Mechanisms to control cell division and the cell cycle are essential parts of the cell regulation machinery. These processes are not well understood in unicellular protozoa such as the malaria parasite Plasmodium. Plasmodium undergoes two distinct mitotic processes; one involving repeated DNA duplication, in which karyokinesis occurs after each replication and is associated with asexual proliferation and the other involving endoreduplication, with three rounds of replication prior to the simultaneous formation of eight microgamete nuclei during microgametogenesis. Here, we describe a CDC20/CDH1 orthologue in Plasmodium as an important regulator of mitosis during male gametogenesis, but interestingly it has no effect on the mitotic process undergone during schizogony.
Our bioinformatic studies suggest that in Plasmodium
there is only one gene representing CDC20 and its homologue CDH1, and that the protein is a true structural homologue of CDC20/CDH1, even though we could not complement CDC20 function in yeast (data not shown). Although we cannot exclude the possibility that we failed to detect a second highly spliced Plasmodium cdc20/cdh1
homologue, the phylogenetic clustering of all the Plasmodium
CDC20 homologues gives confidence that there is only a single CDC20 orthologue in Plasmodium
species. This suggests that Plasmodium
diverged from other eukaryotes prior to the duplication event that presumably gave rise to CDC20 and CDH1 genes. It is interesting to note that the Plasmodium
cluster is distinct from the Trypanosomatidae
cluster where there is also a single corresponding gene in each genome. Furthermore, this orthologue has a classical KEN box-like domain at the N-terminus and an RVL domain and IR motif at the C-terminus, all of which are required for cyclin degradation and binding to the APC/C core 
. The presence of these domains suggests that CDC20 in Plasmodium
could influence the cell cycle in a similar manner to other systems, such as yeast, mammals and plants 
. The lack of a D-box and presence of a KEN-box are consistent with the structure of CDC20 in humans, with the presence of a KEN-box suggesting that Plasmodium
CDC20 is a prime target for ubiquitination, as suggested in a recent study 
. Alternatively, as Plasmodium
CDC20 is the only orthologue of both CDC20 and CDH1 present in other systems, it is plausible that ubiquitination of CDC20 in Plasmodium
is self-regulating, as CDC20 is known to be degraded by APC/CCDH1
via its KEN-box 
and could therefore act as a “negative feedback” mechanism as seen in human cells 
. The seven conserved WD repeats in the Plasmodium
CDC20 protein also suggests that it does bind an as yet unknown multi-protein complex. Plasmodium
CDC20 shows some differences from the ccs52
homologue reported in plants, such as Medicago sativa
, since it lacks a MAD-binding box and also the D-box that appears to be specific for CDH1 and is not conserved in CDC20 and FZY proteins. It has been reported recently that in Arabidopsis thaliana
there are five isoforms of CDC20, and two of them are functional 
. We did not observe any such expansion of genes for this protein in Plasmodium
Our CDC20-GFP expression studies showed that CDC20 is highly expressed in activated male gametocytes (with gametocytes showing highest expression at the mRNA level, in agreement with previous transcriptomic studies 
) but it is also present throughout the life-cycle and located mainly in the nuclear compartment, with some cytoplasmic localisation, consistent with expression in other systems 
. However, although previous studies have shown cdc20
transcripts and protein to be highly expressed in sporozoites of P. falciparum
, we did not observe high protein expression levels of CDC20-GFP in sporozoites.
