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 4: Fgn Is Sensitive to Acetylation Status of MTs in the Axon(A) Ratio images of acetylated tubulin to total βIII-tubulin displayed in fire-scale pseudo-color (white being the highest intensity and red being the lowest). Fgn siRNA neurons (right) display a decrease of acetylated to total tubulin ratio compared to Ctl siRNA.(B) Same result by western blot analysis with (right) quantification indicating that Ctl siRNA cultures have a higher ratio of acetylated to total tubulin compared to Fgn siRNA (fold change: Ctl siRNA, 2.1 ± 0.29; Fgn siRNA, 1; Student’s t test, p ≤ 0.05).(C–E) Brain immunohistochemistry of acetylated tubulin (C, left and right) and total βIII-tubulin (D, left and right) from E18.5 wild-type mouse and Fgn knockout/reporter mouse, respectively. Knockout brain has a decreased ratio of acetylated to total tubulin (E, right) compared to wild-type (E, left).(F) Quantification is shown as fold change normalized to the value of Fgn knockout ratio value as 1. Such difference was observed in all brain areas analyzed (the ratio value for the wild-type mouse are as follows: olfactory bulb, 2.2; cortex, 2.2; septum, 1.31; thalamus, 2.6; and pretectum, 1.9).(G) Ratio images in fire-scale pseudo-color of neurons treated with 2.5 μM tubacin or DMSO vehicle (Ctl) and immunostained for acetylated tubulin and total tubulin. The ratio of acetylated to total tubulin is higher with tubacin treatment.(H) (Top) Western blotting of parallel cultures also show an increase in acetylated tubulin. (Bottom) RFL-6 fibroblasts prepared for IF visualization of acetylated tubulin after overnight expression of an enzymatically inactive mutant of αTAT-1 (called αTAT-1 D157N) or the wild-type αTAT-1. Wild-type αTAT-1 shows higher staining intensity of acetylated MTs than the mutant. Similar results were obtained on neurons (data not shown).(I) Morphological quantification of experiments similar to those from Figure 2, except that MT acetylation was manipulated by either tubacin treatment or αTAT-1 overexpression. Ctl for the former and latter were DMSO vehicle and the αTAT-1 D157N, respectively. Neurons depleted of Fgn treated with vehicle (DMSO) have significantly longer axons (175 ± 11.04 μm; p ≤ 0.05, one-way ANOVA) and a higher number of minor processes (10 ± 0.9; 1 p ≤ 0.05, one-way ANOVA) compared to DMSO Ctl siRNA neurons (average axon length, 105 ± 8.29 μm; process number, 5 ± 0.49). With tubacin treatment, there were no morphological differences between Ctl (average axon length, 117 ± 6.80 μm; process number, 4.85 ± 0.37) and Fgn siRNA (average axon length, 117 ± 9.00 μm; process number, 6.26 ± 0.64; p > 0.05, one-way ANOVA for both measurements). Neurons depleted of Fgn expressing αTAT-1 D157N have longer axons (146 ± 9.78 μm; p ≤ 0.05, one-way ANOVA) and a higher number of minor processes (10 ± 0.86; p ≤ 0.05, one-way ANOVA) compared to αTAT-1 D157N Ctl siRNA neurons (average axon length, 96 ± 8.62 μm; process number, 6 ± 0.63). With αTAT-1 expression, there were no morphological differences between Ctl (average axon length, 98 ± 8.80 μm; process number, 6 ± 0.71) and Fgn siRNA (average axon length, 110 ± 7.21 μm; process number, 7 ± 0.79; p > 0.05, one-way ANOVA for both measurements).(J) The effects on morphology when GFP-Fgn-overexpressing neurons were treated with tubacin (2.5 μM). Neurons ectopically expressing GFP-Fgn and treated with vehicle (DMSO) show reduced minor process number (3.4 ± 0.27) and axon length (81 ± 6.04 μm) compared to neurons expressing GFP in the presence or absence of tubacin (minor process numbers, 5.1 ± 0.38 and 4.8 ± 0.38, respectively; axon lengths, 111 ± 6.32 and 115 ± 7.64, respectively; p < 0.05, one-way ANOVA). Scale bars represent 10 μm (A, left and G, right) and 100 μm (B, left). *p value ≤ 0.05; data are expressed as average ± SEM.
Cultures (Ctl-siRNA or Fgn-siRNA) double-labeled for IF visualization of total or acetylated tubulin in MTs revealed that total MT levels were elevated in the Fgn-depleted cultures, but acetylated MT levels remained roughly the same as in Ctl neurons (Figure 4A). This same reduction in the ratio of acetylated to total tubulin relative to Ctls was observed by western blotting of the cultured neurons (Figure 4B) and by immunohistochemistry on embryonic day (E)18.5 brain of the Fgn knockout/reporter mouse (Figures 4C–4F).