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)被全球动态平衡的威胁所激活。作为回应，肾上腺髓质嗜铬细胞分泌一种强效儿茶酚胺和神经肽的混合物，储存在致密的核心颗粒中，进入血液循环。根据设计，嗜铬细胞的分泌反应不是固定的，而是可变的，因此释放可以被调节，以驱动可能是生存所必需的代偿性或预见性的生理变化。然而，这种调谐实现如此高的时间保真度和上下文特异性的机制仍然不清楚。嗜铬细胞的分泌反应已经被广泛地用生物物理方法来模拟，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相似的放电速率。我们最近的发现对这一观点提出了挑战。具体地说，我们发现颗粒含有功能上不同的内源性关键钙感受器突触素(SYT)的亚型。这些异构体(SYT-1和SYT-7)赋予原位颗粒不同的钙敏感性，使它们能够对去极化刺激做出不同的反应，并以超过一个数量级的动力学变化释放内容物。因此，支持这项研究的假设是，细胞利用分泌颗粒的分子和功能异质性，基于刺激/钙离子水平来调节释放。与这一假说相关的两个具体目标将被解决：1)不同颗粒群体上SYT-1和SYT-7的存在如何驱动嗜铬细胞的分泌行为；2)不同亚型之间的结构差异，特别是在关键的钙/磷脂结合方面 C2AB结构域，是其功能的基础。在目标1中，将以不同的频率对细胞进行化学刺激，以确定SYT-1和SYT-7颗粒激活要求的差异。“光学膜片钳”将被用来分析和绘制SYT颗粒在钙微域和通道上的分布。最后，我们将使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。在特定的目标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的钙结合环已经交换。利用人造膜和单分子和曲率敏感光学方法的独特组合，我们将在体外将这些蛋白质的功能分配给特定的部分。这些“还原”实验将得到活细胞中嵌合体表达的补充，使我们能够确定SYT亚型的关键属性，这些属性与它们在钙离子触发的胞吐作用中的作用相关，并利用这些属性来操纵释放。总体而言，这些研究将促进我们目前对分泌系统的基本分子组织的理解--这是我的研究项目的广泛兴趣。他们还将揭示激素分泌调节以支持交感神经系统功能的机制，交感神经系统功能对于维持心血管、呼吸和新陈代谢健康至关重要。

 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.