Imaging the Spatio-temporal Dynamics of mRNA Transport and Translation in Response to Synaptic Activity

响应突触活动的 mRNA 运输和翻译的时空动态成像

• 批准号: 0819022
• 负责人: Jason Dictenberg
• 金额: $ 17.5万
• 依托单位: CUNY Hunter College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0819022&HistoricalAwards=false
• 关键词: Imaging; Spatio; temporal; Dynamics; mRNA

Intellectual Merit: Neuronal function requires the precise spatio-temporal control of mRNA translation in dendrites in response to electrical and chemical signaling events between cells. In addition, certain forms of synaptic plasticity, which may be molecular correlates of learning and memory, are mediated by protein-synthesis dependent changes within dendrites. It is known that various mRNAs encoding proteins critical to synapse function are transported into dendrites, where subsequent synapse activity regulates their translation, but the timing and location between synapse activity and these events remains unclear. This project aims to test the hypothesis that localized synaptic stimulation results in the subsequent capture and translation of mRNAs involved in the modification of activated synapses very rapidly, and that their translation rates will be exponentially increased for a brief but significant time after stimulation. Using molecular approaches including live imaging of single mRNA particle movements and translation events in living dendrites, these activities will be quantified in response to spatially restricted activation at synapses of hippocampal neurons. mRNAs known to be critical for changes in synaptic efficacy and localized in response to synaptic stimulation will be used as markers for these transport and translation activities. These approaches represent a significant departure from previous experiments that have used biochemical fractions of subcellular components to assess changes in dendritic mRNA abundance and translation rates since these are often not pure and do not give spatial information about these processes in vivo. Establishment of live cell assays for mRNA transport and translation in neurons will be an important advance for the assessment of gene expression studies in response to localized subcellular activation.Broader Impact: This project is attractive and well suited to training of students interested in pursuing careers in the neurosciences. It is fundamentally intuitive since the experiments are based in live cell imaging and therefore, the highly complex processes of neuronal function can be visualized directly. In addition, the techniques are based in digital fluorescence microscopy, which represents a major new component to biological education at many levels of future curriculum development in the biological sciences. Hunter College is one of the largest public institutions in the United States that serves an underrepresented population of ethnic undergraduate students that originates from around the world. Students will be able to simultaneously take classes in related neurobiological topics while applying them in the laboratory, where they will learn basic molecular techniques coupled to modern cell biological approaches to complex neurobiological problems, such as the molecular regulation of learning and memory function.

智力优点：神经元功能需要对树突中的mRNA翻译的精确时空控制，以响应细胞之间的电信号事件。此外，某些形式的突触可塑性可能是学习和记忆的分子相关性，是由树突中蛋白质合成的依赖性变化介导的。众所周知，编码对突触功能至关重要的蛋白质的各种mRNA被运输到树突中，随后的突触活动调节其翻译，但是突触活动与这些事件之间的时机和位置尚不清楚。该项目旨在检验以下假设：局部突触刺激会导致随后捕获和翻译激活突触修饰的mRNA的捕获和翻译，并且在刺激后，它们的翻译速率将在短暂但很大的时间内成倍增加。使用分子方法，包括单个mRNA颗粒运动的实时成像和活树突中的翻译事件，这些活性将响应于海马神经元突触的空间限制激活而量化。已知对突触功效的变化至关重要的mRNA和对突触刺激的局部定位将被用作这些运输和翻译活动的标志物。这些方法与以前使用亚细胞成分的生化分数来评估树突状mRNA丰度和翻译速率的变化的实验显着偏离，因为这些方法通常不是纯净的，并且不提供有关这些过程的空间信息。建立用于mRNA传输的现场细胞分析和神经元中的翻译将是评估基因表达研究以响应局部亚细胞激活的重要进步。BRODER的影响：该项目很有吸引力，非常适合培训有兴趣从事神经科学职业的学生。它从根本上是直观的，因为实验基于活细胞成像，因此，可以直接将神经元功能的高度复杂过程直接可视化。此外，这些技术基于数字荧光显微镜，这代表了生物科学中许多未来课程发展的生物教育的主要新组成部分。猎人学院是美国最大的公共机构之一，该机构为来自世界各地的民族本科生提供的人数不足。学生将能够在相关的神经生物学主题中同时上课，同时将其应用于实验室，在那里他们将学习与现代细胞生物学方法相连的基本分子技术，以解决复杂的神经生物学问题，例如学习和记忆功能的分子调节。