Supplementary Figure 6: SAS6 is differentially required in neurons and sperm cells.
a, b) SAS6 is essential for centriole assembly in neurons, but is not required for neuronal cilia function. a) Representative electron micrographs show longitudinal and cross sections through the centrioles in olfactory neurons (before ciliogenesis: at 24 h APF) in wild type flies. Note that those centrioles are close to the cell membrane and have the cartwheel. b) i) The experimental setting used to reduce/remove SAS6 during centriole and cilia biogenesis in neurons. ii) Representative images show olfactory and auditory neurons in flies with different genotypes. Cilia in olfactory and auditory neurons were studied using anti-acetylated tubulin (green) and anti-glutamylated tubulin (green) antibody, respectively. PLP (red, centrosomes) and DAPI (blue, DNA). Arrowheads mark BBs and arrows mark cilia. c) Both SAS6 and ANA2 are required for sperm BB elongation, being important for male fertility. i) Schematic representation of the experimental setting used to reduce/remove SAS6 before and after centriole biogenesis in sperm cells. ii) Tukey-Box plot of the number of progeny produced per male with different genotypes (control1, n = 10, control5, n = 10, and SAS6RNAi2, n = 11: **p < 0.001, two-tail Mann-Whitney Test; control3, n = 11, SAS6RNAi3, n = 10 **p < 0.01, ANA2RNAi1, n = 10 *p < 0.05, and BLD10RNAi1, n = 10: **p < 0.01, Kruskal-Wallis Test). iii) Tukey-Box plot of the number of BBs per cell in mature spermatocytes (control1, n = 32, and SAS6mutant, n = 42: **p < 0.0001, two-tail Mann-Whitney Test; control3, n = 62, SAS6RNAi3, n = 56, ANA2RNAi1, n = 40, and BLD10RNAi1, n = 63). iv) Representative images of mature spermatocyte BBs of flies with different genotypes (control3, SAS6RNAi3 and ANA2RNAi1). RFP::PACT (red) marks BBs and Anti-SAS6 antibody (green) stains the proximal part of the centriole. Insets show SAS6 (green) close to the arrowhead (in grey scale). v) Tukey-Box plot of the total amount of SAS6 at the mature spermatocyte BBs of the different genotypes. For v (control3, n = 60, SAS6RNAi3, n = 42 **p < 0.0001, and ANA2RNAi1, n = 40, **p < 0.0001: Kruskal-Wallis Test; n represent the number of BB pairs). All experiments in a, cii, ciii were repeated independently twice, while in b, civ-v were repeated independently thrice with similar results. Scale bars in a, bii,iii and civ represent 100 nm, 5 µm and 1 µm, 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.
