α-tubulin mutations cluster in conserved functionally important region on exterior microtubule surface.

Alignment (A) of C. elegans alpha-tubulin genes mec-12, tba-1, tba-2, tba-7 and human alpha tubulin homologues TUBA1A, TUBA1C, and TUBA4A demonstrate the high degree of conservation at the region corresponding to helix 12 seen in the AlphaFold2 predicted structure for tba-2 UniProt Accession: P34690 (B) which are exposed to the outside surface of the MT when assembled and are involved in MAP (e.g. tau) binding (C) Created in BioRender.com/ol82yg3.

© 2026, Benbow et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Mutations in C. elegans alpha-tubulin genes suppress wildtype tau-induced motility deficits to varying degrees.

Independent tba-2 mutant alleles (D429N and D429A) in high expressing wildtype human tau background (WT-TauH, strain CK144) rescue tau-induced motility deficits to wildtype levels (A and B, respectively), whereas only partial rescue is seen with similar mutations in mec-12 (D431N) (C) (***p<0.001, error bars reflect SEM,). tba-2 (D429N) also rescues mutant tau, Mut-TauH (strain CK10) induced deficits to ∼87% of wildtype levels (D, ***p<0.0001, error bars reflect SEM). (A-B: n ≥ 55, C-D: n ≥142)

Tubulin expression correlates positively with expression level and suppresses in co-pathology models.

Transgenic C. elegans with pan-neuronal over-expressing mutant tba-2 (D429N) were created and compared for level of tau-induced motility deficit suppression. The strain tba-2(Tg-A) showed a lower level of motility deficit suppression as compared to tba-2(Tg-B) (A). qRT-PCR analysis revealed that tba-2(Tg-A) expressed a significantly lower amount of mutant tubulin as compared to tba-2(Tg-B) (B), correlating with the relative difference between the strain in motility rescue. Motility means were compared using a Kruskal-Wallis test with a Dunn’s post-hoc analysis, ***p<0.001, **p<0.01, n ≥ 110, error bars reflect SEM. qPCR data was analyzed using a Student’s T-test p=0.003, n=3, error bars represent SEM. Mutant tubulin partially suppresses motility deficits in worms with co-pathology models. tba-2 (D429N) provides partial suppression of synergistic tau and Aβ pathology in worms expressing both human tau at high levels and toxic human Aβ peptide (C, ***p<0.001, n ≥ 85, error bars reflect SEM). tba-2 partially suppresses synergistic tau and TDP-43 pathology in worms expressing both wildtype human tau at lower levels and human TDP-43 protein (D, *p<0.05, ***p<0.0001 in a Kruskal-Wallis test with a Dunn’s post-hoc analysis, n ≥ 184 error bars reflect SEM).

Mutant tubulins suppress neurodegeneration.

tba-2 (D429N) and mec-12 (D431N) both suppress tau-induced neuron loss to wildtype levels (A) and (B) as visualized by GFP-labeled GABAergic ventral cord neurons (***p<0.001, n ≥ 82 animals error bars reflect SEM, Kruskal-Wallis test with Dunn’s multiple comparison post-hoc analysis). Representative images for tba-2 (D429N)/WT-Tau and control animals (C). Scale bar (50µm).

Tubulin mutations moderately alter total tau levels, without changing levels of tau phosphorylation.

Western blot analysis of total protein from day 1 adult worms demonstrated that mutant tba-2 (D429N) modestly but significantly reduced tau protein levels. However, significant reduction in tau was not observed in worms with mutant mec-12 (D431N) (A and B). Tau phosphorylation was not altered significantly. (**p<0.01, 1-way ANOVA with Tukey post-hoc test, error bars reflect SEM). n = 4 populations (C-E, tubulin loading control in F).

Mutant tubulin does not suppress tau aggregation in worms expressing high levels wildtype human tau.

