Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing

Abstract

An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(βTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by βTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.

Figure 1. Cep68 is degraded in prometaphase in a βTrCP-dependent manner

a) Cep68 protein levels are downregulated in prometaphase. HeLa cells were synchronized by double-thymidine arrest before release into fresh media. Seven hours after release, nocodazole was added to the media to accumulate cells in prometaphase, which were collected by mitotic shake-off. Cells were collected at the indicated times and lysed for immunoblotting as indicated. AS, asynchronous. PM, prometaphase. b) Cep68 is degraded in prometaphase by a cullin-RING ligase. HeLa cells stably expressing Flag-HA-Cep68 were released from double-thymidine arrest. Seven hours after release, nocodazole was added to the media to accumulate cells in prometaphase. Where indicated, MG132 (a proteasome inhibitor) or MLN4924 (a cullin-RING ligase inhibitor) were also added to cells seven hours after release. Where indicated, cells were transfected with βTrCP siRNA. Cells were harvested either in G2 phase (eight hours after release from double-thymidine arrest) or prometaphase (twelve hours after release). c) Cep68 degradation occurs between prophase and prometaphase. U-2OS cells were fixed and stained with DAPI and antibodies recognizing Cep68 (green) and Lamin B (red). The areas in the white boxes are shown at higher magnification directly beside the corresponding image. The graph indicates the percentage of cells that were either Cep68 positive or negative in each stage of mitosis. Prophase: n=110; Prometaphase: n=130; Metaphase: n=102 (from one experiment). d) Cep68 interacts specifically with βTrCP1. HEK293T cells were transfected with either empty vector (EV) or FLAG-tagged centrosomal proteins (CEPs). Cell lysates were immunoprecipitated with anti-FLAG resin, and immunoprecipitates were probed with the indicated antibodies. WCL, whole cell lysate. e) Cep68 is stabilized in prometaphase by βTrCP depletion. HeLa cells were transfected with βTrCP siRNA. Cells were synchronized by double-thymidine arrest and released into fresh media for the indicated times, before processing as in Figure 1A.

Figure 2. PLK1 phosphorylates Cep68 on Ser332 to promote βTrCP-mediated Cep68 degradation

a) Ser332 is necessary for Cep68 degradation in prometaphase. HeLa cells expressing inducible FLAG-tagged Cep68, Cep68(Δ331-337), or Cep68(S332A) were synchronized by double-thymidine arrest. Cells were released and harvested at the G1/S transition, in G2 phase (8 hours after release), or in prometaphase (PM). Cell lysates were immunoblotted as indicated. b) PLK1 inhibition prevents the degradation of endogenous Cep68. HeLa cells were synchronized by double-thymidine arrest, treated with nocodazole seven hours after release, and incubated for a further five hours to allow cells to enter prometaphase (PM). Where indicated, cells were treated with PLK1 inhibitor (BI2536). Cep68 was immunoprecipitated from cell lysates and immunoblotted as indicated. Cep68 interacts with Cep215 both in asynchronous cells and when Cep68 is stabilized by PLK1 inhibition. c) PLK1 depletion, but not Nek2 depletion, prevents the degradation of Cep68. HeLa cells expressing inducible FLAG-Cep68 were transfected with siRNAs to βTrCP, Nek2, or PLK1. Cells in prometaphase were harvested and imunoblotted as indicated. d) PLK1 inhibition prevents phosphorylation of Cep68 on Ser332 in vivo. HeLa cells expressing either inducible FLAG-Cep68 or FLAG-Cep68(S332A) were released from a double-thymidine arrest. Seven hours after release, cells were treated with nocodazole and, where indicated, either BI2536 (a PLK1 inhibitor) or MLN4924 (a CRL inhibitor). Cells were then harvested at the indicated time points. Cep68 or Cep68(S332A) were immunoprecipitated from cell lysates using an anti-FLAG resin. Whole cell lysates (WCL) and immunoprecipitates were immunoblotted as indicated.

Figure 3. Cep68 degradation is not required for centrosome separation in mitosis

a) Cep68(S332A) localizes to the intercentrosomal linker in interphase cells. Cells expressing inducible FLAG-tagged Cep68 or Cep68(S332A) were fixed and analyzed by immunofluorescence using anti-FLAG (red) and anti-PCNT (green) antibodies. The areas in the white boxes are shown at higher magnification directly beside the corresponding image. Scale bars represent 1 μm. b) Centrosome separation occurs before the degradation of endogenous Cep68. HeLa cells were fixed and analyzed by immunofluorescence using antibodies recognizing Cep68 (red) and γ-tubulin (green). The areas in the white boxes are shown at higher magnification directly below the corresponding image. Scale bars represent 1 μm. c) Centrosome separation and γ-tubulin recruitment occur normally in cells expressing Cep68(S332A). Cells expressing HA-tagged Cep68 or Cep68(S332A) were fixed and stained with anti-HA (green) and anti-γ–tubulin (red) antibodies. Cells in interphase, G2 phase, metaphase, and telophase were analyzed by immunofluorescence. The areas in the white boxes are shown at higher magnification directly below the corresponding image. Scale bars represent 1 μm. This experiment was reproduced three times. For each experiment in this figure, approximately 30 cells per condition were analysed.

Figure 4. Cep68 degradation is not required for the displacement of Rootletin or c-Nap1 from mitotic centrosomes

Cells expressing inducible FLAG-Cep68 or FLAG-Cep68(S332A) were stained with an anti-FLAG antibody (green) and either an anti-Rootletin or an anti-c-Nap1 antibody (red). Cells in interphase and mitosis were analyzed. The areas in the white boxes are shown at higher magnification directly beside the corresponding image. Scale bar represents 5 μm. This experiment was independently reproduced two times and each time approximately 30 cells per condition were analysed.

