Figure 1.
A stylised representation of the ePK complement in T. brucei. Protein kinases are grouped together into families defined by amino acid sequence similarities [51]. The node arrangements and lengths of the branches do not have any phylogenetic significance, and are for illustrative purposes only. Red nodes denote PKs, which, when depleted in this study, resulted in a loss of fitness phenotype.
Figure 2.
Protein kinases involved in the cell cycle.
In vitro growth and cell cycle analysis for ATR (A) and CLK1 (B). A: Upper left: growth curves showing cumulative cell counts over time following tetracycline (Tet) induction (+) or not (−) of RNAi cell line in culture. Cell densities were maintained between 105 and 106 cells ml−1. Upper right: DAPI staining. Cells were stained with DAPI and the number of nuclei (N) and kinetoplasts (K) per cell was quantified (n>200) at the time-points indicated. Lower panel: flow cytometry profiles for 50,000 propidium iodide labelled cells at 24 hr or 48 hr post-induction. The DNA content of each peak is indicated. B: legend as for A, but including in upper right panel an analysis of the cytokinesis stage of 2N2K cells following tetracycline (Tet) induction (+) or not (−) of CLK1 RNAi cell line in culture. Lower left panels: flow cytometry profiles at 6 and 12 hours post-induction. Lower right: proliferation of CLK1 RNAi line in mice. 1×105 trypanosomes were inoculated in 4 mice and RNAi was induced with doxycycline (Dox, as indicated) in 2 mice 48 hr later. Uninduced mice were culled as indicated () when their parasitaemias rose above 108 cells ml−1.
Table 1.
Summary of the characteristics of RNAi cell lines displaying loss of fitness phenotypes as determined by Alamar Blue assay and growth curves.
Figure 3.
RDK1 is a repressor of BSF to PCF differentiation.
A: RDK1 RNAi was induced for 72 hours before cells were analysed by (immuno)fluorescence microscopy with DAPI (to stain nuclei and kinetoplasts) and FITC-conjugated anti-EP procyclin. Left panel: DIC images; right panel: merged fluorescence channels - DAPI (blue), EP-procyclin (green). Scale bar: 10 µm. B: quantification of EP procyclin-expressing cells by flow cytometry over time following induction of RDK1 RNAi. C: Growth curves showing cumulative cell counts over time following tetracycline (Tet) induction (+) or not (−) of RDK1 RNAi in culture. Cell densities were maintained between 105 and 106 cells ml−1. Error bars indicate the standard deviations around the means of three technical replicates. Inset: Analysis of RDK1 protein knockdown following RNAi induction. RDK1 RNAi cell line clone 1 expressing RDK1::12Myc from the endogenous locus was analysed by Western blotting with an anti-Myc antibody 24 hr after RNAi induction. Anti-EF1α antibody was used as a loading control. D: predicted domain structure of RDK1. TM: transmembrane domain. E: cell fractionation. Cells expressing RDK1::12myc were fractionated and analysed by Western blotting with anti-Myc antibody to detect RDK1::12myc and anti-OPB, anti-β-tubulin and anti-MCA4 antibodies as cytoplasmic, cytoskeletal and membrane protein controls, respectively. T: total cell lysate; DS: detergent-soluble; DP: detergent-pellet; HS: hypotonic soluble; HP: hypotonic pellet. F: immunofluorescence of RDK1::12Myc expressed in an RDK1 RNAi background. Cells were induced (+) or not (−) with tetracycline (Tet) for 22 hr and fixed, permeabilised and labelled with DAPI (blue) and an anti-Myc antibody (green). Left: DIC image; right: DAPI/Myc merge. Scale bar: 5 µm.
Figure 4.
Analysis of differentiation during RDK1 RNAi.
A: qRT-PCR analysis of PAD1 mRNA expression after 24 hr treatment of uninduced RDK1 RNAi cells with 250 µM 8-pCPT-cAMP. B–D: quantification of EP procyclin-expressing cells by flow cytometry following induction of RDK1 RNAi. RNAi of RDK1 was induced for 24, 48 and 72 hr by addition of tetracycline (Tet) and the percentage of EP-procyclin positive cells detected by flow cytometry after treatment with 250 µM 8-pCPT-cAMP (B), cold shock treatment of cells at 27°C (C) or incubation with 150 µM BZ3 (D) for the final 24 hr of each RNAi induction.
Figure 5.
RDK2 is a repressor of BSF to PCF differentiation.
A: Predicted domain structure of RDK2. PH: pleckstrin homology domain. B: Growth curves showing cumulative cell counts over time following tetracycline (Tet) induction (+) or not (−) of RDK2 RNAi in culture. Cell densities were maintained between 105 and 106 cells ml−1. Error bars indicate the standard deviations around the means of three technical replicates. Inset: qRT-PCR analysis of RDK1 mRNA knockdown following RNAi induction. C: quantification of EP procyclin-expressing cells by flow cytometry following induction of RDK2 RNAi. RNAi of RDK2 was induced for 24 and 48 hr by addition of tet and the percentage of EP-procyclin positive cells detected by flow cytometry after treatment with 250 µM 8-pCPT-cAMP (D), cold shock treatment of cells at 27°C (E) or incubation with 150 mM BZ3 (F) for the final 24 hr of each RNAi induction.
Figure 6.
Schematic representation of the BSF trypanosome cell cycle and differentiation from BSF to PCF.
Protein kinases implicated in cell cycle control and differentiation are indicated (red font: cell cycle function identified in this study; black font: cell cycle function previously identified).