Investigating the fundamental mechanisms of immune cell exocytosis

囊泡胞吐是一种基本的细胞过程，在大脑和免疫系统功能中都起着至关重要的作用。在大脑中，突触小泡胞吐是神经元间交流的基础，使多种功能和认知过程成为可能。在免疫系统中，特殊的免疫细胞如肥大细胞、自然杀伤(NK)细胞和细胞毒性T淋巴细胞(CTL)经历分泌颗粒胞吐，从而促进感染细胞和恶性细胞的清除。神经元和免疫细胞使用类似的蛋白质组来控制依赖钙离子的胞吐作用。然而，与深入研究的神经元胞吐作用相比，免疫细胞胞吐作用的功能机制还很少被描述和研究。 我的实验室在研究神经元和免疫细胞胞吐方面积累了广泛的知识和专业知识，这导致了在理解这两个过程之间的共同和不同方面的协同作用。例如，虽然SNARE蛋白在两种类型的胞吐作用中都扮演着重要的角色，但每种类型似乎都利用了不同的SNARE蛋白亚型。此外，神经元和免疫细胞的胞吐作用也使用不同的蛋白质来诱导胞吐作用，尽管共同的C2结构域似乎用于所需的钙感应作用。 本研究计划的长期目标是阐明免疫细胞胞吐作用的机制，并最终修改和增强免疫细胞胞吐作用，以产生更强大的免疫系统。短期目标是确定介导免疫细胞胞吐的关键SNARE蛋白及其调节蛋白(或启动蛋白)，并探讨免疫细胞胞吐调节的潜在生化机制。基于我们最近的研究，我们提出了以下假设： 1)Synaxin-3和SNAP-23是参与免疫细胞胞吐的关键t-SNARE蛋白。 2)Munc13-4与Munc18-2一起作为胞吐的钙感受器和促进胞吐启动，在分泌颗粒胞吐中起双重作用。 3)Munc13-4的钙敏感功能是通过其C2结构域介导的，而Munc13-4和Munc18-2的启动功能是通过与含有Synaxin-3和SNAP-23的SNARE复合体相互作用来实现的。 我们将通过关注以下具体目标来探讨这些假设： 目的1.什么是免疫细胞胞吐的关键T-SNARE蛋白？ 目的2.潜在的功能获得性Munc13-4和Munc18-2突变体能否增强免疫细胞的胞吐功能，同时增强NK细胞的细胞毒作用？ 目的3.Munc13-4调节功能的生化基础是什么？ 我们的研究将对免疫细胞中颗粒胞吐的机制提供基础性的见解。重要的是，从这项研究中获得的知识有可能揭示提高免疫系统有效性的新策略。

Vesicular exocytosis is a fundamental cellular process that plays an imperative role in both brain and immune system function. In the brain, synaptic vesicle exocytosis is the basis for interneuronal communication, enabling a diversity of functional and cognitive processes. In the immune system, specialized immune cells such as mast cells, natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) undergo secretory granule exocytosis, which facilitates the eradication of infected and malignant cells. Neurons and immune cells use paralogous sets of proteins to control Ca2+-dependent exocytosis. However, the functional machinery involved in immune cell exocytosis is much less characterized and understudied compared to the intensively studied neuronal exocytosis. My lab has developed extensive knowledge and expertise in studying both neuronal and immune cell exocytosis, which has led to a synergy in understanding common and distinct aspects between the two processes. For example, although SNARE proteins play essential roles in both types of exocytosis, each type appears to utilize different isoforms of SNARE proteins. Furthermore, neuronal and immune cell exocytosis also employ distinct proteins for inducing exocytosis, although common C2 domains seem to be used for the Ca2+ sensing action that is required. The long-term objective of the present research program is to elucidate the mechanisms of immune cell exocytosis and ultimately, to modify and enhance immune cell exocytosis to generate a more robust immune system. The short-term objective is to determine the key SNARE proteins and their regulators (or priming proteins) that mediate immune cell exocytosis and to investigate the underlying biochemical mechanisms of immune cell exocytotic regulation. Based on our recent studies, we propose the following hypotheses: 1) Syntaxin-3 and SNAP-23 are the key t-SNARE proteins involved in immune cell exocytosis. 2) Munc13-4 plays a dual role in secretory granule exocytosis by acting as a Ca2+ sensor for exocytosis and facilitating exocytotic priming along with Munc18-2. 3) The Ca2+ sensing function of Munc13-4 is mediated via its C2 domains, while the priming function of Munc13-4 and Munc18-2 is facilitated through interactions with the SNARE complexes containing syntaxin-3 and SNAP-23. We will explore these hypotheses by focusing on the following Specific Aims: Aim 1. What are the critical t-SNARE proteins for immune cell exocytosis? Aim 2. Can potential gain-of-function Munc13-4 and Munc18-2 mutants enhance exocytosis from immune cells as well as increase the cytotoxicity of NK cells? Aim 3. What is the biochemical basis for the regulatory functions of Munc13-4? Our research will provide fundamental insight into the mechanisms of granule exocytosis in immune cells. Importantly, the knowledge that would be obtained from this study has the potential to reveal novel strategies for improving the effectiveness of the immune system.