Imaging the Transport of Individual mRNA Molecules to the Active Synapses

描述（由申请人提供）：通过长时程增强（LTP）在海马体中形成记忆需要编码能够实现突触传递的蛋白质的mRNA的新鲜转录和这些mRNA在激活的突触处的定位翻译。 这些mRNA从索马到突触，作为RNA颗粒上被抑制的货物，通过树突被主动转运。 对激活的突触将颗粒吸引到其结构域，然后激活mRNA货物进行翻译的机制的理解，等待开发方法，该方法将允许在神经元激活的同时对活神经元中的mRNA动力学进行成像。 分子信标探针可以发挥这种作用，因为它们只有在与特定的mRNA序列杂交时才会发出荧光。 使用多个分子信标为每个mRNA种类，我们将图像的树突交通的一组选择的mRNA，被认为是发挥重要作用，在建立LTP。 将使用相同的探针来确定颗粒中这些mRNA的化学计量。 我们也将对mRNA组装成索马中的颗粒以及它们最终在突触处释放进行翻译的过程进行成像。 该任务所需的单分子灵敏度将通过将串联重复的探针结合位点插入每个mRNA种类的非翻译区来获得。 当多个分子信标与这些位点结合时，每个mRNA分子都呈现出强烈的荧光，以至于它可以作为衍射限制点看到。 这些修饰的mRNA将在培养的神经元中表达，并在它们组装成颗粒、穿过树突、停靠在突触处并通过突触的激活从颗粒释放时以单分子灵敏度进行跟踪。 对于不同的mRNA种类同时使用不同的串联重复的探针结合序列将使得两种不同的mRNA种类能够用不同颜色的分子信标区别地标记。 此外，颗粒本身可以通过颗粒的稳定GFP标记的蛋白质组分的共表达而以不同的颜色标记。 该系统的发展，研究mRNA的动态活神经元与单分子的敏感性，将使探索的分子机制，区分激活的突触从幼稚突触。

DESCRIPTION (provided by applicant): The formation of memory in the hippocampus by long term potentiation (LTP) requires both fresh transcription of mRNAs encoding the proteins that enable synaptic transmission and localized translation of these mRNAs at the activated synapses. These mRNAs travel from the soma to the synapses as translationally repressed cargo on RNA granules that are actively transported through the dendrites. An understanding of the mechanisms by which activated synapses draw the granules to their domains, and then activate the mRNA cargo for translation, awaits the development of methods which will permit the imaging of mRNA dynamics in live neurons simultaneously with neuronal activation. Molecular beacon probes can fulfil this role, because they only fluoresce when they are hybridized to a specific mRNA sequence. Using multiple molecular beacons for each mRNA species, we will image the dendritic traffic of a select set of mRNAs that are believed to play significant roles in the establishment of LTP. The same probes will be used to determine the stoichiometry of these mRNAs in the granules. We will also image the assembly of the mRNAs into the granules in the soma and their eventual release for translation at the synapse. The single-molecule sensitivity necessary for this task will be obtained by inserting a tandemly repeated probe binding site into a non-translated region of each mRNA species. When multiple molecular beacons bind to these sites, each mRNA molecule is rendered so intensely fluorescent that it is visible as a diffraction-limited spot. These modified mRNAs will be expressed in cultured neurons and tracked with single-molecule sensitivity as they assemble into granules, travel through the dendrites, dock at the synapses, and are released from the granules by the activation of the synapse. The simultaneous use of different tandemly repeated probe binding sequences for different mRNA species will enable two different mRNA species to be distinctively labeled with differently colored molecular beacons. Moreover, the granules themselves can be labeled in a different color by the co-expression of a stable GFP-tagged protein constituent of the granules. The development of this system to study mRNA dynamics in live neurons with single-molecule sensitivity will enable an exploration of the molecular mechanisms that distinguish activated synapses from naive synapses.