Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei

Abstract

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

Figure 1. Identification of CRK12 as a CYC9 interaction partner using tandem affinity purification.

A. Construction of procyclic CYC9:TAP cell line. CYC9 alleles were replaced sequentially with a neomycin resistance (NEO) cassette and a CYC9:TAP cassette, which included a blasticidin resistance marker (BSD) for selection, thus generating a cell line expressing CYC9:TAP in a CYC9 null mutant background. Light grey boxes: CYC9 UTR sequences; white boxes: tubulin intergenic sequences. PCR (reactions a-e, sizes and positions of products amplified indicated by black bars) was used to verify correct integration of the cassettes. Reactions (a) and (b) (panel (i)), were used to confirm correct integration of the NEO cassette in the single allele CYC9 knockout line; negative control (–): procyclic 427 wildtype. Reaction (c) (panel (ii)) was used to confirm that a wildtype copy of CYC9 no longer existed in the CYC9:TAP cell line; positive control (+): procyclic 427 wildtype cell line transfected with CYC9:TAP cassette. Reactions (d) and (e) (panel (iii)), were used to confirm correct integration of the CYC9:TAP cassette at its 5’ and 3’ ends; positive and negative controls as for (c) and (b), respectively. B. Western blots probed with anti-CBP (left), anti-protein A (right, upper blot) and EF1α (loading control; right, lower blot) antibodies. Lane 1: procyclic 427 transfected with NEO cassette; lane 2: as for lane 1 but also transfected with CYC9:TAP construct. Expected size of CYC9:TAP is 51.6 kDa. Asterisk: degradation product; arrowhead: cross-reacting band that serves as a loading control. C. (i) Immunofluorescence analysis of procyclic CYC9:TAP cell line. Top left: DIC; top right: DAPI; bottom left: anti-protein A (CYC9:TAP); bottom right: DAPI/anti-protein A merge. (ii) Wildtype control. Top: DIC; bottom: DAPI/anti-protein A merge. The configuration of nuclei (N) and kinetoplasts (K) per cell is given. Scale bar: 5 µm. D. Western blot of CYC9:TAP purification, probed with anti-CBP. In: input to the IgG column; FT: flow through; W: washes; E: elution fractions. Expected size of CYC9:CBP (after cleavage of CYC9:TAP from the IgG column with TEV protease) is 35.5 kDa. Asterisk: degradation product.

Figure 2. Co-immunoprecipitation of CRK12 and CYC9.

A. Schematic showing features of procyclic cell lines generated. B–D. Immunoprecipitation (IP) was performed with (B) an irrelevant antibody (anti-GST), (C) anti-TY antibody (for tyGFP:CRK12) and (D) anti-rabbit IgG (for CYC9:TAP). Samples (In: input; FT: flow through; W1: first wash; W4: last wash; E: elution) were analysed by Western blotting with anti-GFP to detect tyGFP:CRK12, anti-PAP to detect CYC9:TAP and anti-oligopeptidase B (anti-OPB) to act as a loading control for the input fractions and control for the stringency of the purification.

Figure 3. CRK12 is an active protein kinase.

A. Anti-TY immunoprecipitates from procyclic cell lysates (1: 427 wildtype; 2: 427 tyGFP:CRK12; 3: 427 CYC9:TAP; 4: 427 tyGFP:CRK12 CYC9:TAP, see Fig. 2A) were subjected to a radiolabelled in vitro kinase assay and analysed by SDS-PAGE and autoradiography. Lane 5: kinase assay buffer only. Bands likely representing phosphorylated tyGFP:CRK12 (113.5 kDa) are indicated. B: Western blot of 427 cell lysates overexpressing ty:CRK12 (active) or kinase dead ty:CRK12 (K358M) in response to tetracycline (tet) induction. A 427 wildtype cell lysate is included as a negative control. Western blots were probed with anti-TY antibody to detect ty:CRK12 or anti-OPB as a loading control, as indicated. C. Immunoprecipitation of ty:CRK12 performed using anti-TY beads and 427 cell lysates overexpressing either ty:CRK12 (active) or kinase dead ty:CRK12 (K358M). 427 wildtype lysates were included as a non-specific binding control. Western blots (left) were performed on the input (In), flow through (FT) and elution (E) fractions with anti-TY antibody to detect ty:CRK12 or with anti-OPB antibody as a control for non-specific binding to the beads, as indicated. Immunoprecipitates were then subjected to a radiolabelled kinase assay (autoradiograph, right). 1: 427 tyGFP:CRK12; 2: 427 wt; 3: 427 ty:CRK12 (active); 4: 427 ty:CRK12 K358M. Bands likely representing phosphorylation of tyGFP:CRK12 (113.5 kDa) or ty:CRK12 (86 kDa) are indicated.

Figure 4. RNAi of CYC9 reveals it is essential for viability in procyclic and bloodstream form trypanosomes.

A. Cumulative growth curves for two independent procyclic CYC9 RNAi clones (clone 1: C1; clone 2: C2) cultured in the presence or absence of tetracycline (tet). B. Real time PCR analysis of CYC9 transcript for procyclic CYC9 RNAi clones 1 and 2 at 48 hours post-induction. Error bars represent standard deviations of three replicates. The percentage of mRNA transcript remaining is indicated. C. Cumulative growth curves for two independent bloodstream CYC9 RNAi clones (clone 1: C1; clone 2: C2) cultured in the presence or absence of tetracycline (tet). D. Real time PCR analysis of CYC9 transcript for bloodstream form CYC9 RNAi clones 1 and 2 at 20 hours post-induction. Error bars represent standard deviations of three replicates.

