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

智力优势：神经元功能需要精确的时空控制树突中的mRNA翻译，以响应细胞之间的电和化学信号事件。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物，由树突内的蛋白质合成依赖性变化介导。已知编码对突触功能至关重要的蛋白质的各种mRNA被转运到树突中，在树突中随后的突触活动调节它们的翻译，但是突触活动和这些事件之间的时间和位置仍然不清楚。该项目旨在验证以下假设：局部突触刺激导致随后非常迅速地捕获和翻译参与激活突触修饰的mRNA，并且它们的翻译速率在刺激后短暂但显著的时间内呈指数级增加。使用分子方法，包括活树突中单个mRNA颗粒运动和翻译事件的实时成像，这些活动将响应于海马神经元突触处的空间限制激活而定量。已知对突触功效的变化至关重要并且响应于突触刺激而定位的mRNA将用作这些转运和翻译活性的标记物。这些方法代表了一个显着偏离以前的实验，使用生化组分的亚细胞成分，以评估树突状mRNA丰度和翻译速率的变化，因为这些往往是不纯的，并没有给这些过程在体内的空间信息。建立活细胞检测mRNA的运输和翻译的神经元将是一个重要的进步，为评估基因表达的研究，在响应本地化的亚细胞activation.Broader影响：这个项目是有吸引力的，非常适合于培养学生有兴趣在神经科学追求的职业生涯。它从根本上是直观的，因为实验是基于活细胞成像，因此，神经元功能的高度复杂的过程可以直接可视化。此外，这些技术是基于数字荧光显微镜，这是一个重要的新的组成部分，生物教育在许多层次的未来课程开发的生物科学。亨特学院是美国最大的公共机构之一，为来自世界各地的少数民族本科生提供服务。学生将能够同时在相关的神经生物学主题的课程，同时在实验室中应用它们，在那里他们将学习基本的分子技术，结合现代细胞生物学方法来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。

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.