FUNCTIONAL ANALYSIS OF CALCIUM STORES IN TETRAHYMENA

四膜虫钙储存的功能分析

• 批准号: 6498689
• 负责人: AARON P TURKEWITZ
• 金额: $ 20.04万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498689
• 关键词: SDS polyacrylamide gel electrophoresis; Tetrahymena; biological signal transduction; calcium binding protein; calcium flux; cell free system; endoplasmic reticulum; exocytosis; homeostasis; ion transport; membrane proteins; molecular cloning; polymerase chain reaction; protein structure function; protein transport; site directed mutagenesis; vesicle /vacuole

Exocytosis of secretory dense-core vesicles in many cell types is triggered by a transient elevation of cytosolic calcium that is mobilized from intracellular reservoirs. The best characterized calcium reservoirs are the endoplasmic reticulum (ER) and specialized ER-like organelles. Both the structure of reservoirs as well as the organization of the proteins within them are likely to contribute to the efficiency and specificity of signaling. One indication of this is that calcium is not uniformly distributed throughout the ER, implying that sub-regions may differ in signaling potential. Calcium-rich domains may be generated by the non-random distribution of specific proteins with the membrane and lumen of these reservoirs. This has not been tested, nor are the bases for such sub-regions known. Our aim is to develop further a system in which individual proteins can be identified and analyzed both in vitro and in vivo, to address these issues. The ciliate Tetrahymena thermophila offers a host of experimental advantages for studying such mechanisms. In this proposal, we focus on an ER-like network in ciliated protists, called the alveoli, that has evolved to facilitate signaling at the cell surface. Alveolar calcium is released when cells undergo stimulation with secretagogues, and the increase in cytosolic calcium triggers exocytosis of regulated secretory vesicles. In Tetrahymena, all such vesicles are tethered at the plasma membrane and undergo synchronous membrane fusion. From the experimental perspective, this provides an ideal read-out of alveolar signaling activity. We propose to study the function of individual alveolar proteins in exocytic signaling in Tetrahymena, taking advantage of homologous recombination for in vivo analysis. To begin, we have developed a cell-free alveolar preparation that is active in calcium transport. Our first aim is to isolate biochemically the calcium buffer proteins (homologs of vertebrate calsequestrins) that reside in the alveolar lumen, and clone the corresponding genes. This will be a starting point for mutational analysis of in vivo function, using gene replacement. Other proteins that modulate calcium flux in alveoli will be identified based on direct or indirect genetic screens. The long-term aim of this work is to develop an understanding of how proteins in intracellular reservoirs contribute to calcium signaling and homeostasis. Such questions are medically important for at least two reasons. First, defects in calcium homeostasis may be a direct cause of muscle necrosis in muscular dystrophy, in which prolonged high cytosolic levels can trigger apoptosis. Secondly, a detailed understanding of alveoli in particular might be a basis for intervention against parasites belonging to the Alveolate lineage, including the organisms responsible for malaria, cryptosporodiosis, and toxoplasmosis.

在许多细胞类型中，分泌性致密核心囊泡的胞吐作用由从细胞内储库动员的胞质钙离子的瞬时升高触发。 最具特征的钙储库是内质网（ER）和专门的ER样细胞器。 水库的结构以及其中蛋白质的组织可能有助于信号传导的效率和特异性。 其中一个迹象是钙在整个ER中分布不均匀，这意味着亚区域的信号传导潜力可能不同。 富钙结构域可以通过特定蛋白质与这些储库的膜和腔的非随机分布来产生。 这一点尚未得到验证，也不知道这些次区域的基础。 我们的目标是进一步开发一种系统，在该系统中，可以在体外和体内识别和分析单个蛋白质，以解决这些问题。 纤毛虫四膜虫thermophila提供了一个主机的实验优势，研究这种机制。 在这个提议中，我们专注于纤毛原生生物中的ER样网络，称为肺泡，它已经进化到促进细胞表面的信号传导。当细胞受到促分泌素刺激时，肺泡钙释放，胞质钙的增加触发受调节的分泌囊泡的胞吐作用。 在四膜虫中，所有这些囊泡都被束缚在质膜上，并进行同步膜融合。 从实验的角度来看，这提供了一个理想的读出肺泡信号活性。我们建议研究单个肺泡蛋白在四膜虫胞外信号传导中的功能，利用同源重组进行体内分析。 开始，我们已经开发了一种无细胞肺泡制剂，是积极的钙转运。 我们的第一个目标是生化分离的钙缓冲蛋白（同源物的脊椎动物钙螯合蛋白），驻留在肺泡腔，并克隆相应的基因。 这将是使用基因置换进行体内功能突变分析的起点。 其他调节肺泡钙流的蛋白质将根据直接或间接的遗传筛选进行鉴定。 这项工作的长期目标是了解细胞内储存库中的蛋白质如何促进钙信号传导和稳态。 这些问题在医学上很重要，至少有两个原因。 首先，钙稳态的缺陷可能是肌营养不良症中肌肉坏死的直接原因，其中长时间的高胞质水平可引发细胞凋亡。 其次，对肺泡的详细了解可能是对属于肺泡谱系的寄生虫进行干预的基础，包括负责疟疾，隐孢子虫病和弓形虫病的生物体。