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A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

Guttery DS, Ferguson DJ, Poulin B, Xu Z, Straschil U, Klop O, Solyakov L, Sandrini SM, Brady D, Nieduszynski CA, Janse CJ, Holder AA, Tobin AB, Tewari R - PLoS Pathog. (2012)

Bottom Line: Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes.Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant.This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology Queens Medical Centre, University of Nottingham, Nottingham, UK.

ABSTRACT
Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

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Global phosphorylation in Δcdc20 and Δmap2 lines.Gametocytes from wild type, Δmap2 and Δcdc20 parasites were purified on 48% Nycodenz and activated for 30 mins in ookinete medium before addition of 32P-orthophosphate for 30 mins. After washing, labelled activated gametocytes were lysed with NP40 and fractionated using anion exchange chromatography on an AKTA system. Individual fractions were then further resolved by SDS-PAGE and labelled bands detected by autoradiography. The Coomassie blue stained gel shows that protein loading was similar between lanes. Several differences in the 32P signal (indicated by arrows) are observed between the three different parasites. Bands C, D, F, G H and J indicate altered phosphorylation status only in the Δmap2 mutant. Bands A, E and I indicate changes only in the Δcdc20 mutant. Only one band (band B) showed a similar change in both the Δmap2 mutant and Δcdc20 mutant.
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ppat-1002554-g007: Global phosphorylation in Δcdc20 and Δmap2 lines.Gametocytes from wild type, Δmap2 and Δcdc20 parasites were purified on 48% Nycodenz and activated for 30 mins in ookinete medium before addition of 32P-orthophosphate for 30 mins. After washing, labelled activated gametocytes were lysed with NP40 and fractionated using anion exchange chromatography on an AKTA system. Individual fractions were then further resolved by SDS-PAGE and labelled bands detected by autoradiography. The Coomassie blue stained gel shows that protein loading was similar between lanes. Several differences in the 32P signal (indicated by arrows) are observed between the three different parasites. Bands C, D, F, G H and J indicate altered phosphorylation status only in the Δmap2 mutant. Bands A, E and I indicate changes only in the Δcdc20 mutant. Only one band (band B) showed a similar change in both the Δmap2 mutant and Δcdc20 mutant.

Mentions: In order to examine whether or not CDC20 has a role in pathways of protein phosphorylation similar to those of the kinase MAP2, we compared the global phosphorylation profile of wild type activated gametocytes with that of Δcdc20 and Δmap2 lines using metabolic labelling with 32P-orthophosphate [40]. This approach employs metabolic labelling of parasites followed by fractionation by ion exchange chromatography. The experiment was performed in triplicate and in each experiment 20 fractions were collected, resolved by SDS-PAGE and an autoradiograph obtained for seven of them to reveal the phosphorylation profile. Shown in Figure 7 are three fractions from the ion exchange fractionation where differences in the phosphorylation profile between the wild type and mutant parasite strains were observed. Importantly, the Coomassie blue stain of the SDS-PAGE gels demonstrated that the overall protein expression profiles of the wild type and mutant parasite lines were very similar (Figure 7). Despite this similarity, there were clear differences in the phosphorylation profile between the parasite lines. The phosphorylated band labelled A in Figure 7 was significantly decreased in the Δmap2 mutant, whereas the Δcdc20 mutant showed increased phosphorylation. Bands C, D, F, G, H and J showed altered phosphorylation status only in the Δmap2 mutant, whereas bands A, E and I were changed only in the Δcdc20 mutant. Only one band (band B) showed a similar change in both the Δmap2 and Δcdc20 mutants. This analysis indicated that although the phosphorylation profile of the parasite was affected by the deletion of map2 and cdc20, the proteins that showed a change in phosphorylation status in the two mutant lines were (with the exception of one protein) different. It seems unlikely therefore that MAP2 and CDC20 regulate the same network of phospho-proteins. We are currently investigating this result further using mass spectrometry-based phosphoproteomic approaches.


