Barrett Lindy E, Sul Jai-Yoon, Takano Hajime, Van Bockstaele Elisabeth J, Haydon Philip G, Eberwine James H
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Nat Methods. 2006 Jun;3(6):455-60. doi: 10.1038/nmeth885.
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.
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,我们使用一种局部转染程序——“光转染”,将Elk1 mRNA引入活的、完整的原代大鼠神经元的特定区域。在树突中引入并翻译Elk1 mRNA会导致细胞死亡,而在细胞体中引入并翻译Elk1 mRNA则不会导致细胞死亡。在树突中翻译的Elk-1被转运到细胞核,并且细胞死亡依赖于转录,这支持了树突印记假说,并突出了树突环境对蛋白质功能的重要性。我们对光转染用于mRNA空间控制导入的效用的证明,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开辟了更广阔的机会。