Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules.
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.
Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA.
- Drosophila Proteins/metabolism*
- Nuclear Proteins/metabolism*
- Amino Acid Sequence
- Animals, Genetically Modified
- Mice, Knockout
- Molecular Sequence Data
© Copyright Policy
Figure 3: Vertebrate Fgn Depletion Increases Labile MT Mass in the Axon but Does Not Alter MT Number(A) Cortical neurons that had been treated for 48 hr with Fgn or Ctl siRNA and then re-plated were treated with nocodazole (0.2 μg/ml) for 0 min, 30 min, or 4 hr. Cultures were pre-extracted to release free tubulin and then prepared for IF or western blotting. Fgn-depleted cultures had 52.75% ± 8.9% greater MT mass compared to Ctl siRNA (one-way ANOVA, p ≤ 0.05). At 30-min nodocazole, MT mass decreased to roughly half (oneway ANOVA, p ≤ 0.05) compared to the nocodazole-free time point (Ctl siRNA, 45.21% ± 1.21% decrease; Fgn siRNA, 54.71% ± 15.92% decrease), with no difference in MT levels between the Ctl siRNA and Fgn siRNA groups (one-way ANOVA, p ≥ 0.05). Fgn-depleted cultures and Ctl cultures also did not differ in MT mass (one-way ANOVA, p ≥ 0.05) after 4 hr in nocodazole (Ctl siRNA, 31.97% ± 5.46%; Fgn siRNA, 34.78% ± 10.27%). Quantification of MT mass in the axon was by IF.(B) Fgn depletion increases the overall MT mass in the axon by 62% (Ctl siRNA, 327.09 ± 28.26; Fgn siRNA, 530.55 ± 34.88; one-way ANOVA, p ≤ 0.05). No differences were observed (one-way ANOVA, p ≥ 0.05) between Fgn siRNA and Ctl siRNA after treatment with nocodazole (0.2 μg/ml) for 30 min (Ctl siRNA, 150.34 ± 11.84; Fgn siRNA, 167.36 ± 17.66).(C) Neurons were transfected at day 0 with Fgn or Ctl siRNA and re-transfected with EB3-GFP at the time of re-plating, so that dynamic plus ends of MTs could be visualized as fluorescent comets (tracked as 1 frame/s) during bouts of MT assembly. The two experimental groups did not differ (Student’s t test, p ≥ 0.05) in terms of comet number (Ctl siRNA, 44.70 ± 14.43; Fgn siRNA, 47.40 ± 14.39), duration (Ctl siRNA, 10.35 ± 0.46 s; Fgn siRNA, 10.82 ± 0.33 s), or speed (Ctl siRNA, 0.25 ± 0.0073 μm/s; Fgn siRNA, 0.23 ± 0.0098 μm/s).(D) Schematic shows interpretation of data. Drosophila Fgn behaves similarly to spastin or katanin, targeting the stable domain of axonal MTs. Knockdown causes an increase in the proportion of the MT that is stable, without altering the length of the MT. Vertebrate Fgn targets the labile domain of axonal MTs, such that knocking down vertebrate Fgn increases the length of the labile domain.
In neurons depleted of Fgn, there was a 53% ± 8.9% increase in MT mass relative to Ctls as assessed by western blotting (Figure 3A), and a 62% ± 8.63% increase in MT mass per unit area of axon as assessed by quantitative immunofluorescence (IF) (Figure 3B). Nocodazole sensitivity was used to discern the stable and labile fractions of the MT mass (Baas and Black, 1990). After 30 min of drug treatment, the MT levels were indistinguishable from the corresponding drug-treated Ctls, as assessed by either western blotting or IF (Figures 3C and 3D), indicating that the MT mass added as a result of Fgn depletion is entirely labile.