Rapid movement of axonal neurofilaments interrupted by prolonged pauses

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Abstract

Axonal cytoskeletal and cytosolic proteins are synthesized in the neuronal cell body and transported along axons by slow axonal transport, but attempts to observe this movement directly in living cells have yielded conflicting results. Here we report the direct observation of the axonal transport of neurofilament protein tagged with green fluorescent protein in cultured nerve cells. Live-cell imaging of naturally occurring gaps in the axonal neurofilament array reveals rapid, intermittent and highly asynchronous movement of fluorescent neurofilaments. The movement is bidirectional, but predominantly anterograde. Our data indicate that the slow rate of slow axonal transport may be the result of rapid movements interrupted by prolonged pauses.

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Figure 1: Cultured sympathetic neurons exhibit naturally occurring gaps in the axonal neurofilament array.
Figure 2: Characterization of the GFP–NFM fusion protein.
Figure 3: Movement of neurofilaments through a gap in the axonal neurofilament array.
Figure 4: Analysis of moving filaments.

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Acknowledgements

We thank R.J. Lasek, D.L. Holzschu, E.W. Dent and P.W. Baas for help and advice; V.M.-Y. Lee for providing the anti-NFL antibody; and S. Aizicovici for technical assistance. This work was funded by grants from the National Institute of Neurological Disorders and Stroke to A.B. and R.K.H.L.

Correspondence and requests for materials should be addressed to A.B.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://www.nature.com/ncb).

Author information

Correspondence to Anthony Brown.

Supplementary information

Supplementary movies These two time-lapse movies show anterograde movement of fluorescent neurofilaments through naturally occurring gaps in the axonal neurofilament array of cultured rat sympathetic neurons. The filaments move rapidly but infrequently, and the movement is highly asynchronous. These movies have been compressed using a lossy compression algorithm to facilitate downloading.

Movie 1 Anterograde movement of four neurofilaments within a naturally occurring gap in the axonal neurofilament array. The movie starts with a short neurofilament in the gap, which moves rapidly out of the gap in an anterograde direction (0–20 s). A second neurofilament moves rapidly through the gap (35–65 s). A third neurofilament moves rapidly into the gap (140–155 s) and then pauses for the remainder of the movie, causing the gap to become narrower. A fourth neurofilament moves rapidly past the third neurofilament (220–235 s) and then also pauses for the remainder of the movie, causing the gap to narrow even further. Images were acquired at 5-s intervals. Time compression=30:1. The first 13 frames (0–60 s) of this movie are shown in Fig. 3 of the paper. (MOV 1625 kb)

Movie 2 Anterograde movement of two neurofilaments within a naturally occurring gap in the axonal neurofilament array. The movie starts with a neurofilament that moves anterogradely in a slow and intermittent manner (0–36 s), then in a rapid and continuous manner (36–56 s), and then in a slow and intermittent manner again for the remainder of the movie. A second ,shorter neurofilament moves rapidly through the gap and past the first filament without pausing (96–136 s). Images were acquired at 4-s intervals. Time compression=24:1. (MOV 613 kb)

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