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Nuclear DBF-2-related kinases are essential regulators of cytokinesis in bloodstream stage Trypanosoma brucei.

Ma J, Benz C, Grimaldi R, Stockdale C, Wyatt P, Frearson J, Hammarton TC - J. Biol. Chem. (2010)

Bottom Line: Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited.Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo.Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection & Immunity, Faculty of Biomedical and Life Sciences and Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

ABSTRACT
Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

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Characterization of bloodstream stage PK53 RNAi cell lines. A, nucleus and kinetoplast configurations of cells over time following induction, as revealed by DAPI staining and fluorescence microscopy (n >200 per time point). N, nucleus; K, kinetoplast. Other cell types predominantly include cells with >2N and >2K. B, characterization of cytokinesis stage of 2N2K cells from RNAi cell lines cultured in the presence or absence of tetracycline (tet) at 10 h post-induction. C, example images of multinucleate and multikinetoplast cells present in induced PK53 RNAi cell lines 15 h post-induction. Left panels, differential interference contrast images; right panels, DAPI images. D, flow cytometry profiles of propidium iodide-stained cells over time after PK53 RNAi induction. The x axis shows fluorescence intensity in the FL2-A channel. Time points and ploidy of peaks are indicated.
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Figure 3: Characterization of bloodstream stage PK53 RNAi cell lines. A, nucleus and kinetoplast configurations of cells over time following induction, as revealed by DAPI staining and fluorescence microscopy (n >200 per time point). N, nucleus; K, kinetoplast. Other cell types predominantly include cells with >2N and >2K. B, characterization of cytokinesis stage of 2N2K cells from RNAi cell lines cultured in the presence or absence of tetracycline (tet) at 10 h post-induction. C, example images of multinucleate and multikinetoplast cells present in induced PK53 RNAi cell lines 15 h post-induction. Left panels, differential interference contrast images; right panels, DAPI images. D, flow cytometry profiles of propidium iodide-stained cells over time after PK53 RNAi induction. The x axis shows fluorescence intensity in the FL2-A channel. Time points and ploidy of peaks are indicated.

Mentions: To determine whether the growth defect upon PK50 or PK53 depletion occurred as a result of a cell cycle defect, and whether these kinases play roles in cytokinesis like MOB1, cell cycle progression following RNAi induction was analyzed by fluorescence microscopy of DAPI-stained cells to monitor the number of nuclei and kinetoplasts per cell and flow cytometry of propidium iodide-stained cells to monitor their ploidy. DAPI staining is a useful tool to monitor cell cycle progression because trypanosomes contain a single mitochondrion whose DNA is organized into a disc termed the kinetoplast; the kinetoplast replicates and divides ahead of the nucleus allowing the classification of cell cycle stage for individual cells (48). The results for both clones for the PK50 and PK53 RNAi cell lines were very similar, and hence only data obtained for clone 1 is presented here. Following depletion of either kinase, DAPI staining revealed an initial increase in cells with two nuclei and two kinetoplasts (2N2K cells), with a concomitant decrease in 1N1K and 1N2K cells (Figs. 2A and 3A). Following PK50 RNAi, 2N2K cells, which constituted around 10% of the population in uninduced cells, rose to over 25% of the population at 9 h post-induction (Fig. 2A). Further examination revealed that nearly 80% of these 2N2K cells had not yet commenced cytokinesis furrowing, around 15% were in the process of furrowing, and only ∼5% were in the final stages of cytokinesis (abscission) (Fig. 2B). In uninduced cultures, the distribution was notably different, with around 60% 2N2K cells yet to commence furrowing, ∼15% with a visible cleavage furrow, and 30% cells having reached abscission (Fig. 2B). This suggests that onset of cytokinesis furrowing is delayed following depletion of PK50. At later time points, cells with >2 nuclei and >2 kinetoplasts were generated (Fig. 2, A, Other cell types, and C), but very few zoids (0N1K cells) or 2N1K cells were formed. Flow cytometry profiles of these cells showed that cells re-replicated their nuclei and kinetoplasts, as demonstrated by the appearance of 6C and 8C peaks following induction (Fig. 2D). These data strongly suggest that PK50 is essential for initiation of cytokinesis, and following its depletion, the delay in entering cytokinesis allows cells to re-replicate their DNA, leading to cell cycle deregulation.


