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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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Depletion of DHC by siRNA. Immunofluorescence indicates depletion of DHC in DHC siRNA-treated sympathetic neurons (B), compared with control siRNA-treated neurons (A). (C) Quantification of total fluorescent intensity within soma revealed the progression of DHC depletion by siRNA (t-test, *, P < 0.05). Western blots of whole cell extracts probed with the DHC antibody (D) confirmed the protein lowering effect. The blot was stripped and reprobed with a MAP1b antibody (E), and the overlay (F) confirmed the specificity of both the DHC antibody and the DHC depletion by siRNA. Arrowheads in D–F indicate the 219-kD molecular mass marker. Bar, 20 μm.
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fig1: Depletion of DHC by siRNA. Immunofluorescence indicates depletion of DHC in DHC siRNA-treated sympathetic neurons (B), compared with control siRNA-treated neurons (A). (C) Quantification of total fluorescent intensity within soma revealed the progression of DHC depletion by siRNA (t-test, *, P < 0.05). Western blots of whole cell extracts probed with the DHC antibody (D) confirmed the protein lowering effect. The blot was stripped and reprobed with a MAP1b antibody (E), and the overlay (F) confirmed the specificity of both the DHC antibody and the DHC depletion by siRNA. Arrowheads in D–F indicate the 219-kD molecular mass marker. Bar, 20 μm.

Mentions: We used a mixture of four different siRNA duplexes that target different regions on the mRNA of DHC. Quantitative immunofluorescence images revealed that more than 80% of neurons in DHC siRNA-treated cultures exhibit a dramatic diminution of the protein (Fig. 1, A and B). Significant protein reduction starts as early as 2 d and proceeds at least until 6 d after siRNA transfection when <20% of the protein is still present (Fig. 1 C). Western blot analyses confirmed the drastic depletion of DHC by siRNA (Fig. 1 D). Furthermore, when stripped and reprobed with a MAP1b antibody, the DHC bands migrated just ahead of the MAP1b bands, as expected (Bloom et al., 1984), and we detected no diminution in the levels of MAP1b as a result of the DHC siRNA (Fig. 1, E and F). Therefore, assuming that the DHC antibody recognizes most or all isoforms of the DHC protein, we are able to specifically knock down at least 80% and perhaps over 90% of the protein in sympathetic neurons.


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)

Depletion of DHC by siRNA. Immunofluorescence indicates depletion of DHC in DHC siRNA-treated sympathetic neurons (B), compared with control siRNA-treated neurons (A). (C) Quantification of total fluorescent intensity within soma revealed the progression of DHC depletion by siRNA (t-test, *, P < 0.05). Western blots of whole cell extracts probed with the DHC antibody (D) confirmed the protein lowering effect. The blot was stripped and reprobed with a MAP1b antibody (E), and the overlay (F) confirmed the specificity of both the DHC antibody and the DHC depletion by siRNA. Arrowheads in D–F indicate the 219-kD molecular mass marker. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171826&req=5

fig1: Depletion of DHC by siRNA. Immunofluorescence indicates depletion of DHC in DHC siRNA-treated sympathetic neurons (B), compared with control siRNA-treated neurons (A). (C) Quantification of total fluorescent intensity within soma revealed the progression of DHC depletion by siRNA (t-test, *, P < 0.05). Western blots of whole cell extracts probed with the DHC antibody (D) confirmed the protein lowering effect. The blot was stripped and reprobed with a MAP1b antibody (E), and the overlay (F) confirmed the specificity of both the DHC antibody and the DHC depletion by siRNA. Arrowheads in D–F indicate the 219-kD molecular mass marker. Bar, 20 μm.
Mentions: We used a mixture of four different siRNA duplexes that target different regions on the mRNA of DHC. Quantitative immunofluorescence images revealed that more than 80% of neurons in DHC siRNA-treated cultures exhibit a dramatic diminution of the protein (Fig. 1, A and B). Significant protein reduction starts as early as 2 d and proceeds at least until 6 d after siRNA transfection when <20% of the protein is still present (Fig. 1 C). Western blot analyses confirmed the drastic depletion of DHC by siRNA (Fig. 1 D). Furthermore, when stripped and reprobed with a MAP1b antibody, the DHC bands migrated just ahead of the MAP1b bands, as expected (Bloom et al., 1984), and we detected no diminution in the levels of MAP1b as a result of the DHC siRNA (Fig. 1, E and F). Therefore, assuming that the DHC antibody recognizes most or all isoforms of the DHC protein, we are able to specifically knock down at least 80% and perhaps over 90% of the protein in sympathetic neurons.

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