The Impact of Synaptotagmin Isoform Structure and Diversity on Dense Core Granule Exocytosis

 描述（由申请人提供）：交感神经系统（SNS）被全局稳态的威胁激活。作为回应，肾上腺髓质嗜铬细胞分泌一种有效的儿茶酚胺和神经肽的混合物，储存在致密的核心颗粒中，进入循环。通过设计，嗜铬细胞的分泌反应不是固定的，而是可变的，因此可以调节释放以驱动生理学上的补偿或预期变化，这可能是生存所必需的。然而，这种调整的机制是实现如此高的时间保真度和上下文特异性仍然不清楚。嗜铬细胞的分泌反应已被广泛使用生物物理方法建模，但一个主要的假设一直是，颗粒具有相同的基本生化成分和放电内容物在类似的速率。我们最近的发现对这一观点提出了挑战。具体而言，我们发现，颗粒港口功能不同的异构体的关键内源性钙离子传感器突触结合蛋白（Syt）。这些亚型（Syt-1和Syt-7）赋予不同的Ca 2+敏感性的颗粒在原位，使他们能够响应差异去极化刺激和释放内容物的动力学变化超过一个数量级。因此，所提出的研究的假设是，细胞利用分泌颗粒的分子和功能的异质性，以修改基于刺激/Ca 2+水平的释放。与此假说相关的两个具体目标将被解决：1）Syt-1和Syt-7在不同颗粒群上的存在如何驱动嗜铬细胞分泌行为; 2）异构体之间的结构差异，特别是在关键的Ca 2 +/磷脂结合中， C2 AB结构域是其功能的基础。在目标1中，将以各种频率对细胞进行化学刺激，以确定Syt-1和Syt-7颗粒活化要求的差异。“光学膜片钳”将用于分析和映射Syt颗粒相对于Ca 2+微区和通道的分布。最后，我们将采用一种新的亲和纯化方案来确定Syt-1和Syt-7颗粒的其他成分是否也不同。在具体目标2中，我们将利用异构体之间的结构差异来产生一系列嵌合蛋白，其中Syt-1和Syt-7的Ca 2+结合环已经交换。使用人工膜和单分子和曲率敏感光学方法的独特组合，我们将在体外功能分配给这些蛋白质的特定部分。这些“还原论”实验将通过在活细胞中表达嵌合体来补充，使我们能够确定与其在Ca 2+触发的胞吐作用中的作用相关的Syt亚型的关键特性，并利用这些特性来操纵释放。总的来说，这些研究将促进我们目前对分泌系统基本分子组织的理解-这是我研究计划的广泛兴趣。他们还将揭示调节激素分泌以支持交感神经系统功能的机制，这对维持心血管，呼吸和代谢健康至关重要。

 DESCRIPTION (provided by applicant): The sympathetic nervous system (SNS) is activated by threats to global homeostasis. In response, adrenomedullary chromaffin cells secrete a cocktail of potent catecholamines and neuropeptides, stored within dense core granules, into the circulation. By design, the chromaffin cell secretory response is not fixed, but mutable, so that release can be tuned to drive the compensatory or anticipatory changes in physiology that may be necessary for survival. However, the mechanisms by which this tuning is achieved with such high temporal fidelity and context specificity remain unclear. The chromaffin cell secretory response has been modeled extensively using biophysical methods, but a major assumption has always been that granules have the same basic biochemical constituents and discharge contents at similar rates. This idea is challenged by our recent findings. Specifically, we discovered that granules harbor functionally different isoforms of the key endogenous Ca2+ sensor Synaptotagmin (Syt). These isoforms (Syt-1 and Syt-7) confer different Ca2+ sensitivities to the granules in situ, enabling them to respond differentially to depolarizing stimli and to release contents with kinetics that vary by more than an order of magnitude. Thus, the hypothesis underlying the proposed studies is that cells exploit the molecular and functional heterogeneity of secretory granules to modify release based on stimulation/Ca2+ levels. Two Specific Aims related to this hypothesis will be addressed: 1) how the presence of Syt-1 and Syt-7 on different granule populations drives chromaffin cell secretory behavior; 2) how the structural differences between isoforms, particularly within the critical Ca2+/phospholipid binding C2AB domains, underlies their function. In Aim 1, cells will be chemically stimulated at various frequencies to define differences in the activation requirements of Syt-1 and Syt-7 granules. "Optical patch-clamping" will be used to analyze and map the distribution of Syt granules with respect to Ca2+ microdomains and channels. Finally, we will employ a novel affinity purification scheme to determine whether other constituents of Syt-1 and Syt-7 granules also differ. In Specific Aim 2, we will exploit structural differences between isoforms to generate a series of chimeric proteins in which the Ca2+ binding loops of Syt-1 and Syt-7 have been exchanged. Using artificial membranes and a unique combination of single-molecule and curvature-sensitive optical approaches, we will assign in vitro functions to specific parts these proteins. These "reductionist" experiments will be complemented by expression of chimeras in living cells, allowing us to identify the key properties of the Syt isoforms that are relevant to their roles in Ca2+-triggered exocytosis and to exploit those properties to manipulate release. Overall, the studies will advance our current understanding of the basic molecular organization of secretory systems - a broad interest of my research program. They will also reveal the mechanisms by which hormone secretion is tuned to support sympathetic nervous system function, which is essential for maintaining cardiovascular, respiratory, and metabolic health.