We have analyzed the dynamics of neuronal intermediate filaments in living neurons by using the method of photobleaching of fluorescently-labeled neurofilament L protein and immunoelectron microscopy of incorporation sites of biotinylated neurofilament L protein. Low-light-level imaging and photobleaching of growing axons of mouse sensory neurons did not affect the rate of either axonal growth or the addition of intermediate filament structures at the axon terminal, suggesting that any perturbations caused by these optical methods would be minimal. After laser photobleaching, recovery of fluorescence did occur slowly with a recovery half-time of 40 min. Furthermore, we observed a more rapid fluorescence recovery in growing axons than in quiescent ones, indicating a growth-dependent regulation of the turnover rate. Incorporation sites of biotin-labeled neurofilament L protein were localized as numerous discrete sites along the axon, and they slowly elongated to become continuous arrays 24 h after injection. Collectively, these results indicate that neuronal intermediate filaments in growing axons turn over within the small area of the axoplasm possibly by the mechanism of lateral and segmental incorporation of new subunits.

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