MECHANISM OF REGULATED EXOCYTOSIS IN TETRAHYMENA

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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.