A test of microtubule translocation during neurite elongation

Abstract

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.