MECHANISM OF REGULATED EXOCYTOSIS IN TETRAHYMENA

专门用于调节分泌的真核细胞利用致密的 核心颗粒（DCG）在蛋白质输出的途径。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DCG内，直到细胞外刺激触发颗粒融合 与质膜接触，导致内容物的释放。在 与组成性蛋白质输出的替代途径相反，颗粒- 介导释放使合成和分泌分离，这是一种适应， 细胞快速调节大分子的分泌， 环境变化。DCG是协调的一个基本特征， 高等真核生物 刺激依赖性分泌不限于多细胞 然而，有机体和致密的核心颗粒在纤毛虫中丰富， 原生动物基于最近的发现包括我们在四膜虫中的工作 颗粒在纤毛虫中与高等纤毛虫相似， 真核生物，这使得调节分泌的机制有可能 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 和用于组成型分泌的酵母酿酒酵母。 我们的长期目标是了解分子间的相互作用， 是致密核心颗粒有规律融合的基础。两项技术进步 于T.嗜热菌现在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，它可以重建细胞质- 依赖性颗粒与质膜融合，以及第二， 通过同源重组的转化已经到位。作为 为理解规范融合的基础，我们提出 具体目标如下：1.体外复溶表征 四膜虫的颗粒融合。我们将具体确定 依赖于胞质溶胶提取物钙和其他潜在的辅因子。 这些结果将成为评估该进程各阶段的基础。2. 使用体外融合试验的细胞溶质要求来鉴定 促进钙依赖性颗粒融合的细胞质因子。 通过将抗血清升高至开始来表征这些蛋白质的作用 体外和体内的描述性和功能性分析。3.探索 草履虫作为融合因子的异源来源。检测细胞质， 草履虫和四膜虫中胞吐缺陷突变体，包括 新分离的突变体，以确定其中的外表型可以是 是细胞质缺乏造成的这将使我们能够聚焦细胞质 其重要性已明确确定的因素的分馏 vivo. 4.克隆与胞吐作用相关的蛋白质的基因片段（见5）。 这两种蛋白质都是通过特异性目标2鉴定的， 预测参与胞吐作用的蛋白质。5.测试 通过基因中断指导克隆蛋白质的体内功能， 克隆的基因片段，并进行修饰，包括插入 表位标签插入感兴趣的蛋白质中。

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.