包括生化、分子和单细胞在内的多学科方法 嗜铬细胞的电生理学方法。这个项目将加强我们的 理解小泡收回过程中的小泡recvclin2过程 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.