Novel optogenetic method for control of protein synthesis

项目摘要 局部蛋白质合成允许蛋白质的就地和按需快速表达，而不是通过蛋白质合成。 从其他地方运输预先存在的蛋白质。在神经元中，mRNA的位点特异性翻译提供了 用于将基因表达限制在远离细胞体的单个结构的手段，例如 突触和生长锥蛋白质合成失调与多种神经发育相关 和认知障碍然而，尽管局部翻译对正常神经元功能的重要性， 大多数广泛使用的操纵蛋白质合成的方法都存在空间或时间分辨率低的问题。在 这个建议将产生新的光遗传学方法来调节单个mRNA的局部翻译 分子。这些方法将广泛适用于不同的mRNA，用于各种生物体 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局部翻译，而 第二个目标是产生一种互补的功能获得方法，以急性刺激局部蛋白质合成。 作为原理证明，我们将使用这些方法来确定局部β-的要求和充分性 肌动蛋白翻译的突触可塑性使用原代海马神经元。响应神经元活动，β- 肌动蛋白mRNA在树突内迅速转运到活性突触，在那里它被翻译，新生的β-肌动蛋白mRNA在树突内迅速转运到活性突触， 肌动蛋白特异性地保留在活性树突棘内。在本提案中，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确控制， 亚细胞区室

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.