FUNCTIONAL ANALYSIS OF CALCIUM STORES IN TETRAHYMENA

在许多细胞类型中，分泌致密核囊泡的胞外分泌是由细胞内蓄水池动员的胞质钙的短暂升高引起的。最具特征的钙储存库是内质网（ER）和特化的ER样细胞器。储存库的结构以及其中蛋白质的组织都可能有助于信号传导的效率和特异性。这方面的一个迹象是，钙在整个内质网中的分布并不均匀，这意味着子区域的信号传导电位可能不同。富钙结构域可能是由特定蛋白质与这些储层的膜和管腔的非随机分布产生的。这一点尚未得到检验，这些分区域的基础也不为人所知。我们的目标是进一步开发一种系统，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。纤毛虫嗜热四膜虫为研究这一机制提供了许多实验优势。在本研究中，我们将重点放在纤毛原生生物的内质网样网络上，称为肺泡，它已经进化到促进细胞表面的信号传导。当细胞受到促分泌剂的刺激时，肺泡钙被释放，胞质钙的增加引发受调节的分泌囊泡的胞吐。在四膜动物中，所有这些囊泡都系在质膜上并进行同步的膜融合。从实验的角度来看，这提供了肺泡信号活动的理想读数。我们建议利用同源重组的方法在体内研究单个肺泡蛋白在四膜虫胞外信号传导中的功能。首先，我们已经开发了一种无细胞的肺泡制剂，它在钙运输中是活跃的。我们的第一个目标是生物化学分离肺泡腔内的钙缓冲蛋白（脊椎动物钙螯合蛋白的同源物），并克隆相应的基因。这将是使用基因替代进行体内功能突变分析的起点。其他调节肺泡钙通量的蛋白质将根据直接或间接的遗传筛选来确定。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体内平衡。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中肌肉坏死的直接原因，其中长期的高细胞质水平可触发细胞凋亡。其次，对肺泡的详细了解可能是干预肺泡谱系寄生虫的基础，包括导致疟疾、隐孢子虫病和弓形虫病的生物。

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.