Figure 7.

The binding of trichoplein to AurA in centrioles is indispensable for suppression of primary cilia assembly. (A–D) Schematic (A) of human trichoplein full length (FL) and deletion constructs showing the ability to bind AurA (B), to localize to the centriole (C), and to prevent primary cilia assembly (C and D). Numbers indicate human trichoplein amino acids. TPHD indicates a trichohyalin and plectin homology domain in trichoplein. (B) Interactions were determined by GST pull-down assays using each purified protein. (C and D) Centriolar localization and primary cilia inhibition (also see Fig. S5 A) were tested as follows. RPE1 cells were transiently transfected with each GFP fusion protein. 3 h after transfection, the medium was changed to serum-free medium. (C) Cells were incubated for additional 48 h and then subjected to the immunostaining with anti–acetylated tubulin (red) or anti–γ-tubulin (not depicted). Exogenous trichoplein FL and deletion mutants were visualized by GFP luminescence (green). Nuclei were also stained with DAPI (blue). Higher magnification images of centrioles with or without detectable GFP luminescence are indicated in top right or bottom left insets, respectively. (D) To quantify data shown in C, we analyzed 100 GFP-positive cells per group and calculated the percentages of cells with primary cilia (n = 3). (E–G) Each Tet-ON RPE1 cell line was incubated as described in the legend to Fig. 1 (C and D) and then subjected to immunoblotting (E) and the quantification (E, bottom; as described in Fig. 2 E), with a slight modification. (F) 10 ng/ml Dox was used for the induction of the GFP-trichoplein 1–130 fragment, which was visualized by GFP luminescence (green). (top) Magnifications of insets are shown. (G) The quantification data were obtained as described in the legend to Fig. 1 D. (H and I) GST pull-down assays using GST-AurA (H) or –Odf2-β (I) in the presence of purified 1–130 or 1-130DD (Fig. S5 E) in vitro. As a negative control, GST was used instead of each GST-tagged protein (−). (J) HeLa cells were transfected with Flag-tagged AurA wildtype (+) or a kinase-dead mutant (KM) in the presence of Myc-tagged trichoplein FL, 1–130, or 1-130DD as described in the legend of Fig. 6 B. (K and L) AurA recruitment/activation and primary cilia inhibition were tested by expression of each trichoplein 1–130 fragment as a GFP fusion in RPE1 cells. The cells were transiently transfected and incubated as described in A–D. Then, the cells were immunostained with anti–acetylated tubulin (Ac-Tub.). Cells were simultaneously stained with DAPI (K), anti-AurA (L, left), or anti-pAurA (L, right). Tricho., trichoplein; IB, immunoblot; CBB, Coomassie brilliant blue; exo., exogenous; endo., endogenous; mag., magnification. Data are means ± SD. Bars: (C [main images], F [bottom], and K [top]) 10 µm; (C [insets], F [top], K [bottom], and L) 1 µm.