Synthesis of β-Tubulin, Actin, and Other Proteins in Axons of Sympathetic Neurons in Compartmented Cultures
Synthesis of β-Tubulin, Actin, and Other Proteins in Axons of Sympathetic Neurons in Compartmented Cultures
复制标题
隔室培养物中交感神经元轴突中 β-微管蛋白、肌动蛋白和其他蛋白质的合成
作者：
H. Eng;K. Lund;R. Campenot
一般认为轴突生长和维持所需的蛋白质在细胞体中合成并通过顺行运输递送至轴突。然而，最近的报告表明，一些蛋白质也可以在轴突内合成。我们使用[35S]蛋氨酸代谢标记来研究新生大鼠交感神经元隔室培养物中轴突蛋白的合成。将远端轴突与[35S]蛋氨酸一起孵育 4 小时，在 SDS-PAGE 上产生高度特异性的标记轴突蛋白模式，在 43-55 kDa 范围内有 4 个显着条带。轴突中的标记蛋白质不在细胞体中合成，因为它们也是在细胞体被去除后由轴突产生的。其中两种蛋白质通过免疫沉淀鉴定为肌动蛋白和β-微管蛋白。轴突合成的肌动蛋白和微管蛋白占细胞体内合成并转运到轴突的<1%，放线菌酮和嘌呤霉素对轴突蛋白合成的75-85%抑制未能抑制轴突伸长。尽管如此，轴突细胞骨架主要蛋白质的特定产生表明轴突蛋白质合成源于特定机制，并且可能具有生物学意义。一种假设的情况涉及体内具有长轴突的神经元，其中轴突运输过程中周转造成的损失可能会限制细胞体合成蛋白质对远端轴突的可用性。在这种情况下，很大一部分轴突蛋白可能由轴突合成提供，因此，轴突合成在轴突维持、再生和发芽中发挥重要作用。
The proteins needed for growth and maintenance of the axon are generally believed to be synthesized in the cell bodies and delivered to the axons by anterograde transport. However, recent reports suggest that some proteins can also be synthesized within axons. We used [35S]methionine metabolic labeling to investigate axonal protein synthesis in compartmented cultures of sympathetic neurons from newborn rats. Incubation of distal axons for 4 hr with [35S]methionine resulted in a highly specific pattern of labeled axonal proteins on SDS-PAGE, with 4 prominent bands in the 43–55 kDa range. The labeled proteins in axons were not synthesized in the cell bodies, because they were also produced by axons after the cell bodies had been removed. Two of the proteins were identified by immunoprecipitation as actin and β-tubulin. Axons synthesized <1% of the actin and tubulin synthesized in the cell bodies and transported into the axons, and 75–85% inhibition of axonal protein synthesis by cycloheximide and puromycin failed to inhibit axonal elongation. Nonetheless, the specific production by axons of the major proteins of the axonal cytoskeleton suggests that axonal protein synthesis arises from specific mechanisms and likely has biological significance. One hypothetical scenario involves neurons with long axons in vivo in which losses from turnover during axonal transport may limit the availability of cell body synthesized proteins to the distal axons. In this case, a significant fraction of axonal proteins might be supplied by axonal synthesis, which could, therefore, play important roles in axonal maintenance, regeneration, and sprouting.