Microtubule reconfiguration during axonal retraction induced by nitric oxide

Axonal retraction is induced by different types of physiological cues and is responsible for the elimination of mistargeted axons. There is broad agreement that alterations in the cytoskeleton underlie axonal retraction. The prevailing view is that axonal retraction involves a wholesale depolymerization of microtubules and microfilaments. However, axons retracting physiologically display a very different morphology than axons induced to retract by experimental depolymerization of microtubules. Experimental depolymerization of microfilaments actually prevents retraction rather than causing it. We have proposed an alternative hypothesis, namely that axonal retraction involves a backward retreat of cytoskeletal elements rather than their wholesale depolymerization. In the present study, we sought to test this hypothesis with regard to microtubules. When a donor of nitric oxide was applied to cultured chick sensory neurons, the majority of axons retracted dramatically within 30-60 min. Retracting axons were characterized by an enlarged distal region, a thin trailing remnant, and sinusoidal bends along the shaft. Quantitative immunofluorescence analyses showed no detectable loss of microtubule mass during retraction, even with regard to the most labile microtubules. Instead, microtubules were reconfigured into coiling and sinusoidal bundles to accommodate the shortening of the axon. Stabilization of microtubules by taxol did not prevent the retraction, even at concentrations of the drug that actually caused microtubule levels to increase. The retractions induced by nitric oxide were remarkably similar to those observed when motor proteins are manipulated, suggesting that these retractions may result from alterations in the activities of the motors that configure microtubules.