FUNCTIONAL ANALYSIS OF CALCIUM STORES IN TETRAHYMENA

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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.