Fig. 8

The microtubule-binding activity of FSD1 is essential for ciliogenesis. a A representative sequence of amino-acid alignment of the FSD1-SPRY domain was shown. The dotted box indicates six conserved arginine residues. b Expression of GFP-FSD1 WT, but not FSD1 3RE (R332E, R335E, and R337E) mutant, induced microtubule bundles. Scale bars, 5 μm (main image) and 1 μm (magnified region). c Mutation of three conserved arginine residues to glutamic acid (R332E, R335E, and R337E) abolishes the microtubule-binding activity of FSD1-SPRY domain. Purified FSD1-SPRY-WT, but not FSD1-SPRY-3RE, was co-pelleted with Taxol-stabilized microtubules. d Expression of GFP-FSD1 WT, but not 3RE mutant, rescued MT aster formation defects caused by FSD1 depletion. RPE-1 cells transfected with indicated siRNA and plasmids were subjected to a microtubule regrowth assay after nocodazole release 20 min. e Expression of GFP-FSD1 WT, but not 3RE mutant, rescued ciliogenesis defects caused by FSD1 depletion in quiescent cells. f Expression of GFP-FSD1 WT, but not 3RE mutant, rescued Cep290 localization at centriolar satellites in cycling cells. g Expression of GFP-FSD1 WT, but not 3RE mutant, rescued TMEM67 localization at centrioles in quiescent cells. h Expression of GFP-FSD1 WT, but not 3RE mutant, rescued NPHP8 localization at centrioles in quiescent cells. i Model of MT aster promoting ciliary transition zone assembly. FSD1 and other subdistal appendage proteins, such as Ninein, Kif3a, facilitate the formation of MT asters by anchoring MTs at the mother centriole. MT asters keep Cep290 at the centriolar satellites and promote transition zone assembly during ciliogenesis. MC mother centriole, MT microtubule, TZ transition zone, CV ciliary vesicle. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. **P < 0.01, ***P < 0.001