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
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Figure 2: Studies on Vertebrate Fgn Expression in Rodent Neurons(A) Amino acid (aa) alignment of Fgn orthologs from rat (759 aa), mouse (759 aa), and Drosophila (523 aa). The two vertebrate orthologs are 99% identical, but notably different from Drosophila Fgn (see Results).(B) X-gal staining of Fgn knockout/reporter mouse fetus at E12.5. Fgn is highly expressed in CNS regions, such as brain and eye (left) and spinal cord (right). (C–E)MT immunostaining (anti-βIII-tubulin antibody) in cortical neurons expressing either GFP or GFP-Fgn. Fgn-overexpressing neurons have statistically shorter axons (70.04 ± 6.95 μm) and fewer minor processes (3.33 ± 0.163) compared to GFP-expressing Ctl neurons (axon length, 99.44 ± 8.0 μm; minor process number, 5.22 ± 0.35). Quantification is shown in (E) (Student’s t test, p ≤ 0.05). No difference in growth cone morphology or relative MT mass was observed as a result of Fgn overexpression (D, left and right). Similarly, there was no significant difference in MT mass in the axon (Student’s t test, p ≥ 0.05; E, third graph from left) between Ctl GFP (484 ± 69.42 AFU) and Fgn-expressing neurons (552 ± 46.95). Scale bars represent 0.5 mm (B, left), 2 mm (B, right), 10 μm (C, right), and 5 μm (D, right).(F–J) The effect on cortical neurons of Fgn siRNA pool. (F–H) Immunostains for βIII-tubulin. (F, left) and (G, left) are stage 2 neurons from Ctl and Fgn siRNA groups, respectively, while (F, right) and (G, right) are stage 3 neurons from the same experimental groups. Fgn depletion increases axon length (158.59 ± 14.90 μm; G, right and I, right), minor process number (10 ± 0.183; G, left and right and I, left), and rate of polarization (stage 1, 30% ± 5.6%; stage 2, 40% ± 3.5%; stage 3, 31% ± 2.5% (H, left and I, left) compared to Ctl siRNA neurons (F, left and right and H, left; axon length, 102.77 ± 5.95 μm; process number, 6 ± 0.47; polarization stage 1, 45% ± 4.4%; stage 2, 42% ± 3.5%; stage 3, 14% ± 1.7%) (Student’s t test p ≤ 0.05). Quantification is shown in (I). Validation of Fgn siRNA is shown in (J) where RFL-6 cells were co-transfected with GFP-Fgn and either Ctl siRNA (left) or Fgn siRNA (right). Fgn siRNA reduced (Student’s t test, p ≥ 0.05) by more than 70% the GFP-Fgn expression observed with Ctl siRNA, as evaluated by western blotting (fold change: Ctl siRNA, 1 ± 0.104; Fgn siRNA, 0.28 ± 0.194; J and immunocytochemistry [data not shown]). Scale bars represent 20 μm (F, right) and 50 μm (H, right).
Fgn was discovered in vertebrates as a gene spontaneously mutated in a mouse strain that displayed a fidgeting phenotype (Cox et al., 2000). As shown in Figure 2A, vertebrate Fgn is larger than Drosophila Fgn, with a region of over 300 amino acids toward the N terminus that is absent from the fly ortholog. The Walker A motif in the AAA region is the same as in fly, but the Walker B has unusual amino acid substitutions. Multiple attempts at developing Fgn antibodies in the past have failed for unknown reasons (Yang et al., 2005). Here, a line of mice that knocks out Fgn by replacing most of the Fgn gene for LacZ was purchased, so that Fgn’s expression pattern could be observed by staining for β-galactosidase. Fgn expression was observed in various tissues, but was especially high in developing nervous tissue (Figure 2B).