Figure 5. Cep68 stabilization increases the amount of PCNT at metaphase centrosomes, but does not affect its removal at the end of mitosis

a) Cep68(S332A) expression results in increased levels of PCNT on metaphase centrosomes. Cells expressing pBABE-HA-tagged Cep68 or Cep68(S332A) were fixed and stained with anti-HA and anti-PCNT antibodies. PCNT levels decrease in telophase centrosomes, as previously reported, regardless of the expression of Cep68. The areas in the white boxes are shown at higher magnification directly above the corresponding image. This phenomenon was observed in >3 experiments. The graph on the right shows the volume of PCNT-positive material in metaphase cells expressing Cep68 or Cep68(S332A) measured using Volocity 3D image analysis software. Bars represent the mean ± standard deviation (S.D.). Cep68: n=32 centrosomes; Cep68(S332A):n=33 centrosomes. P< 0.0001. b) The biochemical interaction between stable Cep68 and PCNT increases in metaphase, and decreases at the end of mitosis. In contrast, the interaction between stable Cep68 and Cep215 persists throughout mitosis. Cells expressing doxycycline (DOX)-inducible FLAG-tagged Cep68 or Cep68(S332A) were collected from asynchronous conditions (AS), prometaphase (PM), or late mitosis/early G1 phase (the latter obtained by releasing cells from nocodazole arrest for two hours). Cep68 or Cep68(S332A) were immunoprecipitated from cell lysates with anti-FLAG resin, and whole cell lysates (WCL) and immunoprecipitates were immunoblotted as indicated. Total Cep215 levels do not change following release from mitosis. The asterisk denotes a non-specific band.

Figure 6. Retention of Cep68 or PCNT in late mitosis prevents the removal of Cep215

a) Expression of non-degradable Cep68(S332A) results in the retention of Cep215 on centrosomes in late mitosis. Cells expressing Cep68 or Cep68(S332A) were fixed and stained with antibodies recognizing Cep68 (red) and anti-Cep215 (green) antibodies. Cells in interphase and metaphase expressing HA-tagged Cep68 constructs were analyzed by immunofluorescence. The localization of Cep215 in telophase in cells expressing FLAG-tagged Cep68 constructs was analyzed by three-dimensional structured-illumination microscopy (3D-SIM). Scale bars represent 1 μm. The graph below shows the relative intensity of Cep215 quantified in metaphase and telophase cells expressing HA-tagged Cep68 or Cep68(S332A). The bars represent the mean ± standard deviation (S.D.). Cep68 metaphase: n=30 centrosomes; Cep68(S332A) metaphase: n=26 centrosomes; Cep68 telophase: n=30 centrosomes; Cep68(S332A) telophase: n=32 centrosomes. ****P < 0.0001. b) Expression of non-cleavable PCNT(R2231A) in late mitosis prevents Cep215 removal. HeLa cells were transiently transfected with FLAG-PCNT or FLAG-PCNT(R2231A). Cells were fixed and stained with antibodies to FLAG (red) and Cep215 (green). The localization of Cep215 in telophase cells expressing FLAG-tagged PCNT constructs was analyzed by 3D-SIM. The areas in the white boxes are shown at higher magnification directly above the corresponding image. Scale bars represent 1 μm. This experiment was reproduced three times.

Figure 7. Cep68 degradation promotes centriole separation in a Cep215-dependent manner

a) The distance between mother and daughter centrioles in late mitosis is restricted in cells expressing non-degradable Cep68(S332A). Cells expressing HA-Cep68 or HA-Cep68(S332A) were synchronized by double-thymidine arrest and released into fresh media. Cells in metaphase or cytokinesis were analyzed by immunofluorescence using antibodies recognizing HA (green) or Centrin 2 (red). The areas in the white boxes are shown at higher magnification directly above the corresponding image. Scale bars represent 1 μm. b) Measurement of the distance between mother and daughter centrioles in late mitosis. Cells expressing inducible FLAG-tagged Cep68 or Cep68(S332A) were transfected with Cep215 siRNA. Cells were synchronized by double-thymidine arrest and released into fresh media. Cells in metaphase or cytokinesis were analyzed by immunofluorescence. The distance between centrioles (CP110 foci) was measured using the SoftWorx measure tool and is displayed as a scatter plot. Bars represent the mean distance ± standard deviation (S.D.). The n value is pooled from at least three independent experiments. c) Cep68 degradation is not required for centriole disengagement. In contrast, non-cleavable PCNT(R2231A) prevents centriole disengagement. Left panels: Cells expressing Cep68 or Cep68(S332A) were synchronized by double-thymidine arrest and released into fresh media for 14 hrs. Cells in G1 phase were stained with for c-Nap1 (red) and CP110 (green) and analyzed by three-dimensional structured-illumination microscopy (3D-SIM). c-Nap1 can be resolved into two populations in cells expressing Cep68 and Cep68(S332A). Scale bars represent 1 μm. Right panels: Cells expressing wild-type FLAG-PCNT or non-cleavable FLAG-PCNT(R2231A) were synchronized by double thymidine arrest and released into fresh media until they entered G1 phase. Cells were stained with antibodies to FLAG (green) and c-Nap1 (red) and analyzed by 3D-SIM. The graph on the right shows the quantification of cells containing 2 c-Nap1 dots in cells expressing Cep68 or Cep68(S332A). > 10 cells per experiment were analysed per experiment. Bars represent the mean of three experiments ± standard deviation (S.D.). Non-cleavable PCNT served as the positive control in two out of the three experiments and is shown without error bars. ns= non-significant.

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 pattern. 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^, , after which the C-terminus of PCNT (which contains the Cep215-binding region) is degraded via the N-end rule. 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.