Microtubule behavior in PC12 neurites: variable results obtained with photobleach technology

We have examined the effects of various means of photobleaching on the recovery of fluorescence, movement, and morphology of the microtubules in the neurites of rhodamine-tubulin-injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low-power beam for an extended period, fluorescence in polymer recovers very little after 1 hour. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high-power laser, for 0.5-5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate-stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high-intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20-30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extend through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in PC12 neurites, and both are likely to be involved in neurite maintenance and growth.