The assembly of SNARE proteins into a ternary core complex is essential for neurotransmitter release. Precise regulation of SNARE complex assembly ultimately determines the site and dynamics of the exocytotic event. Our objective is to understand the mechanisms that regulate temporal and spatial assembly of these SNARE complexes. Tomosyn is a protein that is critical in setting the level effusion- competent SNARE complexes. Its regulatory action has been proposed to be primarily mediated by its interaction with the Q-SNARE syntaxinl A, which results in the formation of non-fusogenic SNARE complexes. The goal of the research proposed is to provide an understanding of the molecular mechanisms and signal transduction pathways governing the assembly/disassembly of tomosyn-SNARE complexes in the regulated release of neurotransmitter. Our general hypothesis is that tomosyn-SNARE complex formation is promoted by Rho-GTPase signaling pathways and antagonized by protein kinase A-dependent pathways, with the balance of activation of these pathways fine-tuning the level of fusion-competent SNARE complexes. Aim 1 will test the hypothesis that the formation of tomosyn-SNARE complexes is under dynamic control by secretory agonists to regulate secretion. In addition, we will identify N-terminal tomosyn domains important for this regulation, and quantity effects of endogenous tomosyn on granule priming and exocytosis. Aim 2 will test the hypothesis that the level of tomosyn-SNARE complexes and their functional effects on the exocytotic pathway are finely regulated by a balance between the activation state of Rho, to promote tomosyn-SNARE complex assembly, and PKA, to antagonize tomosyn-SNARE complex assembly. Aim 3 will define the spatial properties of tomosyn-SNARE complexes on the plasma membrane and determine if RhoA and PKA mediate spatially delimited effects on the assembly and disassembly of these complexes. Experiments will be performed using a combination of biochemical, optical and electrophysiological approaches on adrenal chromaffin cells, a highly characterized cell model for neurotransmitter release. Understanding the regulation of neurotransmitter release is essential to understanding the function of the nervous system and fundamental to development of therapeutic treatments in the many psychiatric and neurological conditions typified by an imbalance of particular neurotransmitters.