Supplementary Figure 4: CEP290 localises to the TZ in 9-fold symmetric manner and is required to form cilia in all Drosophila ciliated cells.
a-c) Localisation analysis of GFP tagged CEP290 proteins at different TZs. a) Representative SIM images of the ciliary bases of olfactory neurons marked using acetylated α-tubulin (red) and ectopically expressing CEP290::GFP (Gal4cha19b/UAS-CEP290::GFP) or GFP::CEP290 (Gal4cha19b/UAS-GFP::CEP290). SIM analysis shows CEP290::GFP localises towards the lumen and on the MTs and GFP::CEP290 localises towards the ciliary membrane in the olfactory TZ. b) Schemes show the method of measuring the distance (d) between the MT or the hook and ciliary membrane at the different TZs. c) (i) Schemes show methods of measuring the inter-distance between the outer (do) and inner (di) tips of adjacent MT-membrane linkers. Representative STED micrographs of GFP::CEP290 in the cross-section of the TZs of olfactory (i: do = 99 ± 23 nm; n = 194), auditory (ii: do = 100 ± 24 nm; n = 59) and spermatocyte (iii: do = 90 ± 23 nm; n = 112) cilia. While the white arrowheads mark the resolved GFP foci, empty arrowheads (with dotted border) indicate the postulated missing foci´s position. d-h) CEP290 is required for all cilia assembly in the fly. d) i) The scheme shows the odour repulsion test using the T-tube to measure the ability of adult flies to detect a repulsive odour (Benzaldehyde). ii) Quantification of the percentage of flies that are in the compartment with repulsive odor. A null mutant of Orco, a co-receptor essential for olfaction, was used as a positive control (control1, n = 60 and Orcomutant, n = 60: **p < 0.0001, two-tail Mann-Whitney Test; control2, n = 80 and CEP290RNAi1, n = 90: **p < 0.0001, two-tail Mann-Whitney Test). e) i) Scheme depicts the bang assay and the vertical tube used to test the gravitaxis ability of adult flies. ii) Quantification of the time taken by ≥80% of the flies to successfully climb the half height mark of the tube (18 cm long) (control1, n = 70, control2, n = 60 and CEP290RNAi1, n = 60: **p < 0.0001, two-tail Mann-Whitney Test). iii) Representative kymographs of ten flies with respective genotypes followed for the first 5 seconds after the bang. f) i) Representative pictures of olfactory cilia (marked using Acetylated α-Tubulin) in flies with different genotypes. ii) Representative images of different types of olfactory shafts. iii) Quantification of ciliary defects in flies with different genotypes (control1, n = 36, Cep290mutant, n = 27, and CEP290RNAi1, n = 25). g) i) Representative electron micrographs of cross sections of scolopale in second antennal segments of flies with different genotypes. ii) Quantification of percentage of scolopale with two or more cilia in flies with different genotypes (control1, n = 48, and CEP290RNAi1, n = 34). h) i) Quantification of number of progeny produced per male with different genotypes (control1, n = 10, control3, n = 11, and CEP290RNAi2, n = 11: **p < 0.0001, two-tail Mann-Whitney Test). ii) Representative pictures of the BBs in spermatids marked using BLD10, a centriolar protein, in flies with different genotypes. DNA, BB and sperm flagella are marked by DAPI (blue), BLD10 (green) and acetylated tubulin (red), respectively. iii) Quantification of the length of BBs marked using BLD10 as shown in (ii) (control3, n = 153, and CEP290RNAi2, n = 90: **p < 0.0001, two-tail Mann-Whitney Test). iv) Representative cross-section micrographs show the axoneme bundle of the elongating flagella in different flies. Notably, while 9 + 2 arrangement of the MTs was normal in control3 flies, the MT arrangement was defective in CEP290RNAi2 flies (see insets). All experiments were repeated independently with similar results: a (thrice), c (twice), d (thrice), e (thrice), f (twice), g (twice), h (twice). Scale bars in a, c, eiii, f, g, hii and Hiv represent 1 µm, 100 nm, 1 cm, 10 µm, 500 nm, 10 µm and 500 nm, respectively. In each Tukey-box plot, centre line indicates median and error bars indicate full range of variation (from minimum-to-maximum) and dots are outliers. For different variables of each Tukey-box plot and fly genotypes see Supplementary Table 4 and Supplementary Tables 1,3, respectively.