Nuclear DBF-2-related kinases are essential regulators of cytokinesis in bloodstream stage Trypanosoma brucei.

Ma J, Benz C, Grimaldi R, Stockdale C, Wyatt P, Frearson J, Hammarton TC - J. Biol. Chem. (2010)

Characterization of bloodstream stage PK53 RNAi cell lines. A, nucleus and kinetoplast configurations of cells over time following induction, as revealed by DAPI staining and fluorescence microscopy (n >200 per time point). N, nucleus; K, kinetoplast. Other cell types predominantly include cells with >2N and >2K. B, characterization of cytokinesis stage of 2N2K cells from RNAi cell lines cultured in the presence or absence of tetracycline (tet) at 10 h post-induction. C, example images of multinucleate and multikinetoplast cells present in induced PK53 RNAi cell lines 15 h post-induction. Left panels, differential interference contrast images; right panels, DAPI images. D, flow cytometry profiles of propidium iodide-stained cells over time after PK53 RNAi induction. The x axis shows fluorescence intensity in the FL2-A channel. Time points and ploidy of peaks are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2865264&req=5

Figure 3: Characterization of bloodstream stage PK53 RNAi cell lines. A, nucleus and kinetoplast configurations of cells over time following induction, as revealed by DAPI staining and fluorescence microscopy (n >200 per time point). N, nucleus; K, kinetoplast. Other cell types predominantly include cells with >2N and >2K. B, characterization of cytokinesis stage of 2N2K cells from RNAi cell lines cultured in the presence or absence of tetracycline (tet) at 10 h post-induction. C, example images of multinucleate and multikinetoplast cells present in induced PK53 RNAi cell lines 15 h post-induction. Left panels, differential interference contrast images; right panels, DAPI images. D, flow cytometry profiles of propidium iodide-stained cells over time after PK53 RNAi induction. The x axis shows fluorescence intensity in the FL2-A channel. Time points and ploidy of peaks are indicated.
Mentions: To determine whether the growth defect upon PK50 or PK53 depletion occurred as a result of a cell cycle defect, and whether these kinases play roles in cytokinesis like MOB1, cell cycle progression following RNAi induction was analyzed by fluorescence microscopy of DAPI-stained cells to monitor the number of nuclei and kinetoplasts per cell and flow cytometry of propidium iodide-stained cells to monitor their ploidy. DAPI staining is a useful tool to monitor cell cycle progression because trypanosomes contain a single mitochondrion whose DNA is organized into a disc termed the kinetoplast; the kinetoplast replicates and divides ahead of the nucleus allowing the classification of cell cycle stage for individual cells (48). The results for both clones for the PK50 and PK53 RNAi cell lines were very similar, and hence only data obtained for clone 1 is presented here. Following depletion of either kinase, DAPI staining revealed an initial increase in cells with two nuclei and two kinetoplasts (2N2K cells), with a concomitant decrease in 1N1K and 1N2K cells (Figs. 2A and 3A). Following PK50 RNAi, 2N2K cells, which constituted around 10% of the population in uninduced cells, rose to over 25% of the population at 9 h post-induction (Fig. 2A). Further examination revealed that nearly 80% of these 2N2K cells had not yet commenced cytokinesis furrowing, around 15% were in the process of furrowing, and only ∼5% were in the final stages of cytokinesis (abscission) (Fig. 2B). In uninduced cultures, the distribution was notably different, with around 60% 2N2K cells yet to commence furrowing, ∼15% with a visible cleavage furrow, and 30% cells having reached abscission (Fig. 2B). This suggests that onset of cytokinesis furrowing is delayed following depletion of PK50. At later time points, cells with >2 nuclei and >2 kinetoplasts were generated (Fig. 2, A, Other cell types, and C), but very few zoids (0N1K cells) or 2N1K cells were formed. Flow cytometry profiles of these cells showed that cells re-replicated their nuclei and kinetoplasts, as demonstrated by the appearance of 6C and 8C peaks following induction (Fig. 2D). These data strongly suggest that PK50 is essential for initiation of cytokinesis, and following its depletion, the delay in entering cytokinesis allows cells to re-replicate their DNA, leading to cell cycle deregulation.

Bottom Line: Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited.Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo.Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection & Immunity, Faculty of Biomedical and Life Sciences and Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

ABSTRACT
Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

Show MeSH
Related in: MedlinePlus