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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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DHC depletion results in Golgi dispersion and partial vesicle transport inhibition. Golgi-58K protein immunostaining revealed compact Golgi apparatus in a control siRNA-treated neuron (A) and dispersed Golgi apparatus in a DHC siRNA-treated neuron (B) 6 d after siRNA transfection. (C) Quantification of Golgi area relative to soma area revealed a gradual increase of the Golgi size in DHC siRNA-treated neurons (t-test, *, P < 0.05) starting from 4 d after siRNA transfection. Bar, 10 μm. (D) Left panel shows an anterogradely moving vesicle (arrowhead) with elapsed time in seconds. Distance traversed is depicted by closed circles in graph. Right panel shows a retrogradely transported vesicle (arrowhead), with movement depicted by red squares in the graph. Graph shows cumulative distance plots of four vesicles that underwent transport during 60 s (4 d DHC siRNA). Each plot shows the position of the moving vesicle in successive frames relative to the starting position. Black and red plots depict anterogradely and retrogradely moving vesicles, respectively. Both anterogradely transported vesicles and one of the retrogradely moving vesicles (red squares) undergo relatively sustained movement, whereas the other retrogradely moving vesicle spends more time paused than moving. (E) Summary of results of DHC depletion on vesicle transport frequencies and processivity (t-test, *, P < 0.05). See Results and discussion and Table S1 for details.
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fig2: DHC depletion results in Golgi dispersion and partial vesicle transport inhibition. Golgi-58K protein immunostaining revealed compact Golgi apparatus in a control siRNA-treated neuron (A) and dispersed Golgi apparatus in a DHC siRNA-treated neuron (B) 6 d after siRNA transfection. (C) Quantification of Golgi area relative to soma area revealed a gradual increase of the Golgi size in DHC siRNA-treated neurons (t-test, *, P < 0.05) starting from 4 d after siRNA transfection. Bar, 10 μm. (D) Left panel shows an anterogradely moving vesicle (arrowhead) with elapsed time in seconds. Distance traversed is depicted by closed circles in graph. Right panel shows a retrogradely transported vesicle (arrowhead), with movement depicted by red squares in the graph. Graph shows cumulative distance plots of four vesicles that underwent transport during 60 s (4 d DHC siRNA). Each plot shows the position of the moving vesicle in successive frames relative to the starting position. Black and red plots depict anterogradely and retrogradely moving vesicles, respectively. Both anterogradely transported vesicles and one of the retrogradely moving vesicles (red squares) undergo relatively sustained movement, whereas the other retrogradely moving vesicle spends more time paused than moving. (E) Summary of results of DHC depletion on vesicle transport frequencies and processivity (t-test, *, P < 0.05). See Results and discussion and Table S1 for details.

Mentions: Golgi dispersion is a reliable indicator of dynein inhibition. In control siRNA-treated neurons, the Golgi apparatus appears as a compact, multi-tubule structure located near the cell center (Fig. 2 A). In DHC-depleted neurons, the Golgi apparatus became elongated, less compact, and distributed throughout the cell body after 4 d of siRNA treatment (Fig. 2 B). Quantification of Golgi size revealed that the dispersion became significant from 4 d and progressed until 6 d after siRNA transfection (Fig. 2 C). We also examined vesicle transport behaviors in living sympathetic neurons treated with control or DHC siRNA. Neurons were incubated with rhodamine-dextran to label a subfraction of vesicular structures arising from endocytosis (Hollenbeck, 1993). The moving behaviors of fluorescent vesicles were recorded by time-lapse imaging and analyzed for both the frequency and the persistence of their movements within the axon. We found that by 2 d after transfection, the number of anterogradely and retrogradely moving vesicles per axon per minute was not affected by DHC siRNA treatment (t-test; Fig. 2 E and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200407191/DC1). In addition, the percentage of vesicles that exhibit processive or sustained motion (for definition see Materials and methods; Fig. 2 D) was not affected in either direction at 2 d of DHC siRNA treatment (t-test; Fig. 2 E and Table S1). However, by 4 d after DHC siRNA transfection, the frequencies of vesicle movements were significantly diminished in both anterograde and retrograde directions, and to similar extent (Fig. 2 E and Table S1). The percentage of sustained movements was decreased, but not significantly, in the anterograde direction (13% decrease; P = 0.07). However, the percentage of labeled vesicles exhibiting sustained retrograde movements was significantly decreased by DHC depletion (43% decrease; P = 0.01; Fig. 2 E and Table S1).


