Figure 8. Cep215 mediates the effects of non-cleavable PCNT(R2231A) on centriole duplication.

a) Representative Edu incorporation patterns for early S-phase, mid S-phase, and late S-phase cells. HeLa cells were incubated with Edu for one hour prior to fixation to identify cells that were actively undergoing DNA replication, then stained using the Click-it Edu imaging system (red) together with antibodies to CP110 (green) and FLAG (blue). The distinct stages of S-phase were identified according to the Edu incorporation pattern⁵⁵. Cells were considered to be in early S-phase if they contained numerous small Edu foci, distributed evenly throughout the nucleus. Cells were considered to be in mid S-phase if the Edu was incorporated at perinuclear or perinucleolar sites. Late S-phase was scored if cells contained very large Edu-positive foci. The areas in the white boxes are shown at higher magnification directly above the corresponding image. Scale bar represents 1 μm.

b) Cep215 depletion rescues the centriole duplication delay caused by expression of non-cleavable PCNT(R2231A). HeLa cells expressing doxycycline-inducible FLAG-tagged Cep68 or Cep68(S332A) were induced for 48 hours with doxycycline. Where indicated, cells were transfected with Cep215 siRNA at the time of transient transfection of FLAG-tagged PCNT(R2231A). Centriole numbers (CP110 foci) were scored. The average of three independent experiments is shown ± standard error of the mean (SEM). The n value is pooled over the three experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

c) Model. Top panel: Cep68 is degraded in a βTrCP- and PLK1-dependent manner in early mitosis. PCNT is cleaved by Separase in anaphase^{21, 22}, after which the C-terminus of PCNT (which contains the Cep215-binding region) is degraded via the N-end rule²². Cep215 is subsequently removed from the centrosome. Middle panel: Stabilization of Cep68 at the centrosome in mitosis is not required for centrosome separation and bipolar spindle assembly. Instead, Cep68 stabilization in late mitosis results in the retention of Cep215, which prevents centriole separation following disengagement. Bottom panel: Expression of non-cleavable PCNT results in the retention of a different population of Cep215, which prevents centriole disengagement and subsequent licensing.