Figure 5. Satellite component MNR promotes centriole duplication by localizing WDR62 to the centrosome.
(A) We immunoprecipitated endogenous WDR62 and MNR from HeLa total cell lysates. Co-precipitation was detected using antibodies specific to WDR62 and MNR. (B) SC, MNR #1, and MNR #2 siRNA transfected S-phase HeLa cells were co-stained with MNR (red) and Centrin (‘c’, green). (C) Total cell lysate of SC, MNR #1, MNR #2 siRNA transfected HeLa cells were analyzed by immunoblotting with antibodies to MNR. The asterisk marks the band specific to MNR, which sits below a non-specific band. Actin served as a loading control. (D) Percentage of S-phase SC, MNR #1, MNR #2 siRNA treated HeLa cells with four Centrin foci. (E) S-phase SC and MNR siRNA-transfected HeLa cells were co-stained for Centrin (‘c’, green) and CEP63 (red). (F) SC and MNR siRNA treated S-phase HeLa cells were co-stained for WDR62 (red) and Centrin (‘c’, green). (G) Total cell lysates of SC and MNR-depleted cells were analyzed by immunoblot with antibodies to WDR62 and MNR. Actin served as a loading control. 20 μg of protein lysate was loaded per lane. (H) SC and MNR #1-depleted S-phase cells were co-stained with Centrin (‘c’, green), CDK5RAP2 (red), CEP152 (red), and WDR62 (red). (I) Our findings indicate that MNR localizes WDR62 to the centrosome, which in turn recruits CEP63. For all quantifications at least 100 cells were counted per experiment (n = 3), p < 0.005 (paired t-test). Scale bars indicate 5 μm for all images.
Figure 5—figure supplement 1. MNR is required to localize WDR62 and CEP63 to the centrosome.
Figure 5—figure supplement 2. WDR62 interacts with CEP152 and CEP63 in a MNR-dependent manner.
