Investigating the fundamental mechanisms of immune cell exocytosis

水疱胞吐是一个基本的细胞过程，在大脑和免疫系统功能中起着至关重要的作用。在大脑中，突触囊泡胞吐是神经元间通讯的基础，使功能和认知过程的多样性成为可能。在免疫系统中，肥大细胞、自然杀伤细胞（NK）和细胞毒性t淋巴细胞（ctl）等特化免疫细胞会分泌颗粒胞吐，有助于清除感染细胞和恶性细胞。神经元和免疫细胞使用相似的蛋白质集来控制Ca2+依赖性胞吐。然而，与神经细胞胞吐的深入研究相比，免疫细胞胞吐的功能机制却很少被表征和研究。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，这使得我们在理解这两个过程之间的共同和不同方面产生了协同作用。例如，尽管SNARE蛋白在两种类型的胞吐作用中都起着重要作用，但每种类型似乎都利用不同的SNARE蛋白同工型。此外，神经元和免疫细胞胞吐也使用不同的蛋白质来诱导胞吐，尽管常见的C2结构域似乎用于所需的Ca2+感应作用。本研究计划的长期目标是阐明免疫细胞胞吐的机制，并最终修改和增强免疫细胞胞吐，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蛋白及其调节因子（或启动蛋白），并研究免疫细胞胞吐调节的潜在生化机制。根据我们最近的研究，我们提出以下假设：1)Syntaxin-3和SNAP-23是参与免疫细胞胞吐的关键t-SNARE蛋白。2) Munc13-4在分泌颗粒胞吐过程中发挥双重作用，作为胞吐的Ca2+传感器，并与Munc18-2一起促进胞吐启动。3) Munc13-4的Ca2+感应功能是通过C2结构域介导的，而Munc13-4和Munc18-2的启动功能是通过与含有syntaxin-3和SNAP-23的SNARE复合物相互作用来促进的。我们将通过以下具体目标来探讨这些假设：目标1。免疫细胞胞吐的关键t-SNARE蛋白是什么？目标2。潜在的功能获得Munc13-4和Munc18-2突变体是否可以增强免疫细胞的胞外分泌以及增加NK细胞的细胞毒性？目标3。Munc13-4调控功能的生化基础是什么？我们的研究将为免疫细胞中颗粒胞吐的机制提供基本的见解。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.