Genetic analysis of regulated exocytosis during Drosophila development

摘要 受调节的胞吐作用是一个基本和关键的细胞生物学过程，在此过程中， 分泌细胞响应特定刺激释放货物蛋白。这一进程具有广泛的 对人类健康的影响，因为受调节的胞吐功能障碍导致许多疾病， 包括糖尿病中胰岛素分泌受损的突出例子。发育 生长障碍、哮喘和甲状腺功能障碍也是由调节的 胞吐作用然而，尽管这对人类健康至关重要，但在受管制的 胞吐途径仍然知之甚少。特别是缺乏对 调节运载货物的分泌颗粒成熟的机制，这是使 这些专门的细胞器负责胞吐作用，并确保分泌的货物是 生物活性。进展受到抑制，主要是因为缺乏遗传背景， 研究分泌颗粒的成熟;以前的研究主要依赖于视觉观察， 电镜（EM）和分泌颗粒的生化分离和纯化。 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 上皮细胞该项目的长期目标是鉴定和表征新的蛋白质，包括 霍比特人，这是所需的调节胞吐。本申请的总体目标是 了解霍比特人和霍比特人依赖的颗粒成熟在调节过程中的作用 胞吐作用我们的中心假设是hobbit，一种调节胞吐作用的新成分， 途径，与ESCRT机制和Rab蛋白合作，调节肿瘤的进展。 分泌颗粒通过成熟的过程。我们打算检验中心假设， 为了实现这一建议的总体目标，我们将努力实现以下两个具体目标：1）什么是 霍比特人在调节胞吐作用中的功能; 2）定义霍比特人 功能协调发展的目的1的重点是揭示霍比特人在分泌颗粒成熟中的功能。的 目标2的重点是确定霍比特人依赖性分泌途径的关键成员，并确定 其他调节胞吐作用的新调节剂。我们的贡献是重要的，因为我们已经确定， 一种以前没有特征的分泌颗粒成熟调节剂，使我们能够从遗传学上 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， 对受调节的胞吐途径的深入了解，这将对我们的研究产生重要影响。 了解与分泌物相关的疾病（包括糖尿病）的病因。

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.