Fig 1.
Schematic overview of the experimental workflow.
A. An early G1 population was selected from an asynchronous culture of SILAC labelled cells using counter-flow centrifugal elutriation [19] and cultured prior to harvest at various time points, with position in the cell cycle recorded by flow cytometry of DNA content. B. Samples were grouped into recognisable cell cycle time points, and differentially labelled time points were combined with a medium-labelled asynchronous control. The combined sample was processed to tryptic peptides and subjected to Fe-IMAC to separate the peptides from phosphopeptides, which were then processed in parallel. High pH reverse phase separation was used to fractionate the sample, and concatenated to 5 fractions prior to LC-MS/MS analysis. DDA–Data dependent acquisition; MSA–Multi-stage activation.
Fig 2.
Cell cycle regulated phosphorylation sites and proteins.
A. Species ordered in increasing peak time (tpeak) and rendered as a heat map of fold change scaled to the unit integer. B. CCR species projected on a polar plot of peak time tpeak (angle of polar coordinate) versus the maximum fold change FCMax.(distance from the centre). Five phosphorylation sites showing > 100-fold change were excluded for clarity.
Fig 3.
Comparison of cell cycle regulated phosphorylation sites and proteins.
A. Venn diagram of overlap at the protein level (proteins and phosphoproteins). B. Scatter plot of overlap at the phosphorylation site level (proteins versus phosphorylation sites); Grey, not CCR; Purple, phosphorylation site CCR; Orange, protein CCR; Red, protein and phosphorylation site CCR.
Fig 4.
Hierarchical clustering of cell cycle regulated phosphorylation sites.
A. Unrestrained hierarchical clustering was performed using Euclidean distance of the complete linkage, generating 30 clusters (Phos-01 to Phos-30). The relationship between the clusters is rendered as a tree, and the profiles are rendered as a heat map. B. Gene Ontology (GO) enrichment analysis of clusters Phos-01 –Phos-06 (red bars), containing >50% of the CCR phosphorylation sites, identifies GO terms related to cell cycle and mRNA. C. Individual phosphorylation sites profiles by cluster; number of cluster members is given in brackets, and the profiles are coloured by the Euclidean distance from the centre (mean profile).
Fig 5.
Cell cycle regulated protein kinases and RNA binding proteins.
Changes in protein or phosphorylation site abundance relative to EG1. Orange–protein; Purple hues–phosphorylation sites. A. CRK3 protein, Y34 and T33. B. CYC6 protein, S13, S17, S111, S47 and S59. C. WEE1 S101. D. PLK protein, S338 and T469. E. RCK protein and S344. F. MAPK6 proteins and Y176, G. PUF9 protein and S617. H. RMM1 protein and S98. I. SLBP2 S221, T3 and T400. For clarity only CCR phosphorylation sites are shown.
Fig 6.
Phosphorylation of the kinetochore is temporally regulated.
Schematic of predicted kinetochore proteins complexes [38], with CCR phosphorylation sites and proteins rendered as a heat map of fold change scaled to the unit integer. KKT–kinetoplastid kinetochore; KKIP–kinetoplastid kinetochore interacting protein; red–CCR; black–not CCR, grey–not observed.
Fig 7.
Dynamic phosphorylation of the translational machinery.
A. Schematic of eIF4F complexes with CCR phosphorylation sites rendered as a heat map of fold change scaled to the unit integer. The components bound to DED1 have not been identified in T. brucei. B. Cumulative growth curve of tetracycline inducible RNAi of DED1.2 in two independent clones, with qRT-PCR inset confirming ablation of DED1.2 mRNA at 48h. C. Flow cytometry analysis of DNA content of DED1.2 RNAi time course shows accumulation of a sub-G1 population. D. Microscopy of DAPI stained DED1.2 RNAi time course shows disintegration of nuclear integrity.
Fig 8.
Cell cycle regulated PSP1-C terminal domain containing proteins.
Schematic representation of protein showing the PSP1-C domain and CCR phosphorylation sites, with CCR proteins and/or phosphorylation sites rendered as a heat map of Log2 Fold change. CSBPII33 –Tb927.11.7140; CSBPI45 –Tb927.5.760; PCD1 –Tb927.11.14750; PCD2 –Tb927.11.4180; PCD3 –Tb927.10.9910; PCD4 –Tb927.10.9910; PCD5 –Tb927.10.11630; PIE8 –Tb927.6.2850.
Fig 9.
PSP1-C domain containing proteins are CCR but not essential.
Left panel–CCR changes in phosphorylation site and protein abundance relative to early G1 (EG1). Centre panel–Expression profile of tagged proteins in cells synchronised in early G1 by centrifugal elutriation; flow cytometry of PI stained cells for cell cycle position, and corresponding analysis of protein expression by flow cytometry (mNeonGreen) or western blot (HA). Right panel–cumulative cell growth with and without induction of RNAi by addition of tetracycline for two independent cell lines; insets show western blot of HA-tagged proteins and KMX1 tubulin loading control.
Fig 10.
Immunoprecipitation of the T. brucei CSBPII complex proteins.
A. IP of HA-TbCSBPII-45 with anti-HA beads from SILAC labelled cells (M or H) versus parental cells (L), biological replicates with label swap. B. IP of HA-TbCSBPII-33 with anti-HA beads from SILAC labelled cells (M or H) versus parental cells (L), biological replicates with label swap. C. Comparison of IP with HA-TbCSBPII-33 and HA-TbCSBPII-45 using averaged SILAC ratios from biological replicates. Grey–proteins identified in both replicates, Red–ribosomal proteins, Blue—TbCSBPII-45, Green–TbCSBPII-33, Purple–PCD4, Yellow–PABP2.