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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS053978-05
Application #
7837574
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Talley, Edmund M
Project Start
2006-08-20
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
5
Fiscal Year
2010
Total Cost
$324,635
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Physiology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Cazares, Victor A; Njus, Meredith M; Manly, Amanda et al. (2016) Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn. J Neurosci 36:11208-11222
Ben-Simon, Yoav; Rodenas-Ruano, Alma; Alviña, Karina et al. (2015) A Combined Optogenetic-Knockdown Strategy Reveals a Major Role of Tomosyn in Mossy Fiber Synaptic Plasticity. Cell Rep 12:396-404
Hoerauf, Widmann W; Cazares, Victor A; Subramani, Arasakumar et al. (2015) Efficient transfection of dissociated mouse chromaffin cells using small-volume electroporation. Cytotechnology 67:573-83
Bielopolski, Noa; Lam, Alice D; Bar-On, Dana et al. (2014) Differential interaction of tomosyn with syntaxin and SNAP25 depends on domains in the WD40 ?-propeller core and determines its inhibitory activity. J Biol Chem 289:17087-99
Lavi, Ayal; Sheinin, Anton; Shapira, Ronit et al. (2014) DOC2B and Munc13-1 differentially regulate neuronal network activity. Cereb Cortex 24:2309-23
Barak, Boaz; Okun, Eitan; Ben-Simon, Yoav et al. (2013) Neuron-specific expression of tomosyn1 in the mouse hippocampal dentate gyrus impairs spatial learning and memory. Neuromolecular Med 15:351-63
Bar-On, Dana; Wolter, Steve; van de Linde, Sebastian et al. (2012) Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters. J Biol Chem 287:27158-67
Bar-On, Dana; Nachliel, Esther; Gutman, Menachem et al. (2011) Dynamic conformational changes in munc18 prevent syntaxin binding. PLoS Comput Biol 7:e1001097
Anantharam, Arun; Bittner, Mary A; Aikman, Rachel L et al. (2011) A new role for the dynamin GTPase in the regulation of fusion pore expansion. Mol Biol Cell 22:1907-18
Williams, Antionette L; Bielopolski, Noa; Meroz, Daphna et al. (2011) Structural and functional analysis of tomosyn identifies domains important in exocytotic regulation. J Biol Chem 286:14542-53

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