Western blot analysis of sequentially extracted fractions from day 1 so adult worm populations to isolate tau aggregates showed that mutant tubulin (tba-2 (D429N)) reduced total tau levels but did not alter levels of tau aggregates (representative blots in A and quantitation in B, and C). FA shows formic acid soluble tau compared to total tau in whole worm lysates. (*p<0.05, Student’s T-test, n = 4, error bars reflect SEM)

Mutation in tba-2 changes taxol driven microtubule mass sedimentation.

Capillary western performed using Peggy SueTM showing microtubule (MT) sedimentation resulted in reduced microtubule mass present after taxol incubation in the tba-2/WT TauH worm extracts as compared to extracts from WT TauH worms. No microtubule mass was detected with either extract when incubated with nocodazole (A). Data from 4 replicate capillary westerns were quantified in (B). Signal from taxol pellets was normalized to the corresponding pre-incubated input showing the reduction in sedimented MT mass seen with tba-2/WT TauH extracts was statistically significant (***p < 0.0001, unpaired T-test, n = 4, error bars represent SEM)

Mutant tubulin does not change binding affinity between Tau and soluble tubulin.

Biolayer interferometry was used to determine the binding affinities between 6x-His tagged wildtype human 4R1N tau (His-Tau) and isolated wildtype (WT) or tba-2(D42N) C. elegans tubulin. The calculated Kd between tau and tba-2 (D429N) tubulin (1.38E-09 M) was not statistically different from tau-WT tubulin Kd (1.23E-09 M) (A, Student’s T-test, n≥3, error bars represent standard deviation). Panel B shows representative binding curves for His-tau and WT tubulin (blue) versus His-tau and tba-2(D429N) (green), with corresponding theoretical fitted curves in red. Supplementary Figure 7 shows representative acrylamide gels showing isolated C. elegans tubulin preparations.

Mutant tubulin Alleles identified in forward genetic screening

Strain List

List of antibodies and dilutions used

tba-2(V433D) shows moderate levels of suppression in a tau-transgenic strain expressing medium levels of wildtype human 4R1N tau (CK1443).

(***p<0.0001, Kruskal-Wallis ANOVA with Dunn’s comparison, n≥114, error bars represent SEM).

Mutant tba-1 moderately suppresses tau-induced motility deficits.

tba-1(E437K) shows moderate levels of suppression in a tau-transgenic strain expressing medium levels of wildtype human 4R1N tau (CK1443). (***p<0.0001, Kruskal-Wallis ANOVA with Dunn’s comparison, n≥230, error bars represent SEM).

Truncated tubulin does not rescue tau-induced phenotypes.

The tubulin truncation of tba-7 at Q230 does not rescue tau-induced motility deficits in worms expressing wildtype human tau at moderate expression levels (CK1443). Kruskal-Wallis ANOVA with Dunn’s post-hoc test, ***p<0.0001, n ≥ 146, error bars reflect SEM.

Tubulin mutations are strong semi-dominant suppressors of tau toxicity.

C. elegans homozygous for tau, but heterozygous for the tba-2(D429N) were tested for homozygous tau suppression in a swimming assay. Population is low due to post-swim confirmation of heterozygosity. n ≥ 15, Kruskall-Wallis ANOVA with Dunn’s post-hoc test, *p<0.05 error bars reflect SEM.

Pan-tissue control for total tba-2 mRNA in qPCR experiments.

Primers amplified endogenous tba-2 expressed across all worm tissues which remains unchanged since overexpression of mutant tubulin is specific to neurons that make up only a small portion of total C. elegans cells.

A. Workflow schematic for sedimentation assay [Created in BioRender.com/fqyruiy]. Biological replicates of capillary westerns data showing input tubulin, and sedimented microtubule mass after incubation with taxol or nocodazole.

© 2026, Benbow et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Representative Coomassie stained acrylamide gel showing approximately 0.2mg/ml tubulin isolated from tba-2 (D429N) and WT Non-Tg animals.