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Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments.

He Y, Francis F, Myers KA, Yu W, Black MM, Baas PW - J. Cell Biol. (2005)

Bottom Line: To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon.The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected.These results support a cargo model for NF transport and a sliding filament model for MT transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA.

ABSTRACT
Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.

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Anterograde MT transport is suppressed in DHC-depleted neurons. (A) Time-lapse images reveal a MT moving in the anterograde direction through the photobleached region. Red arrows mark the leading and trailing ends of the MT. (B) The frequencies (events/min) of anterograde MT transport were significantly decreased in DHC siRNA-treated axons (x2, *, P < 0.05) both at 4 and 7 d after siRNA transfection. However, frequencies of retrograde movements were not significantly affected. (C) Histogram showing that there is no significant difference between control and DHC-depleted neurons with regard to lengths of the moving MTs. (D and E) Histograms depict mean velocity distributions of MT movements. (F and G) Histograms depict instantaneous velocity distributions of randomly chosen MTs from the population depicted in D and E, respectively. See Results and discussion and Table S3 for details. Bar, 5 μm.
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fig4: Anterograde MT transport is suppressed in DHC-depleted neurons. (A) Time-lapse images reveal a MT moving in the anterograde direction through the photobleached region. Red arrows mark the leading and trailing ends of the MT. (B) The frequencies (events/min) of anterograde MT transport were significantly decreased in DHC siRNA-treated axons (x2, *, P < 0.05) both at 4 and 7 d after siRNA transfection. However, frequencies of retrograde movements were not significantly affected. (C) Histogram showing that there is no significant difference between control and DHC-depleted neurons with regard to lengths of the moving MTs. (D and E) Histograms depict mean velocity distributions of MT movements. (F and G) Histograms depict instantaneous velocity distributions of randomly chosen MTs from the population depicted in D and E, respectively. See Results and discussion and Table S3 for details. Bar, 5 μm.

Mentions: To assay MT transport, we used the photobleach method of Wang and Brown (2002), except that we expressed EGFP-tubulin rather than injecting rhodamine-tubulin. We analyzed MT transport behaviors after 4 d of siRNA transfection, by which time the decrease of DHC level had significantly inhibited dynein function, as revealed by Golgi distribution, vesicle transport, and NF transport. In neurons cotransfected with control siRNA, the majority of moving MTs was transported in the anterograde direction, with an anterograde to retrograde frequency ratio of ∼2:1 (see Table S3 for details, available at http://www.jcb.org/cgi/content/full/jcb.200407191/DC1). In DHC siRNA-treated neurons, the frequency of anterograde MT movements is diminished (x2, P < 0.05), whereas that in the retrograde direction remains unchanged, resulting in a drop of anterograde to retrograde movement ratio to 1:1 (Table S3). At day 7, a similar decrease is observed in the anterograde frequency, as well as the anterograde to retrograde ratio of MT movements. There is a small diminution in the frequency of retrograde transport of MTs at day 7, but this did not achieve statistical significance (Fig. 4 B). Combining the data from days 4 and 7, the frequency of anterograde MT movements is significantly diminished upon dynein inhibition by RNAi (x2, P < 0.05), but the frequency of retrograde MT movements is not significantly affected. Comparing populations of control and DHC-depleted axons, there was no significant difference between the instantaneous velocities (Fig. 4, F and G), but there was a small but statistically significant decrease in the average velocities of both anterograde (2-tailed t-test, P < 0.0001) and retrograde movements (2-tailed t-test, P < 0.05; Fig. 4, D and E) in DHC siRNA-treated axons due to a prolongation of the period of time MTs spend in pausing (Table S3).


Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments.

He Y, Francis F, Myers KA, Yu W, Black MM, Baas PW - J. Cell Biol. (2005)

Anterograde MT transport is suppressed in DHC-depleted neurons. (A) Time-lapse images reveal a MT moving in the anterograde direction through the photobleached region. Red arrows mark the leading and trailing ends of the MT. (B) The frequencies (events/min) of anterograde MT transport were significantly decreased in DHC siRNA-treated axons (x2, *, P < 0.05) both at 4 and 7 d after siRNA transfection. However, frequencies of retrograde movements were not significantly affected. (C) Histogram showing that there is no significant difference between control and DHC-depleted neurons with regard to lengths of the moving MTs. (D and E) Histograms depict mean velocity distributions of MT movements. (F and G) Histograms depict instantaneous velocity distributions of randomly chosen MTs from the population depicted in D and E, respectively. See Results and discussion and Table S3 for details. Bar, 5 μm.
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Related In: Results  -  Collection

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fig4: Anterograde MT transport is suppressed in DHC-depleted neurons. (A) Time-lapse images reveal a MT moving in the anterograde direction through the photobleached region. Red arrows mark the leading and trailing ends of the MT. (B) The frequencies (events/min) of anterograde MT transport were significantly decreased in DHC siRNA-treated axons (x2, *, P < 0.05) both at 4 and 7 d after siRNA transfection. However, frequencies of retrograde movements were not significantly affected. (C) Histogram showing that there is no significant difference between control and DHC-depleted neurons with regard to lengths of the moving MTs. (D and E) Histograms depict mean velocity distributions of MT movements. (F and G) Histograms depict instantaneous velocity distributions of randomly chosen MTs from the population depicted in D and E, respectively. See Results and discussion and Table S3 for details. Bar, 5 μm.
Mentions: To assay MT transport, we used the photobleach method of Wang and Brown (2002), except that we expressed EGFP-tubulin rather than injecting rhodamine-tubulin. We analyzed MT transport behaviors after 4 d of siRNA transfection, by which time the decrease of DHC level had significantly inhibited dynein function, as revealed by Golgi distribution, vesicle transport, and NF transport. In neurons cotransfected with control siRNA, the majority of moving MTs was transported in the anterograde direction, with an anterograde to retrograde frequency ratio of ∼2:1 (see Table S3 for details, available at http://www.jcb.org/cgi/content/full/jcb.200407191/DC1). In DHC siRNA-treated neurons, the frequency of anterograde MT movements is diminished (x2, P < 0.05), whereas that in the retrograde direction remains unchanged, resulting in a drop of anterograde to retrograde movement ratio to 1:1 (Table S3). At day 7, a similar decrease is observed in the anterograde frequency, as well as the anterograde to retrograde ratio of MT movements. There is a small diminution in the frequency of retrograde transport of MTs at day 7, but this did not achieve statistical significance (Fig. 4 B). Combining the data from days 4 and 7, the frequency of anterograde MT movements is significantly diminished upon dynein inhibition by RNAi (x2, P < 0.05), but the frequency of retrograde MT movements is not significantly affected. Comparing populations of control and DHC-depleted axons, there was no significant difference between the instantaneous velocities (Fig. 4, F and G), but there was a small but statistically significant decrease in the average velocities of both anterograde (2-tailed t-test, P < 0.0001) and retrograde movements (2-tailed t-test, P < 0.05; Fig. 4, D and E) in DHC siRNA-treated axons due to a prolongation of the period of time MTs spend in pausing (Table S3).

Bottom Line: To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon.The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected.These results support a cargo model for NF transport and a sliding filament model for MT transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA.

ABSTRACT
Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.

Show MeSH