Center for Brain Research, Medical University of Vienna, 1090 Vienna, Austria.
J Neurosci. 2010 Mar 17;30(11):4160-70. doi: 10.1523/JNEUROSCI.3537-09.2010.
Localization of mRNAs to postsynaptic sites and their subsequent translation is thought to contribute to synapse-specific plasticity. However, the direct visualization of dendritic RNA transport in living neurons remains a major challenge. Here, we analyze the transport of Alexa-labeled RNAs microinjected into mature hippocampal neurons. We show that microinjected MAP2 and CaMKIIalpha RNAs form particles that localize into dendrites as their endogenous counterparts. In contrast, nonlocalizing RNAs or truncated CaMKIIalpha, lacking the dendritic targeting element, remain in the cell body. Furthermore, our microinjection approach allowed us to identify a novel dendritically localized RNA, Septin7. Time-lapse videomicroscopy of neurons injected with CaMKIIalpha and Septin7 RNAs demonstrates fast directional movement along the dendrites of hippocampal neurons, with similar kinetics to Staufen1 ribonucleoprotein particles (RNPs). Coinjection and simultaneous visualization of two RNAs, as well as double detection of the corresponding endogenous RNAs, reveal that neuronal transcripts are differentially sorted in dendritic RNPs.
mRNA 定位于突触后部位及其随后的翻译被认为有助于突触特异性可塑性。然而，在活神经元中直接可视化树突 RNA 运输仍然是一个主要挑战。在这里，我们分析了将 Alexa 标记的 RNA 显微注射到成熟海马神经元中的运输情况。我们发现，注射的 MAP2 和 CaMKIIalpha RNA 形成颗粒，其在体内对应物定位到树突中。相比之下，非定位 RNA 或缺乏树突靶向元件的截断的 CaMKIIalpha 留在细胞体中。此外，我们的显微注射方法使我们能够鉴定出一种新型的树突定位 RNA，即 Septin7。用 CaMKIIalpha 和 Septin7 RNA 注射的神经元的延时视频显微镜观察表明，沿着海马神经元的树突快速定向运动，与 Staufen1 核糖核蛋白颗粒（RNP）的动力学相似。两种 RNA 的共注射和同时可视化，以及相应内源性 RNA 的双重检测，揭示了神经元转录物在树突 RNP 中差异分拣。
