Genetic analysis of regulated exocytosis during Drosophila development

摘要 调节性胞吐是一种基本和关键的细胞生物学过程，在此过程中 分泌细胞对特定刺激做出反应，释放货物蛋白。这一过程具有广阔的前景 对人类健康的影响，因为调控的胞吐功能障碍会导致许多疾病， 包括糖尿病患者胰岛素分泌受损的明显例子。发展中 生长障碍、哮喘和甲状腺功能障碍也是由调节基因异常引起的 胞吐。然而，尽管这对人类健康至关重要，但受监管的主要步骤 胞吐途径目前仍知之甚少。尤其是对……缺乏了解 调控载货分泌颗粒成熟的机制，这一步骤是 这些专门的细胞器能够进行胞吐作用，并确保分泌的货物 生物活性的。进展受到抑制，很大程度上是因为缺乏一种 来研究分泌颗粒的成熟；以前的研究主要依赖于通过 分泌颗粒的电子显微镜和生化分离纯化。 重要的是，我们已经确定了一种新的、高度保守的分泌颗粒成熟调节因子， 命名为霍比特人，在神经内分泌和神经内分泌的调节胞吐方面存在严重缺陷 上皮细胞。该项目的长期目标是识别和表征新的蛋白质，包括 霍比特人，这是调节胞吐所必需的。此应用程序的总体目标是 理解霍比特人和依赖霍比特人的颗粒成熟在调节过程中的作用 胞吐。我们的中心假设是霍比特人，一种调节胞吐作用的新成分 途径，与ESCRT机制和Rab蛋白合作，调节 分泌颗粒经过成熟的过程。我们打算检验中心假说和 通过追求以下两个具体目标来实现本提案的总体目标：1)什么是 霍比特人在调节胞吐作用中的作用；以及2)定义霍比特人 功能。目标1的重点是揭示霍比特人在分泌颗粒成熟中的作用。这个 目标2的重点是识别依赖霍比特人的分泌途径的关键成员，并识别 其他调控胞吐作用的新调节剂。我们的贡献是重大的，因为我们已经确定了 一种以前未知的分泌颗粒成熟调节器，允许我们从基因上 剖析调控这一过程的分子途径。我们希望这项工作能提供新的机制 对调控的胞吐作用途径的洞察，这将对我们的 了解分泌物相关疾病的病因学，包括糖尿病。

ABSTRACT Regulated exocytosis is a fundamental and critical cell biological process during which specialized secretory cells release cargo proteins in response to specific stimuli. This process has broad implications for human health, as dysfunction of regulated exocytosis results in many disorders, including the conspicuous example of impaired insulin secretion in diabetes mellitus. Developmental growth disorders, asthma, and thyroid dysfunction are also caused by abnormalities in regulated exocytosis. However, despite this critical importance for human health, major steps in the regulated exocytosis pathway remain poorly understood. There is a particular lack of understanding of mechanisms regulating maturation of cargo-carrying secretory granules, a step that is critical to render these specialized organelles competent for exocytosis and to ensure that the secreted cargo is biologically active. Progress has been inhibited largely because of a lack of a genetic context in which to study secretory granule maturation; previous studies have relied largely on visual observations via electron microscopy (EM) and biochemical separation and purification of secretory granules. Importantly, we have identified a novel, highly-conserved regulator of secretory granule maturation, named hobbit, with associated severe defects in regulated exocytosis in both neuroendocrine and epithelial cells. The long-term goal of this project is to identify and characterize novel proteins, including hobbit, that are required for regulated exocytosis. The overall objective of this application is to understand the function of hobbit and hobbit-dependent granule maturation during regulated exocytosis. Our central hypothesis is that hobbit, a novel component of the regulated exocytosis pathway, cooperates with the ESCRT machinery and Rab proteins to regulate the progression of secretory granules through the process of maturation. We intend to test the central hypothesis and accomplish the overall objective of this proposal by pursuing the following two specific aims: 1) What is the function of hobbit in regulated exocytosis; and 2) Define the molecular pathway in which hobbit functions. The focus of Aim 1 is to uncover the function of hobbit in secretory granule maturation. The focus of Aim 2 is to identify critical members of the hobbit-dependent secretory pathway, and to identify other new regulators of regulated exocytosis. Our contribution is significant because we have identified a previously uncharacterized regulator of secretory granule maturation, allowing us to genetically dissect the molecular pathway regulating this process. We expect this work to provide new mechanistic insights into the regulated exocytosis pathway, which will have important implications for our understanding of the etiology of secretion-related diseases, including diabetes.