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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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Retrograde NF transport is suppressed in DHC-depleted neurons. A–D show GFP-NFH fluorescence in living neurons treated with control siRNA (A) or DHC siRNA for 4 d (B) or 6 d (C and D). The insets in C and D show the accumulation and disorganization of NFs in the axonal tip at higher magnification. Bars: (A–E) 8 μm; (insets) 25 μm. (E) Selected frames from a sequence showing anterograde (upper left-to-lower right) translocation of two NFs. Time in seconds is indicated above each frame. The bracket in the 0 s frame identifies a gap in the fluorescent NF array generated by photobleaching. The arrows and arrowheads identify the leading and trailing end, respectively, of a moving NF. The front of this fluorescent NF enters the photobleached gap early in the sequence, and can be seen at 15 s. It continues moving into the gap over the next 15 s, pauses for a while, and then moves out of the gap. Only the trailing end of this NF can be seen at 60 and 75 s. A second, shorter NF enters at 90 s (arrowhead with asterisk) and moves steadily through the gap over the next 20–25 s. (F) Histograms of the frequency of anterograde and retrograde NF movements in neurons treated for the indicated times with control or DHC siRNA.
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fig3: Retrograde NF transport is suppressed in DHC-depleted neurons. A–D show GFP-NFH fluorescence in living neurons treated with control siRNA (A) or DHC siRNA for 4 d (B) or 6 d (C and D). The insets in C and D show the accumulation and disorganization of NFs in the axonal tip at higher magnification. Bars: (A–E) 8 μm; (insets) 25 μm. (E) Selected frames from a sequence showing anterograde (upper left-to-lower right) translocation of two NFs. Time in seconds is indicated above each frame. The bracket in the 0 s frame identifies a gap in the fluorescent NF array generated by photobleaching. The arrows and arrowheads identify the leading and trailing end, respectively, of a moving NF. The front of this fluorescent NF enters the photobleached gap early in the sequence, and can be seen at 15 s. It continues moving into the gap over the next 15 s, pauses for a while, and then moves out of the gap. Only the trailing end of this NF can be seen at 60 and 75 s. A second, shorter NF enters at 90 s (arrowhead with asterisk) and moves steadily through the gap over the next 20–25 s. (F) Histograms of the frequency of anterograde and retrograde NF movements in neurons treated for the indicated times with control or DHC siRNA.

Mentions: Cytoplasmic dynein has been shown to interact with NF proteins (Wagner et al., 2004) and to move NFs in relation to MTs in vitro (Shah et al., 2000). To investigate effects of dynein depletion on NF transport in axons, we performed the NF transport assay as previously described (Roy et al., 2000; Francis et al., 2005) on neurons exposed to DHC siRNA. The distribution of EGFP-NFH revealed a distal accumulation of NFs in DHC siRNA-treated neurons (Fig. 3, A–D) from day 4 (but not day 2). In controls, we observed fast moving NFs in natural gaps or in photobleached regions in axons, similar to that reported previously (Roy et al., 2000; Wang et al., 2000; Fig. 3 E). These movements occur in both anterograde and retrograde directions, with a frequency ratio of roughly 1:1 (see Table S2 for details). However, in neurons treated with DHC siRNA for 5 to 8 d, the frequency of retrograde movements was dramatically decreased, whereas that of anterograde movements increased (Table S2), resulting in a large increase of the anterograde to retrograde movement ratio (∼24:1; Fig. 3 F).


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)

Retrograde NF transport is suppressed in DHC-depleted neurons. A–D show GFP-NFH fluorescence in living neurons treated with control siRNA (A) or DHC siRNA for 4 d (B) or 6 d (C and D). The insets in C and D show the accumulation and disorganization of NFs in the axonal tip at higher magnification. Bars: (A–E) 8 μm; (insets) 25 μm. (E) Selected frames from a sequence showing anterograde (upper left-to-lower right) translocation of two NFs. Time in seconds is indicated above each frame. The bracket in the 0 s frame identifies a gap in the fluorescent NF array generated by photobleaching. The arrows and arrowheads identify the leading and trailing end, respectively, of a moving NF. The front of this fluorescent NF enters the photobleached gap early in the sequence, and can be seen at 15 s. It continues moving into the gap over the next 15 s, pauses for a while, and then moves out of the gap. Only the trailing end of this NF can be seen at 60 and 75 s. A second, shorter NF enters at 90 s (arrowhead with asterisk) and moves steadily through the gap over the next 20–25 s. (F) Histograms of the frequency of anterograde and retrograde NF movements in neurons treated for the indicated times with control or DHC siRNA.
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Related In: Results  -  Collection

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fig3: Retrograde NF transport is suppressed in DHC-depleted neurons. A–D show GFP-NFH fluorescence in living neurons treated with control siRNA (A) or DHC siRNA for 4 d (B) or 6 d (C and D). The insets in C and D show the accumulation and disorganization of NFs in the axonal tip at higher magnification. Bars: (A–E) 8 μm; (insets) 25 μm. (E) Selected frames from a sequence showing anterograde (upper left-to-lower right) translocation of two NFs. Time in seconds is indicated above each frame. The bracket in the 0 s frame identifies a gap in the fluorescent NF array generated by photobleaching. The arrows and arrowheads identify the leading and trailing end, respectively, of a moving NF. The front of this fluorescent NF enters the photobleached gap early in the sequence, and can be seen at 15 s. It continues moving into the gap over the next 15 s, pauses for a while, and then moves out of the gap. Only the trailing end of this NF can be seen at 60 and 75 s. A second, shorter NF enters at 90 s (arrowhead with asterisk) and moves steadily through the gap over the next 20–25 s. (F) Histograms of the frequency of anterograde and retrograde NF movements in neurons treated for the indicated times with control or DHC siRNA.
Mentions: Cytoplasmic dynein has been shown to interact with NF proteins (Wagner et al., 2004) and to move NFs in relation to MTs in vitro (Shah et al., 2000). To investigate effects of dynein depletion on NF transport in axons, we performed the NF transport assay as previously described (Roy et al., 2000; Francis et al., 2005) on neurons exposed to DHC siRNA. The distribution of EGFP-NFH revealed a distal accumulation of NFs in DHC siRNA-treated neurons (Fig. 3, A–D) from day 4 (but not day 2). In controls, we observed fast moving NFs in natural gaps or in photobleached regions in axons, similar to that reported previously (Roy et al., 2000; Wang et al., 2000; Fig. 3 E). These movements occur in both anterograde and retrograde directions, with a frequency ratio of roughly 1:1 (see Table S2 for details). However, in neurons treated with DHC siRNA for 5 to 8 d, the frequency of retrograde movements was dramatically decreased, whereas that of anterograde movements increased (Table S2), resulting in a large increase of the anterograde to retrograde movement ratio (∼24:1; Fig. 3 F).

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