Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching

Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move alon g axons in a rapid, intermittent, and highly asynchronous manner. In contra st, studies on axonal neurofilaments using laser photobleaching have not de tected movement. Here, we describe a modified photobleaching strategy that does pern-Lit the direct observation of neurofilament movement. Axons of cu ltured neurons expressing GFP-tagged neurofilament protein were bleached by excitation with the mercury arc lamp of a conventional epifluorescence mic roscope for 12-60 s. The length of the bleached region ranged from 10 to 60 mum. By bleaching thin axons, which have relatively few neurofilaments, we were able to reduce the fluorescent intensity enough to allow the detectio n of neurofilaments that moved in from the surrounding unbleached regions. Time-lapse imaging at short intervals revealed rapid, intermittent, and hig hly asynchronous movement of fluorescent filaments through the bleached reg ions at peak rates of up to 2.8 mum/s. The kinetics of movement were very s imilar to our previous observations on neurofilaments moving through natura lly occurring gaps, which indicates that the movement was not impaired by t he photobleaching process. These results demonstrate that fluorescence phot obleaching can be used to study the slow axonal transport of cytoskeletal. polymers, but only if the experimental strategy is designed to ensure that rapid asynchronous movements can be detected. This may explain the failure of previous photobleaching studies to reveal the movement of neurofilament proteins and other cytoskeletal proteins in axons.