Supplementary Figure 7: SAS6 and ANA2 cooperate to elicit ectopic neuronal BB elongation, leading to defects in sensory behaviour.

a) Representative electron micrographs of longitudinal sections through the ciliary base of olfactory neurons in flies with different genotypes. Empty arrow head marks the proximal region of the dBB without cartwheel. b) Representative electron micrographs of the dBB cross sections of olfactory neurons in flies with either no or simultaneous ectopic expression of SAS6 and ANA2. c) Quantification of the time taken by ≥80% of the flies to successfully climb the half way mark of the 18 cm long tube (SAS6::GFP/GFP::ANA2: −/−, n = 70, +/−, n = 80: (ns-not significant)p > 0.05, −/+, n = 70: (ns)p > 0.05, and +/+, n = 60: **p < 0.0001, Kruskal-Wallis Test). d) The method of measuring the dBB length (L_BB). e) Representative SIM images present the longitudinal view of the BBs in flies with different genotypes. PACT (red) marks the neuronal BBs. Insets show PACT (in grey scale) close to the arrowhead. Quantification of BB length (ii), olfactory reception (iii) and gravitaxis (iv) behaviour in flies with either no or ectopic expression of both SAS6 and ANA2 using a driver that only expresses in pre-neuronal cells (Gal4^neur). In ii (SAS6::GFP/GFP::ANA2: −/−, n = 47, and +/+, n = 45: **p < 0.0001, two-tail Mann-Whitney Test), iii (SAS6::GFP/GFP::ANA2: −/−, n = 70, and +/+, n = 60: **p < 0.0001, two-tail Mann-Whitney Test) and iv (SAS6::GFP/GFP::ANA2: −/−, n = 60, and +/+, n = 60: **p < 0.0001, two-tail Mann-Whitney Test). Note that upon ectopic expression of both SAS6 and ANA2 in pre-neuronal cells, we observed one cilium per cell, in most of the olfactory neurons (98%: shown in figure), as observed in controls. In the remainder olfactory neurons (not shown), we observed that cells were forming two cilia, each with one BB. (v) Representative electron micrographs show longitudinal sections through the ciliary base and the distal BB cross sections of olfactory neurons in flies with different genotypes. The empty arrow head marks the proximal region of the dBB without cartwheel. Electron micrographs of the dBB cross sections present the features (including 9-fold symmetric doublet MTs) of n ≥ 7 neurons for each genotype. vi) Tukey-Box plot of dBB length in olfactory neurons (SAS6::GFP/GFP::ANA2: −/−, n = 7, and +/+, n = 8: **p < 0.0001, two-tail Mann-Whitney Test). EM (a, b, dv-vi), SIM (ei-ii), and behaviour (c, eiii-iv) experiments were repeated independently twice, thrice and thrice with similar results, respectively. Scale bars in a, b, ei, and ev represent 0.5, 0.2, 1 and 0.1 µm, respectively. In each Tukey-box plot, centre line indicates median and error bars indicate full range of variation (from minimum-to-maximum). For different variables of each Tukey-box plot and fly genotypes see Supplementary Table 4 and Supplementary Tables 1,3, respectively.