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

对引发的囊泡池进行成像：研究囊泡引发的新工具

• 批准号: 7751984
• 负责人: Joyce Go Rohan
• 金额: $ 5.01万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2012-07-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7751984
• 关键词: Address; Adrenal Glands; Binding; Biochemical; Biological Assay; Biological Models; Biotechnology; Calcium; Cell membrane; Cell secretion; Cells; Chimeric Proteins; Chromaffin Cells; Complex; Development; Dimensions; Disease; Endocrine; Exocytosis; Fluorescence; Fluorescence Microscopy; Future; Hippocampus (Brain); Hormones; Image; Imagery; Individual; Kinetics; Knock-out; Knowledge; Label; Laboratories; Lead; Life; Mediating; Medical; Membrane; Methods; Microscope; Modeling; Molecular; Monitor; Mutation; Neurons; Neurotransmitters; Non-Insulin-Dependent Diabetes Mellitus; Optics; Output; Parkinson Disease; Pharmaceutical Preparations; Play; Process; Production; Protein Binding; Proteins; Reporter; Research; Resolution; Role; SNAP receptor; Secretory Cell; Sequence Homologs; Speed; Stimulus; Testing; Training; Vesicle; Work; base; cell fixing; insight; novel; novel strategies; overexpression; photoactivation; prevent; protein expression; stoichiometry; tool; trafficking

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.

描述(申请人提供)：荷尔蒙和神经递质储存在内分泌和神经细胞中的小包称为囊泡。当细胞受到刺激时，会发生分泌，小泡将其内容物排出细胞。然而，只有当囊泡经历了被称为启动的生化变化时，才会发生分泌。因此，启动决定了分泌量和分泌速度。对启动的了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，在这些疾病中，分泌太少。此外，它可能有助于增加从培养细胞中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启动的囊泡。该方法是基于Chow实验室之前的工作，该工作表明蛋白质络合蛋白对启动至关重要。复合素与SNARE复合体紧密结合，SNARE复合体是一种由三种蛋白质组成的异源低聚物，在分泌过程中共同触发囊泡和质膜的融合。通过用荧光标记复合素，我们将能够识别(用单独的荧光标记标记的)与囊泡相关的圈套复合体结合的复合蛋白。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启动的囊泡的共存，并将使用该方法来评估和区分其他蛋白质(MUNC 13和CAPS)在调节启动动力学中的作用。此外，我们还将解决有多少陷阱复合体(使用标记的复合蛋白作为记者)与单个囊泡在物理上相关的谜题。这将通过使用一种名为荧光光活化定位显微镜(FPALM)的新的超分辨率方法来实现。FPALM将活细胞或固定细胞的光学分辨率提高到近分子尺寸(已完成30-50 nm)。新的启动囊泡标记和FPALM的结合将加快与启动相关的其他蛋白质的鉴定，并为研究详细的蛋白质-蛋白质结合动力学提供了一种手段。它将为治疗分泌物太少的疾病提供可能的方法。使用我们的新方法确定囊泡启动中涉及的蛋白质的作用可能会导致新药或其他治疗方法的开发，以补偿参与囊泡启动的蛋白质表达的变化。此外，在了解如何操作囊泡引发的研究中获得的知识可以用于几种生物技术应用，在这些应用中，分泌产品的生产是限制步骤。