Functional studies using a gene deletion strategy showed that CDC20 controls male gamete development and deletion mutants are impaired during transmission of the parasite to the mosquito vector. Further in-depth analysis of these mutants using a cross fertilisation approach showed that this defect is limited to male gamete differentiation (exflagellation) and formation since Δcdc20
macrogametocytes are fully capable of cross fertilization with microgametes from donor strains. Hence, CDC20 has an essential function for the transition of male gametocytes to gametes. Gametogenesis in Plasmodium
involves three rounds of mitotic division in male gametocytes resulting in eight gametes 
. We have previously shown that CDPK4 is involved in cell cycle progression to S phase and MAP2 may be essential for replication and mitosis to be completed before cytokinesis commences 
(although it is important to note that MAP2 is essential for asexual development in P. falciparum
, so there may be species-specific differences in the roles of different kinases). As cdc20
mRNA levels are up regulated in both Δcdpk4
mutants, this suggests that CDC20 may be interlinked with these kinases and orchestrates the process of male gametogenesis and is perhaps up-regulated to compensate for the loss of these two kinases, but this suggestion requires further investigation. The cdc20
deletion mutants formed axonemes and mitotic spindles but failed to undergo karyokinesis or cytokinesis and also did not form motile, flagellar gametes, a phenotype similar to what we have observed with map2
deletion mutants. The requirement for CDC20 during karyokinesis is consistent with the known function of CDC20 and CDH1 in other systems 
. As described earlier, CDC20 is active during early mitosis in other cells and its up-regulation in gametocytes suggests that it has an essential role in the multiple rounds of DNA replication and the chromosome separation specifically associated with this process. However, mutant cdc20
parasites do not arrest during asexual proliferation and this suggests that Plasmodium
CDC20 is specifically required for microgametogenesis.
Functional studies in human systems have shown that a deficiency of CDH1 results in delayed mitotic exit as well as an accumulation of mitotic errors and difficulty in completion of cytokinesis 
, similar to what is observed in our cdc20
mutants. Therefore we suggest that CDC20 in Plasmodium
fulfils the function of both CDC20 and CDH1. Moreover, loss of cdc20
results in arrest during metaphase to anaphase transition 
, with sister chromatids failing to form. How the single CDC20 protein may fulfil the roles of both CDC20 and CDH1 requires further investigation. Our ultrastructure studies for both Δcdc20
lines, reported for the first time to our knowledge; show that these mutants have a similar arrest in cytokinesis and karyokinesis detected by EM, with defects in nuclear spindle/kinetochore movement and chromatin condensation, confirming our initial light microscopy observation of Δmap2
. Unlike the Δmap2
line, we never observed any exflagellation in the Δcdc20
line. As suggested before 
, classical spindle checkpoints are not present in Plasmodium
since blockage of microtubule organisation does not appear to block DNA synthesis. Therefore, MAP2 and CDC20 may be involved in a critical cell cycle checkpoint during microgametogenesis that controls DNA replication and mitosis, prior to karyokinesis and cytokinesis and is summarised in .
Summary of phenotypes in mutants of cdpk4, map2 and cdc20.
DNA replication in the Δcdc20
line was similar to that in the Δmap2
line, with both mutants undergoing octoploidy 8 mpa, but not undergoing karyokinesis. As a result, we analysed whether phosphorylation of CDC20 could be involved in mitotic progression during microgametogenesis. In other systems, phosphorylation of CDC20 can be achieved by BUB1, CDK1, MAPK 
and also NEK2 
, another protein kinase required for zygote development in Plasmodium
and this modification is an essential step for CDC20 inhibition by the SAC 
. Here, we have shown that CDC20 is more phosphorylated in activated gametocytes and ookinetes (i.e. sexual stages) compared to schizont (asexual stages), which suggests that phosphorylation of CDC20 may be a possible mechanism involved in gametogenesis.
Interestingly, the global phosphorylation profile of Δcdc20 parasites suggests that CDC20 regulates the phosphorylation of specific proteins within the gametocytes. The proteins that are regulated by CDC20 are; however, largely different from those that appear to be regulated by MAP2. This would suggest that at the level of phosphorylation CDC20 and MAP2 regulate different pathways. It would be interesting in future studies to dissect out the proteins regulated by MAP2 and CDC20 and in this way build a network of phospho-proteins that regulate male gametogenesis.
In conclusion, this study identified significant differences in the control of mitosis during asexual development compared to microgametogenesis in the malaria parasite. We have also shown that CDC20 and MAP2 may play independent but essential roles in the mitotic division associated with microgametogenesis but are not essential for mitosis during asexual stages in the malaria parasite.