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既需要编码突触传递蛋白的mrna的新转录，也需要在激活的突触中对这些mrna进行本地化翻译。这些mrna作为翻译抑制的货物从胞体运输到突触，装载在RNA颗粒上，通过树突主动运输。激活的突触将颗粒吸引到它们的区域，然后激活mRNA货物进行翻译的机制的理解，等待着能够在神经元激活的同时对活神经元中的mRNA动力学进行成像的方法的发展。分子信标探针可以发挥这一作用，因为它们只有在与特定mRNA序列杂交时才会发出荧光。使用每个mRNA物种的多个分子信标，我们将对一组被认为在LTP建立中起重要作用的mRNA的树突交通进行成像。同样的探针将用于确定颗粒中这些mrna的化学计量。我们还将成像mrna在胞体颗粒中的组装以及它们最终在突触中释放并进行翻译。这项任务所需的单分子灵敏度将通过在每个mRNA物种的非翻译区插入一个串联重复的探针结合位点来获得。当多个分子信标与这些位点结合时，每个mRNA分子都呈现出如此强烈的荧光，以至于它作为衍射极限点可见。这些修饰的mrna将在培养的神经元中表达，并以单分子灵敏度跟踪它们组装成颗粒，穿过树突，停靠在突触上，并通过突触的激活从颗粒中释放出来。同时对不同的mRNA物种使用不同的串联重复探针结合序列，将使两种不同的mRNA物种被不同颜色的分子信标标记。此外，颗粒本身可以通过颗粒中稳定的gfp标记的蛋白质成分的共同表达来标记为不同的颜色。该系统的发展以单分子敏感性研究活神经元中的mRNA动力学，将有助于探索区分激活突触和原始突触的分子机制。

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.