Profiling Locally Translated mRNAs in Regenerating Axons.
Profiling Locally Translated mRNAs in Regenerating Axons.
复制标题
分析再生轴突中局部翻译的 mRNA。
DOI：
10.1007/978-1-0716-3012-9_8
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Twiss,JefferyL
中科院分区：
文献类型：
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作者：
Sahoo,PabitraK;Twiss,JefferyL
蛋白质表达的时空调控在许多细胞功能中起着重要作用，特别是对于高度极化的细胞类型。虽然亚细胞蛋白质组可以通过重新定位来自细胞其他结构域的蛋白质来改变，但将mRNA转运到亚细胞结构域提供了一种响应不同刺激而局部合成新蛋白质的方法。局部蛋白质合成是神经元的一个关键机制，它使树突和轴突从细胞体延伸到很远的地方。在这里，我们讨论的方法，已开发的研究本地化的蛋白质合成轴突蛋白质合成为例。我们提供了一种深入的方法，使用双荧光恢复后，光漂白可视化网站的蛋白质合成使用报告基因编码两个不同的定位mRNA沿着与扩散限制的荧光报告蛋白。我们展示了如何使用这种方法来确定细胞外刺激和不同的生理状态如何在真实的时间内改变局部mRNA翻译的特异性。
Spatial and temporal regulation of protein expression plays important roles in many cellular functions, particularly for highly polarized cell types. While the subcellular proteome can be altered by relocalizing proteins from other domains of the cell, transporting mRNAs to subcellular domains provides a means to locally synthesize new proteins in response to different stimuli. Localized protein synthesis is a critical mechanism in neurons that extend dendrites and axons long distances from their cell bodies. Here, we discuss methodologies that have been developed to study localized protein synthesis using axonal protein synthesis as an example. We provide an in-depth method using dual fluorescence recovery after photobleaching to visualize sites of protein synthesis using reporter cDNAs that encode two different localizing mRNAs along with diffusion-limited fluorescent reporter proteins. We show how this method can be used to determine how extracellular stimuli and different physiological states can alter the specificity of local mRNA translation in real time.