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. 2013 Nov 25;110(50):E4849–E4857. doi: 10.1073/pnas.1319656110

Fig. 5.

Fig. 5.

Loss of Cep192- or Cep152-dependent interaction with Plk4 induces defects in centriole duplication and cell proliferation. (A–D) U2OS cells stably expressing the indicated constructs were silenced for control luciferase (siGL), Cep192 (siCep192), or Cep152 (siCep152; SI Appendix, Fig. S10 shows details of the experimental procedure). (A) The resulting cells were used for immunoblotting analyses, and the membranes were stained with Coomassie Brilliant Blue (CBB). (B–D) The cells from A were immunostained for further analyses. (B) Relative fluorescence intensities for centrosome-localized Plk4 signals were quantified from three independent experiments (≥50 cells per experiment). Bars indicate mean ± SEM. (C and D) Number of cells with no detectable Cep192 (C) or Cep152 (D) signals was quantified from three independent experiments (≥200 cells per experiment). Bars indicate SD. (E) Number of centrosomal Sas6 signals per cell was quantified from three independent experiments (≥200 cells per experiment). Bars indicate SD. The actual confocal images used for quantification in B and E are provided in SI Appendix, Figs. S13 and S14. (F) The cells obtained in A were quantified 4 d after treatment with siRNAs. Data were obtained from three independent experiments (≥2,000 cells per experiment). Bars indicate SD. (G) Model illustrating the mechanism of how Cep192 and Cep152 promote Plk4 recruitment and centriole biogenesis. In early G1, Cep192 localizes to mother and daughter centrioles, whereas Cep152 localizes only to mother centriole. At this stage, Plk4 mainly associates with mother centriole. Soon after the recruitment of Plk4 to daughter centriole in late G1, Cep152 was amassed at this location (SI Appendix, Fig. S8B). These series of events in turn allow the recruitment of Sas6 to the site of procentriole assembly. As a result of a hierarchical assembly of Cep192 and Cep152, depleting Cep192 delocalizes Cep152 from centrioles, thus resulting in a greatly diminished Plk4 signals (two tiny red Plk4 dots) at centrosomes. On the contrary, depleting Cep152, which is present in centrosomes and cytosol, unleashes Plk4 from the Cep152 tether and allows freed Plk4 to be enriched (two larger red Plk4 dots) at Cep192-associated centrioles. In the absence of Cep192 or Cep152, Sas6 recruitment is disrupted.