Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
Figure 6

Fig. 6 Mitotic cyclin B-dependent phosphorylation sites

(A) Cell synchronisation scheme: to obtain an optimal enrichment in mitotic cells we pre-synchronised B1dd/B2ko cells by serum starvation for 48 hours followed by a release into Thymidine for 24 hours. Cells were then released into S-phase in the presence or absence of DIA and blocked in mitosis by the addition of proTAME. Mitotic cells were collected by shake-off 12 hours following the release.

(B) Phosphoproteomics workflow. Protein extracts were prepared from the indicated cells, digested with trypsin and labelled with 6-plex TMT reagents. The TMT-labelled peptides were then mixed and subjected to fractionation by high pH reverse phase into 24 fractions. From each fraction, 95% was taken for phospho-enrichment and 5% retained for analysis of unmodified peptides. For phospho-enrichment, the 24 fractions were combined into a total of 16 fractions. Samples (unmodified and phospho-enriched) were then analysed by LC-MS/MS on an Orbitrap Fusion instrument using synchronous precursor selection (SPS). This analysis resulted in detection of 11,234 phosphorylation sites, of which 5,670 were quantitated. The number of class I sites are indicated in brackets (site localisation probability > 0.75).

(C) Quantification of cyclin B specific substrates by phospho-proteomics. Phosphorylation sites deemed significantly changing are shown in red (p 0.05, 1.96 * standard deviation fold change cut-offs, see Methods).

(D) Gene ontology analysis of cyclin B-dependent substrates.

(E) Quantification of changes in selected cyclin B-dependent mitotic phosphorylations including Ki-67 (left panel), INCENP/Borealin (middle panel) and Top2B, CDCA2 and Tpx2.

(F) Representative images of Ki-67 immunofluorescent staining of chromosome spreads from control (Ctr) and DIA treated P/A synchronised B1dd/B2ko cells and quantification of Ki-67 staining intensity using cross-sections of chromosomes (Data from three repeats, n=50, shaded area indicates standard deviation, scale bar = 5µm).

(G) Representative images from live-cell imaging of DIA-treated SiR-Tubulin labelled (red) B1dd/B2ko cells expressing FusionRed-H2B (green) and AurB-GFP (white); time is indicated in minutes, scale bar = 5µm. Bottom panel shows frequencies of aberrant AurB localisation in P/A synchronised B1dd/B2ko cells. (Bars indicate mean of three repeats, n=50 cells per repeat, stdv is indicated by error bars).

(H) CDCA2 is excluded from mitotic chromosomes in control cells while this exclusion is lost in DIA treated B1dd/B2ko cells. The upper panel shows immunofluorescence images, for quantification we analysed fluorescence intensity across chromosomes. The data show the mean of 150 cells (from three repeats n=50) and error bars indicate standard deviation.

(I) Top2B levels in mitotic cells are increased in DIA treated B1dd/B2ko cells. The left panel shows immunofluorescence images, for quantification we segmented mitotic DAPI staining, measured the mean intensity of Top2B staining and plotted the ratio between mitotic and interphase nuclei. (Bars indicate median, first and third quartile, data are from three repeats, n=50 cells per repeat.)

(J) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd/B2ko cells. The top panel shows immunofluorescence images, for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell. (Bars indicate median, first and third quartile, data are from three repeats, n=50 cells per repeat). We also analysed the centrosome/spindle distribution of AurA that does not change following DIA treatment.

Data information: For images in (H-J) the scale bar equals 10µm.