Fig. 2

Tau mutations mimicking post-translational modifications to T231 and K274/281 impact touch sensitivity in a single-copy transgenic C. elegans model. a Schematic of TauT4 protein, with the proline-rich domain (PRD), microtubule-binding domain (MTBD), and repeats R1-R4 denoted, along with individual amino acids that were mutated by CRISPR-Cas9 editing. The numbering scheme is based upon Tau-441, the longest of the alternatively spliced human brain isoforms, as is the convention in the field (i.e. T231 is not the 231st amino acid in the 0N4R tau variant, which lacks two N-terminal domains, but is instead positioned at amino acid 173). b List of TauT4 PTM-mimetic and phosphoablation mutant transgenes. All transgenes are translational fusions to photo-convertible protein Dendra2 and they are driven by the mec-7 promoter. 0N4R is the wild type human tau isoform used in this study. CRISPR-Cas9 gene editing was used to introduce phosphomimetic T231E, phosphoablation T231A, and acetylmimetic K274/281Q mutations into the TauT4 ORF. For simplicity, these mutants will be referred to as T231E, T231A and K274/281Q. c, d Touch sensitivity was quantified by measuring responsiveness to light touch in transgenic Dendra2, TauT4, T231A, T231E and K274/281Q mutant strains at day 3 (c) and at day 10 (d) of adulthood (d0 is when the worms enter their reproductive phase). Data were calculated as percent responsiveness following ten repetitive light touches to the anterior body, and are plotted with the mean ± SEM. Each circular point represents a value obtained from a single animal – note that many of the points overlap (N = 20 animals, from two independent biological replicates). e, f TauT4 protein level was measured by quantifying Dendra2 fluorescence from ALM (e) and PLM (f) neurons individually in living animals (N = 60 ± 15, from three independent biological replicates). g The fold change (± SD) in tau mRNA expression relative to the wild type TauT4 strain was assessed by semi-quantitative RT-PCR. The experiment was performed in three biological replicates, with four technical replicates using pmp-4 and act-1 as housekeeping genes for normalization and two sets of primers specific for human tau. Statistical analysis was by one-way ANOVA followed by Tukey’s multiple-comparisons test, with *P < 0.05, **P < 0.01 and ***P < 0.001 denoting significance between bracketed samples