Fig. 5.

Loss of both TtPoc5 and Sfr1 results in cell lethality and exacerbated overproduction of defective BBs. A previously characterized centrin-binding protein, Sfr1, is the second best hPOC5 ortholog in Tetrahymena genome BLAST search, despite lacking the Poc5 box (Heydeck et al., 2016). Beyond sequence conservation, Sfr1 has a similar role in modulating BB production, prompting the generation of double knockout (poc5Δ;sfr1Δ) cells using established methods (Hai et al., 2000). (A) Representative control mated WT, double heterozygous (Het), and double knockout (KO) cells stained with anti-Cen1 antibody after 24 h and 48 h of drug selection following mating. Double KO cell death within 48 h post drug selection is not due to a lack of drug resistance since double heterozygous cells are viable after 48 h in drug. Drug selection is effective since mated WT cells that do not carry drug resistance cassettes die after 48 h in drug. Scale bars: 10 µm. (B) BB density quantification (average BBs/10 µm) at 24 h. Double KO cells have significantly higher BB density relative to mated WT and double heterozygous cells. Results are mean±s.e.m. for n=90 total counts (three counts per cell across 15 cells, in two biological replicates). ***P<0.001; n.s., not sginificant (Student's t-test). (C,D) Electron tomography of double KO BBs. (C) Cross-sectional view showing normal Tetrahymena BB accessory structures [kinetodesmal fiber (KD), transverse microtubules (tMT), post ciliary microtubules (pcMT)] and intact proximal cartwheel (CW). The anterior (A)-posterior (P) axis is indicated with an arrow. Scale bar: 100 nm. (D) Longitudinal views showing BBs that do not template cilia and have varying degrees of transition zone (TZ) formation (left and middle panels). Right panel shows a BB with a templated cilium and a morphologically normal TZ (arrow indicates electron dense axosome). See Movie 1. Scale bars: 100 nm.