A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

Guttery DS, Ferguson DJ, Poulin B, Xu Z, Straschil U, Klop O, Solyakov L, Sandrini SM, Brady D, Nieduszynski CA, Janse CJ, Holder AA, Tobin AB, Tewari R - PLoS Pathog. (2012)

Global phosphorylation in Δcdc20 and Δmap2 lines.Gametocytes from wild type, Δmap2 and Δcdc20 parasites were purified on 48% Nycodenz and activated for 30 mins in ookinete medium before addition of 32P-orthophosphate for 30 mins. After washing, labelled activated gametocytes were lysed with NP40 and fractionated using anion exchange chromatography on an AKTA system. Individual fractions were then further resolved by SDS-PAGE and labelled bands detected by autoradiography. The Coomassie blue stained gel shows that protein loading was similar between lanes. Several differences in the 32P signal (indicated by arrows) are observed between the three different parasites. Bands C, D, F, G H and J indicate altered phosphorylation status only in the Δmap2 mutant. Bands A, E and I indicate changes only in the Δcdc20 mutant. Only one band (band B) showed a similar change in both the Δmap2 mutant and Δcdc20 mutant.
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Related In: Results  -  Collection

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ppat-1002554-g007: Global phosphorylation in Δcdc20 and Δmap2 lines.Gametocytes from wild type, Δmap2 and Δcdc20 parasites were purified on 48% Nycodenz and activated for 30 mins in ookinete medium before addition of 32P-orthophosphate for 30 mins. After washing, labelled activated gametocytes were lysed with NP40 and fractionated using anion exchange chromatography on an AKTA system. Individual fractions were then further resolved by SDS-PAGE and labelled bands detected by autoradiography. The Coomassie blue stained gel shows that protein loading was similar between lanes. Several differences in the 32P signal (indicated by arrows) are observed between the three different parasites. Bands C, D, F, G H and J indicate altered phosphorylation status only in the Δmap2 mutant. Bands A, E and I indicate changes only in the Δcdc20 mutant. Only one band (band B) showed a similar change in both the Δmap2 mutant and Δcdc20 mutant.
Mentions: In order to examine whether or not CDC20 has a role in pathways of protein phosphorylation similar to those of the kinase MAP2, we compared the global phosphorylation profile of wild type activated gametocytes with that of Δcdc20 and Δmap2 lines using metabolic labelling with 32P-orthophosphate [40]. This approach employs metabolic labelling of parasites followed by fractionation by ion exchange chromatography. The experiment was performed in triplicate and in each experiment 20 fractions were collected, resolved by SDS-PAGE and an autoradiograph obtained for seven of them to reveal the phosphorylation profile. Shown in Figure 7 are three fractions from the ion exchange fractionation where differences in the phosphorylation profile between the wild type and mutant parasite strains were observed. Importantly, the Coomassie blue stain of the SDS-PAGE gels demonstrated that the overall protein expression profiles of the wild type and mutant parasite lines were very similar (Figure 7). Despite this similarity, there were clear differences in the phosphorylation profile between the parasite lines. The phosphorylated band labelled A in Figure 7 was significantly decreased in the Δmap2 mutant, whereas the Δcdc20 mutant showed increased phosphorylation. Bands C, D, F, G, H and J showed altered phosphorylation status only in the Δmap2 mutant, whereas bands A, E and I were changed only in the Δcdc20 mutant. Only one band (band B) showed a similar change in both the Δmap2 and Δcdc20 mutants. This analysis indicated that although the phosphorylation profile of the parasite was affected by the deletion of map2 and cdc20, the proteins that showed a change in phosphorylation status in the two mutant lines were (with the exception of one protein) different. It seems unlikely therefore that MAP2 and CDC20 regulate the same network of phospho-proteins. We are currently investigating this result further using mass spectrometry-based phosphoproteomic approaches.

Bottom Line: Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes.Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant.This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology Queens Medical Centre, University of Nottingham, Nottingham, UK.

ABSTRACT
Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

Show MeSH
Related in: MedlinePlus