Figure 5. Depletion of CYC9 in the bloodstream form inhibits cytokinesis.

A. DAPI staining of nuclei (N) and kinetoplasts (K) at the time points indicated (post-induction). >200 cells per time point were classified according to their N/K configuration. B. Cytokinesis stage analysis of 2N2K cells. 2N2K cells (n > 200/timepoint) were scored by differential interference contrast (DIC) microscopy for whether or not they had a visible cleavage furrow (furrow ingression) or were undergoing abscission. Error bars represent the standard deviations from three replicate experiments. C. Flow cytometry analysis of propidium iodide stained cells at the time points indicated (in hours). The ploidies of the peaks are indicated. D. Example images of multinucleate/kinetoplast cells. Left panels: DIC image; right panels: DAPI staining. The N/K configuration of each cell is indicated. Scale bars: 5 µm. Arrows indicate partially ingressed cleavage furrows.

Figure 6. CRK12 is essential for viability in the bloodstream form.

A. Cumulative growth curves for two CRK12 RNAi clones (C1 and C2) cultured in the presence or absence of tetracycline (tet). B. Real time PCR analysis of CRK12 transcript at 18 hours post-induction (% mRNA transcript remaining is indicated). Error bars: standard deviations of four replicates. C. Western blot analysis of CRK12 protein depletion following RNAi induction. Cell lysates (10⁶ cell equivalents/lane) of CRK12 RNAi clone 1 at 0, 12 and 24 hours post-induction were analysed by Western blotting with anti-CRK12 antibody (upper blot). CRK12/ty:CRK12 (85/86 kDa) is indicated. To demonstrate anti-CRK12 monoclonal antibody specificity, procyclic (PCF) and bloodstream form (BSF) cell lysates of 427 wildtype (WT) and 427 pHD449 pHG230 (ty:CRK12 inducible overexpression cell line, OE, induced (+) or not (–) with tetracycline for 24 (bloodstream form) or 48 (procyclic form) hours) were also blotted. As a loading control, blots were probed with anti-EF1α antibody (lower blot). D. Growth curves of CRK12 RNAi cell lines in a mouse model. Mice were inoculated intraperitoneally with 5×10⁵ parasites on day 0; mice 3 and 4 were provided with doxycycline in their drinking water at day 2 (indicated with arrow) to induce the RNAi. Mice 1 and 2 were euthanised on day 3 due to their high parasitaemias. E. DAPI staining of nuclei (N) and kinetoplasts (K) at the time points indicated for CRK12 RNAi clones 1 and 2 induced or not with tetracycline (tet). >200 cells per time point were classified according to their N/K configuration.

Figure 7. Depletion of CRK12 in bloodstream form T. brucei results in a defect in endocytosis.

A and B. Quantification of 1N2K and 2N2K cells, respectively, with normal and abnormal kinetoplast positioning at 12 and 18 hours post-induction with tetracycline (tet). No cells were observed to have abnormal kinetoplast positioning at 0 hours. n >200 cells/time point. C. Visualising enlarged flagellar pockets and mispositioned kinetoplasts. Uninduced cells are shown at the top for comparison. Example images of induced cells with abnormally enlarged flagellar pocket regions (one example indicated by arrow) are shown below. From left to right: DIC image, DAPI image, DIC/DAPI merge. The N/K configuration of each cell is indicated. Note the lateral positioning of the 2 kinetoplasts in some induced cells (indicated by asterisks) compared to the longitudinal positioning in the uninduced cells. Scale bar: 10 µm. D. Example images of cells in abscission with enlarged flagellar pocket regions. From left to right: DIC image, DAPI image, DIC/DAPI merge. Scale bar: 10 µm. E. Transmission electron microscopy (TEM) images of flagellar pockets in CRK12 RNAi cells induced (+) or not (–) with tetracycline (tet) (t = 18 hrs). A: axoneme; BB: basal body; FP: flagellar pocket; K: kinetoplast. Scale bars: 500 nm. F. Quantification of 1N2K (top) and 2N2K (bottom) cells with normal and enlarged flagellar pockets (FP) at 12 and 18 hours post-induction with tetracycline (tet). No cells were observed to have enlarged flagellar pockets at 0 hours. n >200 cells/time point.

Figure 8. CRK12 depleted bloodstream form T.brucei exhibit defective FM4-64 uptake and receptor-linked endocytosis.

A. FM4-64 uptake assay at 4°C and 37°C for CKR12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 18 hours. For each pair of images: left: DIC images; right: DAPI (white)/FM4-64 (red) merge. Two sets of + tet images are shown: those without enlarged flagellar pockets (centre panels) and those with enlarged flagellar pockets (right panels, as indicated by arrows). B. Clathrin heavy chain (CHC) immunofluorescence analysis of CRK12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 12 hours. Left: DIC images; right: DAPI (white)/CHC (green). Induced cells exhibiting normal (upper panels) and enlarged (lower panels, as indicated by arrow) flagellar pockets are shown. C. AF594-transferrin uptake assay at 37°C for CKR12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 18 hours. Left: DIC images; right: DAPI (white)/AF594-transferrin (red) merge. Scale bars: 10 µm. D. Bioluminescent intracellular ATP assay for 427 wildtype and CRK12 RNAi cells (induced or not with tetracycline (tet)). Assays were performed in quadruplicate and the luminescence obtained was averaged and normalised to the wildtype control. Error bars show the standard deviations.