Imaging the Primed Vesicle Pool: A Novel Tool to Study Vesicle Priming

描述（由申请人提供）：激素和神经递质储存在称为囊泡的小包内分泌细胞和神经细胞中。当细胞受到刺激时，分泌发生，囊泡将其内容物排出细胞。然而，只有当囊泡经历了称为引发的生化变化时，分泌才会发生。因此，启动决定了分泌的多少和速度。对启动的理解的增加可能会导致对破坏性疾病的新治疗，如2型糖尿病和帕金森氏症，其中分泌太少。此外，它可能有助于增加从培养细胞分泌的生物技术产品的产量。我建议开发一种新的方法，使人们能够看到单独的启动囊泡。该方法基于Chow实验室先前的工作，该工作表明蛋白质复合蛋白对引发至关重要。复合蛋白与SNARE复合物紧密结合，SNARE复合物是三种蛋白质的异源寡聚体，在分泌过程中共同触发囊泡和质膜的融合。通过用荧光标签标记复合蛋白，我们将能够识别具有结合到囊泡相关SNARE复合物的复合蛋白的囊泡（用单独的荧光标签标记）。我们将使用全内反射荧光显微镜（TIRFM）来检测标记的复合蛋白与引发囊泡的共定位，我们将使用该测定来评估和区分其他蛋白质（Munc 13和CAPS）在调节引发动力学中的作用。此外，我们还将解决的奥秘，有多少陷阱复合物（使用标记的复合物作为报告）是物理上与一个单一的囊泡。这将通过使用一种新的超分辨率方法，称为荧光光活化定位显微镜（FPAL M）。FPALM在活细胞或固定细胞中将光学分辨率提高到近分子尺寸（30-50 nm已完成）。新的引发囊泡标记物和FPALM的组合将加速鉴定引发中涉及的其他蛋白质，并提供研究详细的蛋白质-蛋白质结合动力学的手段。它将为治疗分泌过少的疾病提供可能的方法。使用我们的新方法定义参与囊泡引发的蛋白质的作用可能会导致新药或其他治疗方法的开发，以补偿参与囊泡引发的蛋白质表达的改变。此外，从理解如何操纵囊泡引发的研究中获得的知识可用于几种生物技术应用中，其中分泌产物的产生是限制性步骤。

DESCRIPTION (provided by applicant): Hormones and neurotransmitters are stored in endocrine and nerve cells in small packets called vesicles. When a cell is stimulated, secretion occurs and the vesicles empty their contents out of the cell. However, secretion will occur only if the vesicles have undergone biochemical changes called priming. Priming, therefore, determines how much and how fast secretion can take place. Increased understanding of priming could lead to new treatments in devastating diseases such as type 2 diabetes, and Parkinson's, in which too little secretion occurs. Furthermore, it may help increase the output of biotechnology products that are secreted from cells in culture. I propose to develop a novel method to allow one to see individual primed vesicles. The method is based on previous work of the Chow laboratory that showed that the protein complexin is critical for priming. Complexin binds tightly to the SNARE complex, a hetero-oligomer of three proteins that together triggers the fusion of vesicle and plasma membranes during secretion. By labeling complexin with a fluorescent tag, we will be able to identify the vesicles (labeled with a separate fluorescent tag) that have complexin bound to the vesicle-associated SNARE complex. We will use total internal reflection fluorescence microscopy (TIRFM) to detect co-localization of tagged complexin with primed vesicles and we will use this assay to assess and differentiate the roles of other proteins (Munc 13 and CAPS) in modulating priming kinetics. In addition, we will also address the mystery as to how many SNARE complexes (using tagged complexin as the reporter) are physically associated with a single vesicle. This will be accomplished by using a new super-resolution method called fluorescence photoactivation localization microscope (FPALM). FPALM increases optical resolution to near-molecular dimensions (30-50 nm has been accomplished) in living or fixed cells. The combination of the new primed vesicle marker and FPALM will speed up identification of other proteins involved in priming and provide a means to study detailed protein-protein binding kinetics. It will give insights into possible approaches to treat diseases in which too little secretion occurs. Defining the roles of proteins involved in vesicle priming using our novel approach may lead to developments of new drugs or other treatments to compensate for altered expression of proteins involved in vesicle priming. In addition, the knowledge gained from research in understanding how to manipulate vesicle priming may be used in several biotechnology applications where production of secreted products is the limiting step.