DOI: 10.1038/ncb3076 Supplementary Figure 1 btrcp 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 hours post-transfection. Cells were fixed and analyzed by immunofluorescence using Cep68 antibodies (red) and g-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 btrcp. HeLa cells stably expressing FLAG-HA-Cep68 were transfected with btrcp 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) btrcp 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 btrcp1. PLK1 and PLK4 are known btrcp-interacting proteins. Asterisks denote expression of FLAG-tagged CEPs. f) Different sirna sequences targeting btrcp prevent the downregulation of Cep68 in prometaphase, while sirna targeting Cdc 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 Cdc silencing synchronizes cells in PM, as shown by the phosphorylation state of Cdc27 (see lane 8). WWW.NATURE.COM/NATURECELLBIOLOGY 1 14 Macmillan Publishers Limited. All rights reserved.
Supplementary Figure 2 Mapping of the btrcp 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 btrcp or Cep215 are indicated by the symbol (+). -/+ denotes reduced binding. b) Mapping the btrcp 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 btrcp binding motif in Cep68 orthologs and previously reported btrcp substrates. Critical amino acids required for btrcp binding are highlighted in red. d) Ser332 is required for binding to btrcp1. 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 (BI36), 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 BI36 (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 BI36 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. 2 WWW.NATURE.COM/NATURECELLBIOLOGY 14 Macmillan Publishers Limited. All rights reserved.
Supplementary Figure 3 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(D331-3) 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-a-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. 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 ( 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 µm monastrol. The distance between g-tubulin foci was measured using Softworx software. Cep68: n=28; Cep68(S332A): n=27. Bars represent mean ± standard deviation (S.D.). ns=not significant. WWW.NATURE.COM/NATURECELLBIOLOGY 3 14 Macmillan Publishers Limited. All rights reserved.
Supplementary Figure 4 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 hours with sirnas targeting Cep68 or a non-targeting control sequence. Cells were fixed and stained with antibodies to g-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. 4 WWW.NATURE.COM/NATURECELLBIOLOGY 14 Macmillan Publishers Limited. All rights reserved.
Supplementary Figure 5 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. WWW.NATURE.COM/NATURECELLBIOLOGY 5 14 Macmillan Publishers Limited. All rights reserved.
Supplementary Figure 6 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 doublethymidine arrest. G1 cells were fixed and stained for c-nap1 (green), centrin 2 (red), and Cep68 (a-ha) (blue) to determine their engagement status. The magnification bar represents 1 mm. 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 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. 6 WWW.NATURE.COM/NATURECELLBIOLOGY 14 Macmillan Publishers Limited. All rights reserved.
Figure 1a Figure 1b Figure 1d Figure 1e Cep68 HA 2 1 (Fbxo11 long exp.) βtrcp Cep68 15 phh3(ser10) γ-tubulin 2 Cep215 βtrcp1 2 Cep215 Skp1 15 phh3(ser10) 2 1 Fbxo11 (βtrcp short exp.) CdcA Fbxo5 Cdc27 2 1 Fbxo18 2 1 FLAG Skp1 Supplementary Figure 7 Scans of immunoblots used in the figures. WWW.NATURE.COM/NATURECELLBIOLOGY 7 14 Macmillan Publishers Limited. All rights reserved.
Figure 2a Figure 2c Figure 2d FLAG Cep68 p-cep68(s332a) (IP) γ-tubulin PLK1 FLAG (IP) phh3 (Ser10) phh3(ser10) Nek2 FLAG (WCL) βtrcp α-tubulin (WCL) Figure 2b 2 Cep68 (IP) Cep215 (IP) phh3 (Ser10) Cdc27 (WCL) Centrin 3 (IP) γ-tubulin Cep68 (WCL) 2 Cep215 (WCL) Centrin 3 (WCL) 8 WWW.NATURE.COM/NATURECELLBIOLOGY 14 Macmillan Publishers Limited. All rights reserved.
Figure 5b 1 FLAG (IP) 2 1 Cep215 (IP) 2 PCNT (IP) γ-tubulin(ip) 1 FLAG (WCL) 2 1 Cep215 (WCL) 2 1 PCNT (WCL) γ-tubulin (WCL) 15 PHH3 (Ser10) (WCL) WWW.NATURE.COM/NATURECELLBIOLOGY 9 14 Macmillan Publishers Limited. All rights reserved.
Figure S1b Figure S1c HA HA γ-tubulin phh3(ser10) phh3(ser10) γ-tubulin βtrcp1 CdcA Figure S1e Figure S1f βtrcp Cep68 CdcA 2 1 βtrcp1 FLAG Cdc Cdc27 Skp1 10 WWW.NATURE.COM/NATURECELLBIOLOGY 14 Macmillan Publishers Limited. All rights reserved.
Figure S2b Figure S2d Figure S2e βtrcp1 2 1 βtrcp1 HA phh3 (Ser10) 2 Cep215 FLAG 2 1 FLAG Figure S2g Centrin 3 Figure S2f 1 FLAG (IP) 1 p-cep68 (IP) 1 p-cep68 1 FLAG (WCL) α-tubulin 1 GST PHH3(Ser10) WWW.NATURE.COM/NATURECELLBIOLOGY 11 14 Macmillan Publishers Limited. All rights reserved.
Figure S3a Figure S4b Figure S5b 1 Cep68 Cep68 2 Cep215 (IP) 2 Cep215 PCNT (IP) 1 HA 2 PCNT FLAG (IP) Cdc27 γ-tubutlin 2 Cep215 (WCL) PCNT (WCL) α-tubulin γ-tubutlin (WCL) Cdc27 12 WWW.NATURE.COM/NATURECELLBIOLOGY 14 Macmillan Publishers Limited. All rights reserved.
Table 1. Identification of Cep68 phosphorylation sites in vivo by mass spectrometry. Residue and modification Untreated PLK inhibition Surrounding Sequence T106 1/664 0/972 S G L P P A T M G S G D L S109 17/664 17/972 P P A T M G S G D L L L S S126 1/128 3/286 T K L S S S E E F P Q T S136 0/128 4/286 L S S S E E F P Q T L S L P R S186 0/279 1/482 S V L S P G S A A Q P S S C S S249 0/61 8/113 G L G P R P Q W S P Q P V F S G S268 3/ 6/40 R R L S F Q A E Y W A C V L P D Y273 0/ 8/40 R R L S F Q A E Y W A C V L P D S S285 0/ 5/40 C V L P D S L P P S P D R S332 1/147 0/215 Q D S G V D L D S F S V S P A S T S340 0/147 2/215 Q D S G V D L D S F S V S P A S T S342 9/147 12/215 Q D S G V D L D S F S V S P A S T S354 3/3 6/188 S P T N V S P N C P P A E A T S4 0/144 1/89 S G P R E P S L K Q W P S R S435 2/419 8/4 H L D M G S P Q L R S472 4/ 2/47 W K S E E E V E S D D E Y L A S478 23/ 32/47 W K S E E E V E S D D E Y L A Table 1 Identification of Cep68 phosphorylation sites in vivo by mass spectrometry. FLAG-tagged Cep68 was immunoprecipitated from HEK293T cells that were either untreated or treated with BI36 (a PLK1 inhibitor) for 16 hours. Note that PLK1 inhibition results in prometaphase arrest. The table lists the number of spectra matching phosphorylated peptides as a fraction of total spectra detected for peptides bearing each residue and their corresponding modification percentage. Ser332 was identified as a phosphorylated residue in untreated cells, but not in cells treated with BI36. WWW.NATURE.COM/NATURECELLBIOLOGY 13 14 Macmillan Publishers Limited. All rights reserved.