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

为了使组成有机体的器官和组织的复杂系统保持体内平衡并协调对其生存和健康至关重要的复杂活动，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泡容纳了大量的内容物，细胞使用这些内容物来实现这种细胞与细胞的交流。因此，这些过程的调节是基础细胞生物学领域的一个重要研究领域。有趣的是，这些过程已被证明是强烈影响的囊内室的酸化。记住这一点，我们的长期目标是确定囊泡运输，膜融合和囊泡内酸化在神经元，神经内分泌细胞和免疫细胞中是如何调节的。正如诺贝尔奖获得者发现的那样，SNARE对于囊泡运输和膜融合至关重要。这些SNARE被分类为v-SNARE（例如，小突触泡蛋白），发现于囊泡膜上，或t-SNARE（例如，syntaxin-1，SNAP-25），发现于靶细胞器的膜上。据信，正是这些v-和t-SNARE的缔合调节囊泡运输并成为膜融合起始的基础。作为细胞功能的重要组成部分，v-SNARE和t-SNARE的相互作用似乎受到无数蛋白质的调节，这并不奇怪;然而，这种调节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-ATP酶）的Voa亚基和CAPS 1（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致密核心囊泡运输和神经元突触囊泡的快速胞吐中起关键作用。2)CAPS 1与syntaxin-1（一种关键的t-SNARE蛋白）结合，以促进SNARE复合物的组装和膜融合。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中的细胞器靶向、囊泡内酸化、囊泡运输和胞吐作用中的亚型特异性功能。** 2)阐明参与CAPS 1和syntaxin-1之间物理相互作用的生化因子，并阐明CAPS 1-syntaxin-1相互作用在神经内分泌细胞和生物体中囊泡融合中的生理意义。总之，我们提出的工作将提供基本的，新颖的和令人兴奋的见解，了解V-ATPase和CAPS 1的Voa亚基如何调节囊泡运输，膜融合和囊泡内酸化。

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.*****