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)

DHC depletion results in Golgi dispersion and partial vesicle transport inhibition. Golgi-58K protein immunostaining revealed compact Golgi apparatus in a control siRNA-treated neuron (A) and dispersed Golgi apparatus in a DHC siRNA-treated neuron (B) 6 d after siRNA transfection. (C) Quantification of Golgi area relative to soma area revealed a gradual increase of the Golgi size in DHC siRNA-treated neurons (t-test, *, P < 0.05) starting from 4 d after siRNA transfection. Bar, 10 μm. (D) Left panel shows an anterogradely moving vesicle (arrowhead) with elapsed time in seconds. Distance traversed is depicted by closed circles in graph. Right panel shows a retrogradely transported vesicle (arrowhead), with movement depicted by red squares in the graph. Graph shows cumulative distance plots of four vesicles that underwent transport during 60 s (4 d DHC siRNA). Each plot shows the position of the moving vesicle in successive frames relative to the starting position. Black and red plots depict anterogradely and retrogradely moving vesicles, respectively. Both anterogradely transported vesicles and one of the retrogradely moving vesicles (red squares) undergo relatively sustained movement, whereas the other retrogradely moving vesicle spends more time paused than moving. (E) Summary of results of DHC depletion on vesicle transport frequencies and processivity (t-test, *, P < 0.05). See Results and discussion and Table S1 for details.
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Related In: Results  -  Collection

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fig2: DHC depletion results in Golgi dispersion and partial vesicle transport inhibition. Golgi-58K protein immunostaining revealed compact Golgi apparatus in a control siRNA-treated neuron (A) and dispersed Golgi apparatus in a DHC siRNA-treated neuron (B) 6 d after siRNA transfection. (C) Quantification of Golgi area relative to soma area revealed a gradual increase of the Golgi size in DHC siRNA-treated neurons (t-test, *, P < 0.05) starting from 4 d after siRNA transfection. Bar, 10 μm. (D) Left panel shows an anterogradely moving vesicle (arrowhead) with elapsed time in seconds. Distance traversed is depicted by closed circles in graph. Right panel shows a retrogradely transported vesicle (arrowhead), with movement depicted by red squares in the graph. Graph shows cumulative distance plots of four vesicles that underwent transport during 60 s (4 d DHC siRNA). Each plot shows the position of the moving vesicle in successive frames relative to the starting position. Black and red plots depict anterogradely and retrogradely moving vesicles, respectively. Both anterogradely transported vesicles and one of the retrogradely moving vesicles (red squares) undergo relatively sustained movement, whereas the other retrogradely moving vesicle spends more time paused than moving. (E) Summary of results of DHC depletion on vesicle transport frequencies and processivity (t-test, *, P < 0.05). See Results and discussion and Table S1 for details.
Mentions: Golgi dispersion is a reliable indicator of dynein inhibition. In control siRNA-treated neurons, the Golgi apparatus appears as a compact, multi-tubule structure located near the cell center (Fig. 2 A). In DHC-depleted neurons, the Golgi apparatus became elongated, less compact, and distributed throughout the cell body after 4 d of siRNA treatment (Fig. 2 B). Quantification of Golgi size revealed that the dispersion became significant from 4 d and progressed until 6 d after siRNA transfection (Fig. 2 C). We also examined vesicle transport behaviors in living sympathetic neurons treated with control or DHC siRNA. Neurons were incubated with rhodamine-dextran to label a subfraction of vesicular structures arising from endocytosis (Hollenbeck, 1993). The moving behaviors of fluorescent vesicles were recorded by time-lapse imaging and analyzed for both the frequency and the persistence of their movements within the axon. We found that by 2 d after transfection, the number of anterogradely and retrogradely moving vesicles per axon per minute was not affected by DHC siRNA treatment (t-test; Fig. 2 E and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200407191/DC1). In addition, the percentage of vesicles that exhibit processive or sustained motion (for definition see Materials and methods; Fig. 2 D) was not affected in either direction at 2 d of DHC siRNA treatment (t-test; Fig. 2 E and Table S1). However, by 4 d after DHC siRNA transfection, the frequencies of vesicle movements were significantly diminished in both anterograde and retrograde directions, and to similar extent (Fig. 2 E and Table S1). The percentage of sustained movements was decreased, but not significantly, in the anterograde direction (13% decrease; P = 0.07). However, the percentage of labeled vesicles exhibiting sustained retrograde movements was significantly decreased by DHC depletion (43% decrease; P = 0.01; Fig. 2 E and Table S1).

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
Related in: MedlinePlus