Investigating the coordinated functions of a dual Ca2+ sensor system in C. elegans

摘要 神经系统中的信息处理，对大脑功能至关重要，主要是 由化学神经传递调节。自从Katz和他的同事们第一次揭示钙离子 在突触前末端触发超快的囊泡胞吐，研究集中在其作用上 在这一过程中钙离子感受器蛋白的变化。生化、结构和功能研究 发现了突触素蛋白家族的两个成员，突触素-1和-7(Syt1和Syt1 Syt7)，作为初级和次级钙离子，在突触小泡胞吐中起关键作用 许多脊椎动物突触上的传感器。这些钙感受器的缺失会导致严重的突触 Syt1和Syt7的传递缺陷和突变与多种神经系统疾病有关 精神错乱。尽管在啮齿动物的突触素方面已经做了工作，但仍然有许多问题 关于双钙感受器如何协调调节突触传递的问题尚未解决。在 近20年来，线虫已被广泛应用于分子和细胞研究。 突触传递的机制，并表现出非常保守的突触 机械设备。用行为学和电生理学的方法对C. 优雅，我们最近的工作表明，蠕虫NMJ也使用双钙传感器系统 (SNT-1和SNT-3)调节神经递质的释放。SNT-1(初级钙离子)的丢失 传感器)和SNT-3(二次钙离子传感器)完全消除诱发释放，同时损失 其中任何一种，都有不同的影响。蠕虫和蠕虫的双重钙离子传感器系统 小鼠有许多相似之处，这让我们能够研究突触素是如何在 突触，利用线虫作为遗传模式生物的优势和我们独特的技术 专业知识。在这个项目中，我们提出了三个目标来解决SNT-1和SNT-3如何协同工作 来调节神经传递。AIM1重点研究SNT-1/3与 SNARE复合体和质膜。一些高度保守的SNT-1和SNT-3 在脊椎动物中，已知与圈套和质膜连接的残基将是 突变以测试它们对SV融合的影响。目标2将研究空间和动力学方面的问题 SNT-1和SNT-3在协调突触释放中起作用，并决定 SNT-1、SNT-3和关键启动因子UNC-13。在目标3中，我们将确定SNT-1和 SNT-3在高释放概率条件下调节突触传递。调查结果 应该大大扩展我们对双钙传感器系统如何运行以满足 在基础和升高释放条件下对突触的需求。

SUMMARY Information processing in the nervous system, essential for brain function, is predominantly mediated by chemical neurotransmission. Since Katz and his colleagues first revealed that Ca2+ triggers ultrafast vesicle exocytosis at the presynaptic terminal, studies have focused on the roles of the Ca2+ sensor proteins in this process. Biochemical, structural, and functional studies have found that two members of the synaptotagmin protein family, synaptotagmin-1 and -7 (Syt1 and Syt7), play critical roles in synaptic vesicle exocytosis by acting as primary and secondary Ca2+ sensors at many vertebrate synapses. Deletion of these Ca2+ sensors leads to severe synaptic transmission defects, and mutations in Syt1 and Syt7 have been linked to multiple neurological disorders. Despite the work done on synaptotagmins in rodents, a number of questions remain unresolved regarding how dual Ca2+-sensors coordinate to regulate synaptic transmission. In the last 20 years, the nematode C. elegans has been widely used to study the molecular and cellular mechanisms of synaptic transmission, and exhibit remarkably well-conserved synaptic machinery. Using behavioral and electrophysiological screens of synaptotagmin isoforms in C. elegans, our recent work has shown that the worm NMJs also use a dual Ca2+ sensor system (SNT-1 and SNT-3) to regulate neurotransmitter release. Loss of SNT-1 (the primary Ca2+ sensor) and SNT-3 (the secondary Ca2+ sensor) completely eliminate evoked release, while loss of either alone, has differential effects. The fact that the dual Ca2+ sensor systems of worms and mice share many similarities, allows us to study how synaptotagmins coordinately function at synapses, using the strengths of C. elegans as a genetic model organism and our unique technical expertise. In this project, we propose three aims to address how SNT-1 and SNT-3 work together to regulate neurotransmission. Aim1 focuses on potential interactions between SNT-1/3 with SNARE complexes and the plasma membrane. A number of highly conserved SNT-1and SNT-3 residues known to interface with SNAREs and the plasma membrane in vertebrates, will be mutated to test their effects on SV fusion. Aim 2 will investigate spatial and kinetic aspects of SNT-1 and SNT-3 function in coordinating synaptic release, and determine the interplay between SNT-1, SNT-3 and the key priming factor UNC-13. In Aim 3, we will determine how SNT-1 and SNT-3 regulate synaptic transmission under conditions of high release probability. The findings should significantly expand our understanding of how dual Ca2+ sensor systems operate to meet the demands of synapses under basal as well as elevated-release conditions.