Fig. 4

Mutation of endogenous Drosophila KLC serine-433 to mimic permanent phosphorylation inhibits axonal transport of APP in wing sensory neurons. (a) Alignment showing high conservation of the amino acid sequences encompassing rat KLC1 serine-460 and the homologous Drosophila KLC serine-433 (indicated in red). (b) CRISPR genome editing approach to mutate KLC serine-433 to aspartic acid. Drosophila Klc sequence (upper line) along with the ssODN (lower line) are shown. Serine-433 codon (KLC) and aspartic acid codon (ssODN) are shown in red, the protospacer sequence in grey shade and the PAM site in blue shade. A G-to-A transition is introduced to mutate the PAM. (c) Representative kymographs showing axonal transport of APP-YFP in Klcwt and KlcS433D homozygous backgrounds in 2-day old Drosophila; scale bar and times are indicated. Bar chart shows the relative proportions of stationary, anterograde and retrograde moving APP-YFP in the Klcwt background. (d) Bar charts show total, anterograde and retrograde APP-YFP movement runs in Klcwt and KlcS433D backgrounds. (e) Violin plots show velocities of APP-YFP runs in anterograde and retrograde directions in the different backgrounds. Median and interquartile ranges are indicated by hashed lines. N = 8 wings for each genotype. For velocity studies, N = 577 anterogradely and 347 retrogradely moving APP-YFP in Klcwt, and 343 anterogradely and 247 retrogradely moving APP-YFP in the KlcS433D background. Statistical significance was determined by one-way ANOVA with Holm-Sidak’s multiple comparison test in (c), Mann-Whitney U test in (d) and two-tailed Student’s t test in (e). Error bars are s.e.m.; **p < 0.01; **** p < 0.0001; ns not significant