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Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules.

Leo L, Yu W, D'Rozario M, Waddell EA, Marenda DR, Baird MA, Davidson MW, Zhou B, Wu B, Baker L, Sharp DJ, Baas PW - Cell Rep (2015)

Bottom Line: In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable) MT mass in their axons.Concomitantly, there are more minor processes and a longer axon.Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog) regulates neuronal development by tamping back the expansion of the labile domains of MTs.

View Article: PubMed Central - PubMed

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

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Drosophila Fgn Knockdown Increases Synaptic Connections In Vivo(A–D) Confocal images of third instar larval NMJs, muscles 6 and 7, labeled with α-HRP (white) to detect presynaptic neuronal membranes. The following are shown: (A) a Ctl NMJ; (B) an NMJ from an Fgn-knockdown animal (Dcr2;UAS:Fgn-RNAi;elav-Gal4; note the significant increase in bouton number in Fgn-knockdown animals compared to Ctls); (C) a representative individual bouton from Ctl animals; (D) a bouton with associated satellite boutons from an Fgn-knockdown animal. Arrows show the bouton; arrowheads show satellites. Scale bar represents 10 μm.(E) Quantification of the number of total boutons and satellite boutons in Fgn-knockdown animals compared to Ctls is shown.(F) Quantification of IF intensity of acetylated tubulin (top), total tubulin (middle), and ratio of acetylated to total tubulin (bottom) between Fgn-knockdown animals (Dcr2;UAS:Fgn-RNAi;elav-Gal4) and outcrossed Ctls (Dcr2;;elav-Gal4 and UAS:Fgn-RNAi/+) at both the axonal shaft and distal synapse near the bouton. Note the significant increase of the ratio of acetylated tubulin to total tubulin. Error bars represent SE. *p < 0.05, ***p < 0.001.
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Figure 1: Drosophila Fgn Knockdown Increases Synaptic Connections In Vivo(A–D) Confocal images of third instar larval NMJs, muscles 6 and 7, labeled with α-HRP (white) to detect presynaptic neuronal membranes. The following are shown: (A) a Ctl NMJ; (B) an NMJ from an Fgn-knockdown animal (Dcr2;UAS:Fgn-RNAi;elav-Gal4; note the significant increase in bouton number in Fgn-knockdown animals compared to Ctls); (C) a representative individual bouton from Ctl animals; (D) a bouton with associated satellite boutons from an Fgn-knockdown animal. Arrows show the bouton; arrowheads show satellites. Scale bar represents 10 μm.(E) Quantification of the number of total boutons and satellite boutons in Fgn-knockdown animals compared to Ctls is shown.(F) Quantification of IF intensity of acetylated tubulin (top), total tubulin (middle), and ratio of acetylated to total tubulin (bottom) between Fgn-knockdown animals (Dcr2;UAS:Fgn-RNAi;elav-Gal4) and outcrossed Ctls (Dcr2;;elav-Gal4 and UAS:Fgn-RNAi/+) at both the axonal shaft and distal synapse near the bouton. Note the significant increase of the ratio of acetylated tubulin to total tubulin. Error bars represent SE. *p < 0.05, ***p < 0.001.

Mentions: The effects of knocking down Drosophila Fgn from fly neurons in the neuromuscular junction (NMJ) were investigated first. The Gal4/UAS system (Brand and Perrimon, 1993) was used to express an RNAi hairpin targeting the Drosophila Fgn gene (CG3326, or Fgn) under UAS control (UAS:Fgn-RNAi). Expression was limited to post-mitotic neurons using the Elav-gal4 driver (Campos et al., 1987). The total number of synaptic contacts (boutons) was significantly higher in Fgn-knockdown animals (Figures 1B and 1E) compared to outcrossed control (Ctl) animals (Figures 1A and 1E). The number of satellite boutons, which are small growths of presynaptic membranes that extend out from axonal terminal arbors, in Fgn-knockdown animals (arrows in Figures 1D and 1E) was greater than that of Ctls (Figures 1C and 1E). An increased number of satellite boutons has been observed in flies bearing mutations to other MT-severing proteins (Sherwood et al., 2004; Mao et al., 2014). A significant increase in the ratio of acetylated to total tubulin (measured by quantifying fluorescence intensity) was observed in the axonal shaft and distal synapse, compared to two independent control RNAi lines (Ctl1 and Ctl2) (Figure 1F). This was due to a significant increase in MT acetylation in these areas, as total tubulin levels remained relatively unchanged (Figure 1F). A similar result has been reported in Drosophila neurons with compromised katanin (Mao et al., 2014). These results are consistent with Drosophila Fgn behaving similarly to traditional MT-severing proteins in the neurons of the fly.


Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules.

