Novel optogenetic method for control of protein synthesis

项目摘要 局部蛋白质合成允许蛋白质在现场和按需快速表达，而不是 将预先存在的蛋白质从其他地方运送过来。在神经元中，mRNAs的位置特异性翻译提供了 将基因表达限制在远离细胞体的个别结构上的手段，例如 突触和生长锥体。蛋白质合成失调与多种神经发育有关 和认知障碍。然而，尽管局部翻译对于正常的神经元功能很重要，但 大多数被广泛使用的蛋白质合成操作方法存在着空间或时间分辨率低的问题。在……里面 在这项建议中，我们将产生新的光遗传方法来调节单个mRNA的局部翻译 分子。这些方法将广泛适用于广泛的生物体中使用的不同mRNAs。 和细胞类型。第一个目标是开发一种新的功能丧失方法来抑制本地翻译，而 第二个目标是产生一种互补的功能增益方法，以敏锐地刺激局部蛋白质合成。 作为原则证明，我们将使用这些方法来确定本地β的需求和充分性- 使用原代海马神经元的突触可塑性的肌动蛋白翻译。作为对神经元活动的反应，β- 肌动蛋白mRNA在树突内迅速运输到活性突触，在那里它被翻译，新生的β- 肌动蛋白蛋白被特异性地保留在活跃的树突脊椎中。在这项提议中，我们将开发新的工具 这将为研究人员提供对特定转录后基因调控的精细控制 亚细胞隔间。

Project Summary Local protein synthesis allows for the rapid expression of proteins on-site and on-demand, as opposed to transporting pre-existing proteins from elsewhere. In neurons, the site-specific translation of mRNAs provides a means for restricting gene expression to individual structures that can be far from the cell body, such as synapses and growth cones. Dysregulated protein synthesis is implicated in a variety of neurodevelopmental and cognitive disorders. However, despite the importance of local translation for normal neuronal function, the most widely used methods for manipulating protein synthesis suffer from low spatial or temporal resolution. In this proposal we will generate new optogenetic methods for regulating the local translation of individual mRNA molecules. These methods will be broadly applicable to different mRNAs for use in a wide array of organisms and cell types. The first aim develops a new loss-of-function approach to inhibit local translation, while the second aim generates a complementary gain-of-function method to acutely stimulate local protein synthesis. As a proof of principle, we will use these methods to determine both the requirement and sufficiency of local β- actin translation for synaptic plasticity using primary hippocampal neurons. In response to neuronal activity, β- actin mRNA is rapidly transported within dendrites to active synapses where it is translated, and the nascent β- actin protein is specifically retained within the active dendritic spine. In this proposal, we will develop new tools that will provide researchers with exquisite control over post-transcriptional gene regulation in specific subcellular compartments.