Lim S S, Edson K J, Letourneau P C, Borisy G G
Molecular-Biology Laboratory, University of Wisconsin, Madison 53706.
J Cell Biol. 1990 Jul;111(1):123-30. doi: 10.1083/jcb.111.1.123.
In a previous study using PC-12 cells (Lim, S. S., P. J. Sammak, and G. G. Borisy, 1989. J. Cell Biol. 109:253-263), we presented evidence that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary. However, neurites of PC-12 cells grow slowly, hindering a stringent test of slow axonal transport mechanisms under conditions where growth was substantial. We therefore extended our studies to primary cultures of dorsal root ganglion cells where the rate of neurite outgrowth is rapid. Cells were microinjected with X-rhodamine-labeled tubulin 7-16 h after plating. After a further incubation for 6-18 h, the cells were photobleached with an argon ion laser. Using a cooled charged couple device and video microscopy, the cells were monitored for growth of the neurite and movement and recovery of fluorescence in the bleached zone. As for PC-12 cells, all bleached zones in the neurite recovered their fluorescence, indicating that incorporation of tubulin occurred along the neurite. Despite increases in neurite length of up to 70 microns, and periods of observation of up to 5 h, no movement of bleached zones was observed. We conclude that neurite elongation cannot be accounted for by the transport of a microtubule network assembled only at the cell body. Rather, microtubules turn over all along the length of the neurite and neurite elongation occurs by net assembly at the tip.
在先前一项使用PC-12细胞的研究中（Lim, S. S., P. J. Sammak, and G. G. Borisy, 1989. J. Cell Biol. 109:253 - 263），我们提供了证据表明神经元细胞骨架的微管成分具有不同的动态性但却是静止的。然而，PC-12细胞的神经突生长缓慢，这阻碍了在生长显著的条件下对慢速轴突运输机制进行严格测试。因此，我们将研究扩展到背根神经节细胞的原代培养，其中神经突生长速度很快。接种后7 - 16小时，将细胞显微注射用X - 罗丹明标记的微管蛋白。再孵育6 - 18小时后，用氩离子激光对细胞进行光漂白。使用冷却的电荷耦合器件和视频显微镜，监测细胞神经突的生长以及漂白区域荧光的移动和恢复情况。对于PC-12细胞，神经突中的所有漂白区域都恢复了荧光，表明微管蛋白沿着神经突发生了掺入。尽管神经突长度增加了多达70微米，观察时间长达5小时，但未观察到漂白区域的移动。我们得出结论，神经突伸长不能仅通过仅在细胞体组装的微管网络的运输来解释。相反，微管在神经突的整个长度上都进行周转，神经突伸长是通过在尖端的净组装发生的。
