Fig. 7
MPP9 facilitates the removal of the CEP97-CP110 complex from centrosomes after phosphorylation. a Immunostaining of MPP9 (green) and acetylated-tubulin (Acet-tub, red) or CP110 (red) in hTERT RPE-1 cells after serum starvation and serum re-addition. b Line graph showing the percentage of ciliated hTERT RPE-1 cells, the cells presenting with 4 dots of MPP9, and cells with CP110 or CEP97 on both centrioles. c Immunoblots of lysates from hTERT RPE-1 cells stably overexpressing Flag-tagged MPP9 wild-type (WT) or the indicated unphosphorylatable mutants after serum starvation. Tubulin was used as a loading control. Relative amounts of MPP9 were quantified and normalized to tubulin. d Immunostaining of Arl13B (red, upper) and γ-tubulin (red, upper) or CEP97 (red, lower) in hTERT RPE-1 cell lines stably overexpressing Flag-tagged MPP9 or the indicated unphosphorylatable mutants (green). 2, 629, and 636 A. e Quantification of the percentage of cells with cilia (left) and cells with CEP97 dots on both centrioles (right) from d. f Immunoblots following immunoprecipitation (IP) with an anti-Flag antibody using lysates from HEK293T cells overexpressing the indicated proteins. g Schematic model depicting the function of MPP9 in cilia formation control. After serum starvation, MPP9 is phosphorylated by TTBK2 at S629 and S636, which promotes its ubiquitination at K632 and degradation via UPS. Subsequently, the degradation of MPP9 causes the removal of CEP97 and CP110 from the distal end of the mother centriole and initiates cilia formation. For b, e, bars represent the means ± S.E.M for three independent experiments. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA analysis (e). Scale bars: 1 μm (a, d)
