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

描述（由申请人提供）：激素和神经递质以囊泡的形式储存在内分泌和神经细胞中。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启动的生化变化时，才会发生分泌。因此，启动决定了分泌的数量和速度。对启动的进一步了解可能会导致对2型糖尿病和帕金森氏症等严重疾病的新疗法，这些疾病的分泌量过少。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。我建议开发一种新方法，使人们能够看到单个的启动囊泡。该方法是基于Chow实验室先前的工作，该工作表明蛋白质复合物对启动至关重要。络合蛋白与SNARE复合物紧密结合，SNARE复合物是三种蛋白质的异齐聚物，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络合蛋白，我们将能够识别将络合蛋白与囊泡相关的SNARE复合物结合的囊泡（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）来检测标记的络合蛋白与引物囊泡的共定位，我们将使用该检测来评估和区分其他蛋白质（Munc 13和CAPS）在调节引物动力学中的作用。此外，我们还将解决一个谜题，即有多少SNARE复合物（使用标记络合蛋白作为报告因子）与单个囊泡物理相关。这将通过使用一种称为荧光光激活定位显微镜（FPALM）的新型超分辨率方法来完成。FPALM将活细胞或固定细胞的光学分辨率提高到近分子尺度（30-50纳米已完成）。新的引物囊泡标记物与FPALM的结合将加快对其他参与引物的蛋白质的鉴定，并为研究详细的蛋白质-蛋白质结合动力学提供一种手段。这将为治疗分泌过少的疾病提供可能的方法。利用我们的新方法确定参与囊泡启动的蛋白质的作用可能会导致新药或其他治疗的发展，以补偿参与囊泡启动的蛋白质表达的改变。此外，从了解如何操纵囊泡启动的研究中获得的知识可用于几种生物技术应用，其中分泌产物的生产是限制步骤。

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.