FUNCTIONAL ANALYSIS OF CALCIUM STORES IN TETRAHYMENA

许多细胞类型的分泌型致密核小泡的胞吐作用是由从细胞内储存库中动员的胞内钙的一过性升高引发的。最具特点的钙库是内质网(ER)和特殊的内质网样细胞器。储存库的结构以及储存库内蛋白质的组织都可能对信号的效率和特异性做出贡献。这方面的一个迹象是，钙离子在内质网中的分布并不均匀，这意味着不同的亚区可能在信号潜力上存在差异。富钙区可能是由于特定蛋白质在这些储存库的膜和管腔中的非随机分布而产生的。这还没有经过测试，也不知道这些次区域的基础是什么。我们的目标是进一步开发一种可以在体外和体内识别和分析单个蛋白质的系统，以解决这些问题。嗜热四膜虫的纤毛虫为研究这种机制提供了大量的实验优势。在这个提案中，我们关注纤毛原生动物中一个类似ER的网络，称为肺泡，它已经进化为促进细胞表面的信号传递。当细胞受到促分泌剂的刺激时，肺泡钙被释放，细胞内钙的增加触发调节的分泌囊泡的排泄。在四膜虫中，所有这样的小泡都系在质膜上，并经历同步的膜融合。从实验的角度来看，这提供了一个理想的肺泡信号活动读数。我们建议利用同源重组进行体内分析，研究四膜虫中单个肺泡蛋白在胞外信号中的功能。首先，我们开发了一种在钙转运中活跃的无细胞肺泡制剂。我们的第一个目标是从生物化学的角度分离存在于肺泡腔中的钙缓冲蛋白(脊椎动物钙调素的同源物)，并克隆相应的基因。这将是利用基因替换对体内功能进行突变分析的起点。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对钙信号和动态平衡做出贡献。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，在肌营养不良症中，持续的高胞浆水平可以触发细胞凋亡。其次，特别是对肺泡的详细了解可能是干预属于蜂窝血统的寄生虫的基础，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.