Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor
Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor
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DOI：
10.1038/nmeth885
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发表时间：
2006-06-01
期刊：
影响因子：
48
通讯作者：
Eberwine, James H.
中科院分区：
文献类型：
--
作者：
Barrett, Lindy E.;Sul, Jai-Yoon;Eberwine, James H.
包括E-26样蛋白1（Elk-1）在内的多种核转录因子已在神经元树突中被发现，但这种定位的功能意义尚未得到解释。在这里，我们使用一个焦点转染程序，“phototefection”，引入Elk 1 mRNA到特定区域的活的，完整的原代大鼠神经元。树突中的Elk 1 mRNA的引入和翻译产生细胞死亡，而细胞体中的Elk 1 mRNA的引入和翻译不产生细胞死亡。在树突中翻译的Elk-1被转运到细胞核，细胞死亡依赖于转录，支持树突印迹假说，并强调了树突环境对蛋白质功能的重要性。我们证明了光转染用于空间控制引入mRNA的实用性，这为使用这种方法将选定数量的小分子引入活细胞的离散区域以评估其生物学功能提供了更广泛的机会。
Multiple nuclear transcription factors including E-26-like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.