Unveiling the molecular mechanisms of vesicular trafficking, membrane fusion, and intravesicular acidification

揭示囊泡运输、膜融合和囊泡内酸化的分子机制

• 批准号: RGPIN-2015-06438
• 负责人: Sugita, Shuzo
• 金额: $ 2.77万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614150
• 关键词: Unveiling; molecular; mechanisms; vesicular; trafficking

In order for a complex system of organs and tissues that compose an organism to maintain homeostasis and co-ordinate the intricate set of activities that are vital for its survival and well-being, the cells that comprise these organs and tissues must be able to communicate with and regulate each other. It is the secretory vesicles within these cells that house the vast array of contents that cells use to achieve this cell-to-cell communication. Thus, the regulation of these processes is an important area of inquiry in the field of fundamental cellular biology. Interestingly, these processes have been shown to be strongly influenced by the acidification of the intravesicular compartment. Keeping this in mind, our long-term goal is to determine how vesicular trafficking, membrane fusion, and intravesicular acidification are regulated in neurons, neuroendocrine cells, and immune cells. As described in a Nobel prize-winning discovery, SNAREs are critical for vesicular trafficking and membrane fusion. These SNAREs are classified as either v-SNAREs (e.g., synaptobrevin), found on the vesicular membrane, or t-SNAREs (e.g., syntaxin-1, SNAP-25), found on the membrane of target organelles. It is the association of these v- and t-SNAREs that is believed to regulate vesicular trafficking and to underlie the initiation of membrane fusion. In being important components of cellular function, it is no surprise that the interaction of v-SNAREs and t-SNAREs seems to be regulated by myriad proteins; however, the entire nature of this regulation remains a mystery. Intending to solve this mystery, our short-term goal is to elucidate the roles of two distinct classes of potential SNARE-regulating proteins: the Voa subunit of vacuolar ATPases (V-ATPases) and CAPS1 (Calcium-dependent activator protein for secretion 1). Based on preliminary work of us and others, we hypothesize that: 1) The Voa subunit has critical roles in dense-core vesicle trafficking in neuroendocrine cells and in rapid exocytosis of synaptic vesicles in neurons. 2) CAPS1 binds to syntaxin-1, a key t-SNARE protein, to facilitate SNARE complex assembly and membrane fusion. The Specific Aims of this research proposal are: 1) To determine the isoform-specific function of Voa isoforms in intracellular organelle targeting, intravesiclular acidification, vesicular trafficking, and exocytosis in neuroendocrince cells and neurons. 2) To elucidate the biochemical factors that are involved in the physical interactions between CAPS1 and syntaxin-1 and to elucidate the physiological significance of the CAPS1-syntaxin-1 interaction in vesicular fusion in neuroendocrine cells and in the organism. Taken together, our proposed work will provide fundamental, novel, and exciting insight into how vesicular trafficking, membrane fusion, and intravesicular acidification are regulated by the Voa subunit of V-ATPase and CAPS1.

为了使构成有机体的器官和组织的复杂系统保持动态平衡，并协调对其生存和福祉至关重要的一系列错综复杂的活动，组成这些器官和组织的细胞必须能够相互沟通和调节。正是这些细胞内的分泌小泡容纳了细胞用来实现这种细胞间通信的大量内容。因此，这些过程的调控是基础细胞生物学领域的一个重要研究领域。有趣的是，这些过程已经被证明受到囊泡内室酸化的强烈影响。考虑到这一点，我们的长期目标是确定如何在神经元、神经内分泌细胞和免疫细胞中调节囊泡运输、膜融合和囊泡内酸化。 正如诺贝尔奖获得者的一项发现所描述的那样，陷阱对囊泡运输和膜融合至关重要。这些圈套被分类为v-圈套(例如，突触素)，发现在囊泡膜上，或t-圈套(例如，Synaxin-1，SNAP-25)，发现在靶细胞器的膜上。正是这些v-和t-陷阱的结合被认为调节了囊泡的运输，并奠定了膜融合的基础。作为细胞功能的重要组成部分，v-SNARS和T-SNARS的相互作用似乎受到无数蛋白质的调控，这并不令人惊讶；然而，这种调控的整个性质仍然是一个谜。为了解开这个谜团，我们的短期目标是阐明两类不同的潜在圈套调节蛋白的作用：VOA空泡ATPase亚基(V-ATPase)和CAPS1(分泌钙依赖激活蛋白1)。 根据我们和其他人的初步工作，我们假设： 1)VOA亚单位在神经内分泌细胞的致密核小泡运输和神经元突触小泡的快速吐出中起关键作用。 2)CAPS1与关键的t-SNARE蛋白--Synaxin-1结合，促进SNARs复合体的组装和膜融合。 这项研究建议的具体目标是： 1)确定VOA异构体在神经内分泌细胞和神经元的细胞内细胞器靶向、囊泡酸化、囊泡转运和胞吐中的特异性功能。 2)阐明CAPS1和Synaxin-1相互作用的生化因素，阐明CAPS1-Synaxin-1相互作用在神经内分泌细胞和生物体囊泡融合中的生理意义。 综上所述，我们提出的工作将为囊泡运输、膜融合和囊泡内酸化如何受V-ATPase和CAPS1的VOA亚单位调控提供基本的、新颖的和令人兴奋的见解。