Exploring how cells generate and release distinct subpopulations of dense-core vesicles

项目摘要 当用电子显微镜检查时，可以看到神经元携带着看起来像小黑点的细胞器。 这些黑点被称为致密核囊泡，它们携带许多重要的递质， 神经调节剂，包括神经肽、神经生长因子和单胺如多巴胺和 去甲肾上腺素这种致密核囊泡货物调节广泛的行为，并且这种缺陷在细胞内发生。 货物可能导致许多情绪障碍和其他神经系统疾病。然而， 了解致密核心囊泡如何形成，获得货物和成熟的细胞生物学， 被贩卖到释放地点，最终被释放。因此，神经元中的小黑点实际上是一个大黑点， 盒子增加了这些细胞器的神秘性和复杂性，许多神经元携带多个致密核心， 在同一个细胞中的囊泡货物。这些不同的货物是用同一个密芯包装的吗 囊泡或它们被单独包装在不同的囊泡中？这个问题的答案令人惊讶地众所周知 仅在少数情况下，而且不同货物的共同包装与隔离的一般模式尚不清楚， 但具有关键的生理相关性，因为它决定了不同的致密核心囊泡货物是否 共释放或可独立释放。此外，在不同的致密核心囊泡 已知在同一细胞中存在不同的亚群，但这些不同的种群是如何产生的尚不清楚。 以及货物是如何被不同地分类的。为了开始解决这些理解上的差距，我们的目标是 建立了两个模型系统，用于研究同一细胞中不同的致密核心囊泡群体：ASI 线虫C. elegans和大鼠胰腺β细胞系832/13。我们的初步数据 显示了已知致密核囊泡生物发生和成熟途径Rab 2/EARP的成员 在ASI神经元和832/13细胞中，致密核心囊泡的一个亚群都需要这种信号通路 行，但不是同一细胞中的另一个亚群。在目标1中，我们将进一步确定以下要求： Rab 2/EARP途径成员在不同致密核心囊泡的生物发生和成熟中的作用 ASI神经元和832/13细胞中的亚群。此外，我们将在一个 试图确定Rab 2/EARP独立产生致密核心囊泡所需的因素， 细胞类型。在目标2中，我们将检验致密核心囊泡的不同亚群被标记的假设。 突触结合蛋白家族的不同亚型的钙传感器。我们还将确定这些 突触结合蛋白控制不同致密核心囊泡亚群的释放。总之，该项目将 确定产生和释放致密核心的不同亚群所需的基本分子， 囊泡在同一个细胞，并设置了更多的机制研究这些过程如何 发生.

Project summary When examined by electron microscopy, neurons can be seen to carry organelles that look like little black dots. These black dots are called dense-core vesicles and they carry many important transmitters that act as neuromodulators, including neuropeptides, nerve growth factors, and monoamines such as dopamine and norepinephrine. Such dense-core vesicle cargos regulate a wide array of behaviors, and defects in such cargos can contribute to numerous mood disorders and other neurological conditions. However, little is understood about the cell biology of how dense-core vesicles are made, acquire cargos and mature, are trafficked to release sites, and ultimately released. Thus, the little black dots in neurons are really a big black box. Adding to the mysteries and complexity of these organelles, many neurons carry multiple dense-core vesicle cargos in the same cell. Are these different cargos copackaged together in the same dense-core vesicles or are they packaged separately in distinct vesicles? The answer to this question is surprisingly known in only a few cases, and the general pattern of copackaging versus segregation of distinct cargos is unclear, but is of key physiological relevance as it determines whether different dense-core vesicle cargos are coreleased or can be released independently. Additionally, in cases where distinct dense-core vesicle subpopulations are known to exist in the same cell, it is unclear how these distinct populations are generated and how cargos are differentially sorted. To begin to address these gaps in understanding, here we aim to establish two model systems for the study of distinct dense-core vesicle populations in the same cell: the ASI sensory neuron in the nematode C. elegans and the rat pancreatic beta-cell line 832/13. Our preliminary data show that members of a known dense-core vesicle biogenesis and maturation pathway, the Rab2/EARP pathway, are required for one subpopulation of dense-core vesicles in both the ASI neuron and the 832/13 cell line, but not for another subpopulation in the same cell. In Aim 1, we will further define the requirements for members of the Rab2/EARP pathway in the biogenesis and maturation of distinct dense-core vesicle subpopulations in the ASI neuron and 832/13 cells. Additionally, we will perform candidate screens in an attempt to identify factors required for the Rab2/EARP-independent generation of dense-core vesicles in both cell types. In Aim 2, we will test the hypothesis that different subpopulations of dense-core vesicles are marked by different isoforms of the synaptotagmin family of calcium sensors. We will also determine whether these synaptotagmins control the release of distinct dense-core vesicle subpopulations. In summary, this project will identify the basic molecules required for the generation and release of distinct subpopulations of dense-core vesicles in the same cell, and set the stage for more mechanistic investigations into how these processes occur.