Organization and translation of mRNA in sympathetic axons
Organization and translation of mRNA in sympathetic axons
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DOI：
10.1242/jcs.00745
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发表时间：
2003-11-01
影响因子：
4
通讯作者：
Hollenbeck, PJ
中科院分区：
文献类型：
--
作者：
Lee, SK;Hollenbeck, PJ
许多轴突独立于其细胞体进行大分子的合成，但轴突蛋白质合成的性质、组织和规模尚不清楚。我们已经研究了轴突的这些特征；小鸡交感神经元的细胞培养。原位杂交显示poly(A) mRNA在几乎所有轴突中都是丰富且不均匀分布的。β -肌动蛋白和肌动蛋白解聚因子（ADF）的特异性转录本也存在，并且在轴突中不均匀分布，在生长锥、分支点和轴突变异处的浓度比轴突轴高约100倍。特异性抗体免疫沉淀表明β -肌动蛋白、ADF和神经丝蛋白（NF）在轴突中被翻译；独立于细胞体的对-肌动蛋白和ADF mrna分布的定量分析表明，它们进入轴突的能力可能是整个神经元的特性，而不是单个轴突的特性。为了比较轴突翻译蛋白与mRNA的分布，我们将轴突与细胞体分离，进行了S-35代谢标记。轴突合成的蛋白质分布在整个轴突上，环己亚胺对其合成有抑制作用，而氯霉素对其无抑制作用。主要翻译或只翻译轴突或细胞体的蛋白均通过代谢标记检测。与完整的神经元相比，与细胞体分离的轴突在3小时内合成的蛋白质最多可达5%。由于在我们的培养条件下，轴突含有约50%的神经元非核体积，我们估计交感神经元的轴质每单位体积的蛋白质合成能力相当于细胞体细胞质的10%。
Many axons carry out the synthesis of macromolecules independent of their cell bodies but the nature, organization and magnitude of axonal protein synthesis remain unclear. We have examined these features in axons; of chick sympathetic neurons in cell culture. In situ hybridization showed that poly(A) mRNA is abundant and non-uniformly distributed in nearly all axons. The specific transcripts for beta-actin and actin-depolymerizing factor (ADF) were also present and non-uniformly distributed in axons, with an approximately hundredfold higher concentration in growth cones, branch points and axonal varicosities than in the axon shaft. Immunoprecipitation using specific antibodies indicates that beta-actin, ADF and neurofilament protein (NF) are translated in axons; independently of cell bodies. Quantification of the distribution of beta-actin and ADF mRNAs showed that their ability to enter the axon was likely to be a property of the neuron as a whole rather than of individual axons. To compare the distribution of axonally translated protein to that of mRNA, we performed S-35 metabolic labeling with axons separated from their cell bodies. Axonally synthesized proteins were distributed throughout the axons and their synthesis was inhibited by cycloheximide but not by chloramphenicol. Proteins translated mainly or exclusively in axons or cell bodies were both detected by metabolic labeling. Axons separated from their cell bodies synthesized up to 5% as much protein in a 3-hour period as did intact neurons. Because axons in our culture conditions contain similar to50% of the non-nuclear volume of the neurons, we estimate that axoplasm of sympathetic neurons has a protein synthetic capacity per unit volume equal to 10% that of cell body cytoplasm.