MECHANISM OF REGULATED EXOCYTOSIS IN TETRAHYMENA

专门用于调节分泌的真核细胞利用密集的 蛋白质输出途径中的核心颗粒(DCGS)。在内分泌方面， 外分泌和神经细胞，颗粒是一系列新的- 合成激素、神经肽和消化酶。这些是 储存在DCGS内，直到细胞外刺激触发颗粒融合 与质膜结合，导致内容物释放。在……里面 与构成蛋白输出的另一种途径不同，颗粒- 中介释放使合成和分泌解偶联，一种适应允许 细胞快速调节大分子的分泌以响应 环境变化。DCGS是协调的基本特征 高等真核生物。 依赖刺激的分泌不限于多细胞 然而，有机体和致密的核心颗粒中有大量的纤毛 原生动物。基于最近的发现，包括我们在四膜虫中的工作 纤毛虫中的嗜热菌颗粒与高等纤毛虫中的颗粒相似 真核生物，这使得调节分泌的机制很可能 在整个进化过程中都是保守的。这一原则一直很好。 在过去的十年里，哺乳动物细胞之间的相似性说明了 构成分泌物的酵母菌。 我们的长期目标是了解 致密核心颗粒的规律性融合是基础。两项技术进步 在嗜热性锥虫中，这使其成为研究胞吐作用的独特模型。 首先，我们开发了一种体外系统，它可以重组胞浆- 依赖颗粒与质膜融合，第二，方法 通过同源重组进行的转化现在已经到位。作为一名 为了理解受监管的融合的基础，我们建议 具体目标如下：1.体外重组的特征 四膜虫的颗粒融合。我们将具体确定 依赖胞浆提取钙和其他潜在的辅因子。 这些结果将成为评估这一进程各阶段的基础。2. 用体外融合试验中的细胞溶质要求鉴定 促进钙依赖颗粒融合的细胞质因子。 通过提高抗血清来表征这些蛋白质的作用 体外和体内描述性和功能分析。3.探索 草履虫作为融合因子的异源来源。检测胞浆来自 草履虫和四膜虫的胞吐功能缺陷突变体，包括 新分离的突变体，以确定哪些外表型可以 可追溯到细胞质缺乏。这将使我们能够集中胞浆 对其重要性在#中明确确立的因素进行分级 活着。4.克隆胞吐相关蛋白的基因片段(见5)。 这两种蛋白质都是通过特定目标2鉴定的蛋白质，以及 预测了参与胞吐作用的蛋白质。5.测试 基因阻断克隆蛋白在体内的功能 克隆的基因片段，并进行修改，包括插入 将抗原表位标记成感兴趣的蛋白质。

Eukaryotic cells specialized for regulated secretion make use of dense core granules (DCGs) in a pathway for protein export. In endocrine, exocrine and neuronal cells, granules are reservoirs for a range of newly- synthesized hormones, neuropeptides and digestive enzymes. These are stored within DCGs until an extracellular stimulus triggers granule fusion with the plasma membrane, resulting in release of the contents. In contrast to an alternate pathway of constitutive protein export, granule- mediated release uncouples synthesis and secretion, an adaptation allowing cells to rapidly modulate secretion of macromolecules in response to environmental changes. DCGs are an essential feature of coordination in higher eukaryotes. Stimulus-dependent secretion is not restricted to multi-cellular organisms, however, and dense core granules are abundant in ciliated protozoa. Based on recent discoveries including our work in Tetrahymena thermophila, granules in ciliates appear similar to those in higher eukaryotes, making it likely that mechanisms of regulated secretion have been conserved throughout evolution. This principle has been well illustrated in the last decade by the similarity between mammalian cells and the yeast S.cerevisiae for constitutive secretion. Our long-term goal is to understand the molecular interactions that underlie regulated fusion of dense core granules. Two technical advances in T. Thermophila now make this a unique model for studying exocytosis. First, we have developed an in vitro system which reconstitutes cytosol- dependent granule fusion with the plasma membrane, and second, methods for transformation by homologous recombination are now in place. As a foundation for understanding the basis for regulated fusion, we propose the following specific aims: 1. Characterize the reconstitution in vitro granule fusion in Tetrahymena. We will specifically determine the dependence on a cytosol extract calcium and other potential cofactors. These results will form the basis for evaluating stages in the process. 2. Use the cytosol requirement of the in vitro fusion assay to identify cytoplasmic factors which promote calcium-dependent granule fusion. Characterize the role of these proteins by raising antisera to begin descriptive and functional analysis in vitro and in vivo. 3. Explore Paramecium as a heterologous source for fusion factors. Test cytosol from exocytosis-deficient mutants in Paramecium and Tetrahymena, including newly-isolated mutants, to identify those in which the exophenotype can be traced to a cytosol deficiency. this will allow us to focus cytosol fractionation on factors whose significance is clearly established in vivo. 4. Clone gene fragments (see 5) for proteins related to exocytosis. These will be both proteins identified through Specific Aim 2, and proteins whose involvement in exocytosis is predicted. 5. Test the functions in vivo of cloned proteins by gene interruption directed by cloned gene fragments, and with modifications including insertion of epitope tags into proteins of interest.