Stimulus-induced exocytosis plays a key role in homeostasis and is involved in glandular, synaptic and other secretory systems. Trichocyst release in Paramecium tetraurelia is being studied as a model of stimulus-induced exocytosis. This phenomenon is Ca++ mediated and involves expansion of the secretion granule and membrane fusion. Recently, we have shown that dephosphorylation of a 65,000 Mr phosphoprotein (65kPP) is correlated with one or more of these signatures of secretion. Secretory mutants are available in this cell. In this proposal, we aim to extend our original observations to show that the protein dephosphorylation is involved in one of the regulatory steps between stimulus and secretion in this cell. We then will examine additional mutant strains blocked at different points along the secretory pathway to specify further the part of the pathway correlated with dephosphorylation. Additional secretagogues that may differentiate between content (tmx) expansion and membrane fusion in the mutants will also be studied. Using these secretagogues, we will follow the time course of dephosphorylation and rephosphorylation after a stimulus. Wild type (wt) and mutant cells will be exposed to calmodulin (CaM) antagonists that inhibit stimulus-induced secretion to test whether dephosphorylation is concurrently inhibited. We also hope to obtain a well characterized cell fraction from wt cells that shows an enrichment of a 65kPP, and then to define conditions for the cell fraction under which the protein can be dephosphorylated and rephosphorylated in vitro. Our ultimate objective is a description of the precise sequence of protein modifications and morphological changes that occur locally in the secretory organelle and the cell membrane during stimulus-secretion coupling.
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