Regulation of tomosyn-SNARE complex assembly in neurosecretion

描述（由申请人提供）：SNARE蛋白组装成三元核心复合物对神经递质释放至关重要。SNARE复合物组装的精确调节最终决定了胞吐事件的位点和动力学。我们的目标是了解调节这些SNARE复合物的时空组装的机制。Tomosyn是一种蛋白质，其在设置具有渗出能力的SNARE复合物的水平中是关键的。已经提出其调节作用主要通过其与Q-SNARE突触融合蛋白IA的相互作用介导，这导致非融合SNARE复合物的形成。提出的研究的目标是提供一个了解的分子机制和信号转导途径，在调节释放的神经递质的tomosyn-SNARE复合物的组装/拆卸。我们的一般假设是，tomosyn-SNARE复合物的形成是由Rho-GTdR信号通路促进的，并被蛋白激酶A依赖性通路拮抗，这些通路的激活平衡微调融合能力SNARE复合物的水平。目的1将测试的假设，tomosyn-SNARE复合物的形成是在动态控制下的分泌激动剂调节分泌。此外，我们将确定N-末端tomosyn结构域的重要性，这种调节，和内源性tomosyn颗粒启动和胞吐的数量效应。目的2将检验以下假设：tomosyn-SNARE复合物的水平及其对胞吐途径的功能性影响受到Rho激活状态（促进tomosyn-SNARE复合物组装）和PKA（拮抗tomosyn-SNARE复合物组装）之间的平衡的精细调节。目的3将定义tomosyn-SNARE复合物在质膜上的空间特性，并确定RhoA和PKA是否介导这些复合物的组装和分解的空间限定效应。实验将使用生物化学，光学和电生理方法的组合肾上腺嗜铬细胞，神经递质释放的高度特征化的细胞模型。了解神经递质释放的调节对于了解神经系统的功能是必不可少的，并且对于以特定神经递质失衡为代表的许多精神和神经病症的治疗性治疗的发展是基础。

DESCRIPTION (provided by applicant): 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.