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Vol. 12, Issue 10, 3257-3267, October 2001
Neuroscience Program, Department of Biological Sciences, Ohio
University, Athens, Ohio 45701
Observations on naturally occurring gaps in the axonal
neurofilament array of cultured neurons have demonstrated that
neurofilament polymers move along axons in a rapid, intermittent, and
highly asynchronous manner. In contrast, studies on axonal
neurofilaments using laser photobleaching have not detected movement.
Here, we describe a modified photobleaching strategy that does permit
the direct observation of neurofilament movement. Axons of cultured neurons expressing GFP-tagged neurofilament protein were bleached by
excitation with the mercury arc lamp of a conventional epifluorescence microscope for 12-60 s. The length of the bleached region ranged from
10 to 60 µm. By bleaching thin axons, which have relatively few
neurofilaments, we were able to reduce the fluorescent intensity enough
to allow the detection of neurofilaments that moved in from the
surrounding unbleached regions. Time-lapse imaging at short intervals
revealed rapid, intermittent, and highly asynchronous movement of
fluorescent filaments through the bleached regions at peak rates of up
to 2.8 µm/s. The kinetics of movement were very similar to our
previous observations on neurofilaments moving through naturally
occurring gaps, which indicates that the movement was not impaired by
the photobleaching process. These results demonstrate that fluorescence
photobleaching 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.
The online version of this manuscript contains video
material for Figures 2, 3, 6, and 7. The online version is available at
www.molbiolcell.org.
*
Corresponding author. E-mail address:
brown.2302{at}osu.edu. Present address: Neurobiotechnology Center and
Department of Neuroscience, The Ohio State University, Rightmire Hall,
1060 Carmack Road, Columbus OH 43210.
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