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

为了使一个由器官和组织组成的复杂系统维持体内平衡并协调对其生存和健康至关重要的一系列复杂活动，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囊容纳了细胞用来实现细胞间通信的大量内容物。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，这些过程已被证明受到囊泡内腔室酸化的强烈影响。考虑到这一点，我们的长期目标是确定神经元、神经内分泌细胞和免疫细胞如何调节囊泡运输、膜融合和囊泡内酸化。******正如一项获得诺贝尔奖的发现所描述的那样，SNAREs对囊泡运输和膜融合至关重要。这些SNAREs可分为在囊泡膜上发现的v-SNAREs（如synaptobrevin）和在靶细胞器膜上发现的t-SNAREs（如syntaxin-1， SNAP-25）。正是这些v-和t-SNAREs的关联被认为调节了囊泡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-SNAREs和t-SNAREs的相互作用似乎受到无数蛋白质的调节也就不足为奇了；然而，这一规定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种不同类型的潜在snare调节蛋白的作用：液泡atp酶（V-ATPases）的Voa亚基和CAPS1（钙依赖性分泌激活蛋白1）。******基于我们和其他人的初步工作，我们假设：******1)Voa亚基在神经内分泌细胞的密集核囊泡运输和神经元突触囊泡的快速胞外分泌中起关键作用。***2) CAPS1结合t-SNARE关键蛋白syntaxin-1，促进SNARE复合物组装和膜融合。******本研究计划的具体目的是：******1)确定Voa亚型在神经内分泌细胞和神经元的胞内细胞器靶向、囊泡内酸化、囊泡运输和胞外分泌中的亚型特异性功能。******2)阐明参与CAPS1与syntaxin-1物理相互作用的生化因子，阐明CAPS1-syntaxin-1相互作用在神经内分泌细胞和机体内囊泡融合中的生理意义。******综上所述，我们提出的工作将为v - atp酶和caps1的Voa亚基如何调节囊泡运输、膜融合和囊泡内酸化提供基础的、新颖的和令人兴奋的见解。*****

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.*****