Leo L, Yu W, D'Rozario M, Waddell EA, Marenda DR, Baird MA, Davidson MW, Zhou B, Wu B, Baker L, Sharp DJ, Baas PW - Cell Rep (2015)

Drosophila Fgn Knockdown Increases Synaptic Connections In Vivo(A–D) Confocal images of third instar larval NMJs, muscles 6 and 7, labeled with α-HRP (white) to detect presynaptic neuronal membranes. The following are shown: (A) a Ctl NMJ; (B) an NMJ from an Fgn-knockdown animal (Dcr2;UAS:Fgn-RNAi;elav-Gal4; note the significant increase in bouton number in Fgn-knockdown animals compared to Ctls); (C) a representative individual bouton from Ctl animals; (D) a bouton with associated satellite boutons from an Fgn-knockdown animal. Arrows show the bouton; arrowheads show satellites. Scale bar represents 10 μm.(E) Quantification of the number of total boutons and satellite boutons in Fgn-knockdown animals compared to Ctls is shown.(F) Quantification of IF intensity of acetylated tubulin (top), total tubulin (middle), and ratio of acetylated to total tubulin (bottom) between Fgn-knockdown animals (Dcr2;UAS:Fgn-RNAi;elav-Gal4) and outcrossed Ctls (Dcr2;;elav-Gal4 and UAS:Fgn-RNAi/+) at both the axonal shaft and distal synapse near the bouton. Note the significant increase of the ratio of acetylated tubulin to total tubulin. Error bars represent SE. *p < 0.05, ***p < 0.001.
© Copyright Policy - open-access
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getmorefigures.php?uid=PMC4837332&req=5

Figure 1: Drosophila Fgn Knockdown Increases Synaptic Connections In Vivo(A–D) Confocal images of third instar larval NMJs, muscles 6 and 7, labeled with α-HRP (white) to detect presynaptic neuronal membranes. The following are shown: (A) a Ctl NMJ; (B) an NMJ from an Fgn-knockdown animal (Dcr2;UAS:Fgn-RNAi;elav-Gal4; note the significant increase in bouton number in Fgn-knockdown animals compared to Ctls); (C) a representative individual bouton from Ctl animals; (D) a bouton with associated satellite boutons from an Fgn-knockdown animal. Arrows show the bouton; arrowheads show satellites. Scale bar represents 10 μm.(E) Quantification of the number of total boutons and satellite boutons in Fgn-knockdown animals compared to Ctls is shown.(F) Quantification of IF intensity of acetylated tubulin (top), total tubulin (middle), and ratio of acetylated to total tubulin (bottom) between Fgn-knockdown animals (Dcr2;UAS:Fgn-RNAi;elav-Gal4) and outcrossed Ctls (Dcr2;;elav-Gal4 and UAS:Fgn-RNAi/+) at both the axonal shaft and distal synapse near the bouton. Note the significant increase of the ratio of acetylated tubulin to total tubulin. Error bars represent SE. *p < 0.05, ***p < 0.001.
Mentions: The effects of knocking down Drosophila Fgn from fly neurons in the neuromuscular junction (NMJ) were investigated first. The Gal4/UAS system (Brand and Perrimon, 1993) was used to express an RNAi hairpin targeting the Drosophila Fgn gene (CG3326, or Fgn) under UAS control (UAS:Fgn-RNAi). Expression was limited to post-mitotic neurons using the Elav-gal4 driver (Campos et al., 1987). The total number of synaptic contacts (boutons) was significantly higher in Fgn-knockdown animals (Figures 1B and 1E) compared to outcrossed control (Ctl) animals (Figures 1A and 1E). The number of satellite boutons, which are small growths of presynaptic membranes that extend out from axonal terminal arbors, in Fgn-knockdown animals (arrows in Figures 1D and 1E) was greater than that of Ctls (Figures 1C and 1E). An increased number of satellite boutons has been observed in flies bearing mutations to other MT-severing proteins (Sherwood et al., 2004; Mao et al., 2014). A significant increase in the ratio of acetylated to total tubulin (measured by quantifying fluorescence intensity) was observed in the axonal shaft and distal synapse, compared to two independent control RNAi lines (Ctl1 and Ctl2) (Figure 1F). This was due to a significant increase in MT acetylation in these areas, as total tubulin levels remained relatively unchanged (Figure 1F). A similar result has been reported in Drosophila neurons with compromised katanin (Mao et al., 2014). These results are consistent with Drosophila Fgn behaving similarly to traditional MT-severing proteins in the neurons of the fly.

Bottom Line: In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable) MT mass in their axons.Concomitantly, there are more minor processes and a longer axon.Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog) regulates neuronal development by tamping back the expansion of the labile domains of MTs.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus