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 SCFβTrCP (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.
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Acknowledgements
The authors thank B. D. Dynlacht, J. R. Skaar and M. F. B. Tsou for critical reading of the manuscript; K. Lee and S. Kim for helpful advice; Y. Deng at the NYU SOM Microscopy core for image processing assistance; A. North (Rockefeller University) for use of the DeltaVision OMX V4/Blaze system, supported by award S10RR031855 from the National Center for Research Resources; K. Rhee for the PCNT constructs, and I. Hoffmann for the FLAG–CPAP construct. M.P. is grateful to T. M. Thor for continuous support. This work was funded by grants from the National Institutes of Health (R01-GM057587 and R37-CA076584) and New York State Health Department (NYSTEM-N11G-255) to M.P. and fellowships from the National Health and Medical Research Council of Australia and the Lymphoma Research Foundation to J.K.P. A.S., L.F. and M.P.W. are supported by the Stowers Institute for Medical Research. P.V.J. and M.J.K.J. were supported by NIH grant R01 GM094972 and an award from the Mathers Foundation. M.P. is an Investigator with the Howard Hughes Medical Institute.
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J.K.P. planned and performed most experiments and co-wrote the manuscript. M.P. coordinated the study, oversaw the results, and co-wrote the manuscript. A.M. helped with many biochemical experiments. M.J.K.J. helped with several experiments and provided intellectual advice. P.V.J. provided reagents and intellectual advice. A.S., L.F. and M.P.W. performed the mass spectrometry analyses of the purifications performed by J.K.P. All authors discussed the results and commented on the manuscript.
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Supplementary Figure 2 βTrCP targets Cep68 for degradation during mitosis.
(a) Cep68 immunofluorescence in interphase and metaphase. U-2OS cells were transfected with control siRNA or Cep68 siRNA and analyzed 48 h post-transfection. Cells were fixed and analyzed by immunofluorescence using Cep68 antibodies (red) and γ-tubulin antibodies (green). The areas in the white boxes are shown at higher magnification directly below the corresponding image. Cep68 is absent from mitotic centrosomes. Depletion of Cep68 by siRNA demonstrates that the Cep68 antibody specifically recognizes Cep68. Scale bars represent 1 μm. (b) Cep68 levels during a release from prometaphase. HeLa cells stably expressing FLAG-HA-Cep68 were synchronized in prometaphase with nocodazole. Round, prometaphase cells were collected by mitotic shake-off and released into fresh media. Cells were collected at the indicated times and lysed for immunoblotting as indicated. (c) Cep68 degradation is mediated by βTrCP. HeLa cells stably expressing FLAG-HA-Cep68 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. (d) Multidimensional Protein Identification Technology analysis of a FLAG-Cep68 immunopurification from HEK293T cells, listing the number of spectra, peptides, and the dNSAF (distributed normalized spectra abundance factor) for the indicated co-purifying proteins. (e) βTrCP interacts with Cep68. HEK293T cells were transfected with FLAG-tagged centrosomal proteins (CEPs). Cell lysates were immunoprecipitated with an anti-FLAG resin, and immunoprecipitates were probed with antibodies to βTrCP1. PLK1 and PLK4 are known βTrCP-interacting proteins. Asterisks denote expression of FLAG-tagged CEPs. (f) Different siRNA sequences targeting βTrCP prevent the downregulation of Cep68 in prometaphase, while siRNA targeting Cdc20 does not. HeLa cells were transfected with the indicated siRNA sequences. Prometaphase cells (PM) were harvested by mitotic shake-off after overnight nocodazole treatment. NS, non-synchronized. Please note that Cdc20 silencing synchronizes cells in PM, as shown by the phosphorylation state of Cdc27 (see lane 8).
Supplementary Figure 3 Mapping of the βTrCP degron in Cep68 and identification of PLK1 as the kinase phosphorylating Cep68 degron.
(a) Schematic representation of Cep68 mutants used in mapping experiments. Cep68 mutants found to interact with βTrCP or Cep215 are indicated by the symbol (+). ‘−/+’ denotes reduced binding. (b) Mapping the βTrCP binding region in Cep68 with Cep68 truncation mutants. HEK293T cells were transfected with empty vector (EV), FLAG-tagged Cep68, or the indicated FLAG-tagged Cep68 constructs. Whole cell extracts were immunoprecipitated with an anti-FLAG resin, and immunoprecipitates were immunoblotted with the indicated antibodies. (c) Alignment of the βTrCP binding motif in Cep68 orthologs and previously reported βTrCP substrates. Critical amino acids required for βTrCP binding are highlighted in red. (d) Ser332 is required for binding to βTrCP1. HEK293T cells were transfected with empty vector (EV), FLAG-tagged Cep68, or the indicated FLAG-tagged Cep68 mutants. Cell lysates were immunoprecipitated with an anti-FLAG resin, and immunoprecipitates were probed with the indicated antibodies. (e) PLK1 inhibition prevents Cep68 degradation in prometaphase. HeLa cells expressing FLAG-HA-Cep68 were synchronized by double-thymidine arrest, as in Figure 1b. Where indicated, cells were treated with a PLK1 inhibitor (BI2536), an Aurora kinase inhibitor (VX680), or an Eg5 inhibitor (monastrol) for three hours prior to their collection and analysis by immunoblotting. (f) PLK1 phosphorylates Cep68 on Ser332 in vitro. Recombinant, purified GST-Cep68, GST-Cep68(S332A), or GST alone were incubated with ATP and increasing amounts of purified PLK1. Proteins were detected by immunoblotting as indicated. (g) PLK1 inhibition prevents the in vivo phosphorylation of Cep68 on Ser332. 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) was immunoprecipitated from cell lysates using an anti-FLAG resin. Whole cell lysates (WCL) and immunoprecipitates were immunoblotted as indicated. (h) Analysis of Cep68 binding to PLK1 following BI2536 treatment by Multidimensional Protein Identification Technology. The table lists the total number of spectral counts and the dNSAF values for Cep68 and PLK1 in the untreated and the treated samples.
Supplementary Figure 4 Cep68 degradation does not promote centrosome separation and bipolar spindle assembly.
(a) HA-Cep68 is expressed at near to physiological levels. HeLa cells or HeLa cells stably expressing pBABE-HA-tagged Cep68, Cep68(S332A), or Cep68(Δ331-337) were synchronized by a double-thymidine arrest and harvested at the G1/S transition, in G2 phase (eight hours after release), or in prometaphase (PM). Cell lysates were immunoblotted as shown. (b) Bipolar spindles assemble in the presence of Cep68(S332A). Cells expressing inducible FLAG-tagged Cep68 or Cep68(S332A) were fixed and stained with anti-FLAG and anti-α − tubulin antibodies. Cells in metaphase were analyzed by immunofluorescence. This experiment was reproduced twice. (c) Expression of FLAG-Cep68(S332A) does not prevent centrosome separation in early mitosis. Cells expressing inducible FLAG-tagged Cep68 or Cep68(S332A) were fixed and analyzed by immunofluorescence. The percentage of cells with separated centrosomes in prophase or prometaphase was scored. ≥100 cells were counted for each condition from one experiment. n = cell number. (d) Time spent in mitosis for cells expressing Cep68 or Cep68(S332A). Cells expressing inducible FLAG-tagged Cep68 or Cep68(Δ331-338) were synchronized by double thymidine release. Live cell microscopy was used to calculate time in mitosis (judged from rounded cell morphology). Where indicated, monastrol (50 μm) was added to cells eight hours after release. Bars represent mean ± standard deviation (S.D.). n = 30 cells per condition. ns = not significant. This experiment was performed once. (e) Centrosome disjunction is not inhibited by expression of Cep68(S332A). Cells expressing inducible FLAG-Cep68 or FLAG-Cep68(S332A) were arrested with monopolar spindles in prometaphase with 100 μm monastrol. The distance between γ-tubulin foci was measured using Softworx software. Cep68: n = 28; Cep68(S332A): n = 27. Bars represent mean ± standard deviation (S.D.). ns = not significant.
Supplementary Figure 5 Cep215 levels are reduced at the intercentrosomal linker in Cep68 depleted cells.
(a) Cep68 depletion results in removal of Cep215 from the intercentrosomal linker in interphase. U-2OS cells were transfected for 72 h with siRNAs targeting Cep68 or a non-targeting control sequence. Cells were fixed and stained with antibodies to γ-tubulin (green) and the specified centrosome cohesion factors (red). After Cep68 silencing, only a small amount of Cep215 is observed on interphase centrioles. The graph represents the relative intensity of Cep215 after Cep68 depletion. Bars represent mean ± standard deviation (S.D.). Control siRNA: n = 10 cells; Cep68 siRNA: n = 11 cells. ∗∗∗P = 0.0006. (b) Cep68 knockdown does not affect the total levels of Cep215. U-2OS cells were transfected with siRNAs targeting Cep68 or Cep215. Seventy-two hours post-transfection, cells were lysed for immunoblotting as indicated.
Supplementary Figure 6 The interaction between Cep215 and Cep68 is not mediated by PCNT.
(a) Cep68 interacts with Cep215 and PCNT independently. Cells expressing doxycycline (DOX)-inducible FLAG-tagged Cep68 or Cep68(S332A) were transfected with the indicated siRNA sequences. Cep68 or Cep68(S332A) were immunoprecipitated from whole cell lysates (WCL) with anti-FLAG resin, and both immunoprecipitates and WCLs were immunoblotted with the indicated antibodies. Cep215 interacts with Cep68 when PCNT is depleted in interphase and mitosis. Likewise, PCNT interacts with Cep68(S332A) when Cep215 is depleted. AS, asynchronous. PM, prometaphase. The asterisk denotes a non-specific band. (b) PCNT and PCNT(R2231A) localization in cytokinesis. HeLa cells were transiently transfected with FLAG-tagged PCNT or PCNT(R2231A). Cells in cytokinesis were stained for immunofluorescence using antibodies recognizing FLAG (red) and CP110 (green). Scale bars represent 1 μm.
Supplementary Figure 7 Cep68 degradation is required for centriole separation after disengagement.
Quantification of cells containing 1 c-Nap1 dot in cells expressing Cep68(S332A) using conventional microscopy. HeLa cells expressing HA-tagged Cep68 or Cep68(S332A) were synchronized by double-thymidine arrest and allowed to progress into the next G1 phase (15 hours after release). Where indicated, cells were transfected with Cep215 siRNA during the first release from double-thymidine arrest. G1 cells were fixed and stained for c-Nap1 (green), centrin 2 (red), and Cep68 (α-HA) (blue) to determine their engagement status. The magnification bar represents 1 μm. The areas in the white boxes are shown at higher magnification directly above the corresponding image. The graph shows the quantification of disengagement. G1 cells with 1:2 ratio of c-Nap1:Centrin 2 foci were scored as engaged. n ≥ 100 cells for each experiment from three independent experiments were analysed. ∗∗P ≤ 0.01. Bars represent the mean ± standard deviation (S.D.) from n = three experiments.
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Pagan, J., Marzio, A., Jones, M. et al. Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing. Nat Cell Biol 17, 31–43 (2015). https://doi.org/10.1038/ncb3076
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DOI: https://doi.org/10.1038/ncb3076
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