Identification of sites for exponential translation in living dendrites
Identification of sites for exponential translation in living dendrites
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DOI：
10.1073/pnas.231485698
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发表时间：
2001-11-06
影响因子：
11.1
通讯作者：
Eberwine, J
中科院分区：
文献类型：
--
作者：
Job, C;Eberwine, J
神经元过程包含 mRNA 和膜结构，某些形式的突触可塑性似乎需要海马神经元树突中的蛋白质合成。为了定量树突蛋白的合成，我们使用多光子显微镜观察活体分离树突中合成的绿色荧光蛋白。用编码绿色荧光蛋白的 mRNA 转染树突，在谷氨酸受体激动剂刺激下，荧光呈指数增加；该反应被翻译抑制剂茴香霉素和依米丁减弱。对整个神经元的类似实验表明，(RS)-3,5-二羟基苯基甘氨酸 0.5 H2O (DHPG) 刺激的荧光在细胞体中呈线性，相对于树突中的指数增加。对树突下荧光分布的详细空间分析揭示了“热点”，即空间稳定的树突平移位点。然而，对这些热点的详细时间分析揭示了翻译率的异质性。核糖体的双标签方案复染表明，“最快”翻译的位点与增加的核糖体密度相关，与启动的核糖体亚基组装一致，这是翻译的第一步。我们认为树突具有专门用于快速翻译的特定位点。
Neuronal processes contain mRNAs and membrane structures, and some forms of synaptic plasticity seem to require protein synthesis in dendrites of hippocampal neurons. To quantitate dendritic protein synthesis, we used multiphoton microscopy of green fluorescent protein synthesized in living isolated dendrites. Transfection of dendrites with mRNA encoding green fluorescent protein resulted in fluorescence that exponentially increased on stimulation with a glutamate receptor agonist; a reaction attenuated by the translation inhibitors anisomycin and emetine. Comparable experiments on whole neurons revealed that (RS)-3,5-dihydroxyphenylglycine 0.5 H2O (DHPG)-stimulated fluorescence was linear in cell bodies relative to the exponential increase seen in dendrites. Detailed spatial analysis of the subdendritic distribution of fluorescence revealed "hotspots," sites of dendritic translation that were spatially stable. However, detailed temporal analysis of these hotspots revealed heterogeneous rates of translation. A double-label protocol counterstaining for ribosomes indicated that sites of "fastest" translation correlated with increased ribosome density, consistent with ribosome subunit assembly for initiation, the first step of translation, We propose that dendrites have specific sites specialized for fast translation.