Mutant/mutation
The mutant lacks expression of a serine/threonine-protein kinase, Nek2 (a NIMA-related protein kinase).

Protein (function)
The NIMA-related protein kinases (Neks) constitute an extended family of eukaryotic mitotic serine/threonine kinases. The best characterized members of the Nek family include NIMA (never in mitosis/Aspergillus), the founding member from the fungus Aspergillus nidulans, and its closest homologue in mammals, Nek2. Initially identified as a kinase essential for mitotic entry in Aspergillus, NIMA has been also shown to participate in nuclear membrane fission. Eleven members of the NIMA kinase family (Nek1-11) have now been identified in various human tissues, and together fulfil a number of cell cycle-related functions in centrosome separation, mitosis, meiosis and checkpoint control. The P. falciparum kinome includes four NIMA-related serine/threonine kinases. Pfnek-1 (PlasmoDB identifier PFL1370w) clusters within the Aspergillus NIMA/human Nek2 branch in phylogenetic trees, while clear orthology to mammalian or yeast Neks could not be assigned for the three other P. falciparum sequences (Pfnek-2, -3 and -4, PlasmoDB identifiers PFE1290w, PFL0080c and MAL7P1.100,respectively).

Phenotype
The phenotype analyses indicate gamete formation and fertilisation are not affected, but development of zygotes is arrested at the 2N zygote stage (see also 'Additional Information').  The analyses suggest that the mutants are unable to properly control pre-meiotic DNA replication.

Additional information
Genetic crosses were used  to determine whether the defect leading to the block in ookinete development was carried by the male or the female gametocytes. Crossing pbnek-2- with pbcdpk4- mutants (RMgm-12), in which male gametocyte exflagellation is abolished and which therefore can produce only female gametes, restored the ookinete development. This indicates that pbnek-2- male gametes are fully functional and can cross-fertilise the pbcdpk4- female gametes. In contrast, crossing the pbnek-2- mutant with a mutant lacking Pbnek-4 (in which female gametocytes carry a defect preventing ookinete development, RMgm-60) did not restore ookinete conversion capacity. Thus, absence of Pbnek-2 causes female gametocytes to be unable to support normal development from zygote to ookinete, similar to the developmental phenotype of  pbnek-4- mutants (RMgm-59, RMgm-60).

In order to investigate in more detail the stage at which the block in ookinete development occurred, the amount of DNA in individual cells was quantified using the DNA-specific fluorescent dye Hoechst 33342. In wild-type parasites, fertilisation is followed by fusion of gamete nuclei and one round of replication, increasing the zygote’s nuclear DNA content to 4C prior to meiosis. Following meiosis four sets of chromosomes are maintained within the ookinetes nucleus, making this stage tetraploid. pbnek-2- parasites appeared to undergo activation and fertilisation like wild-type parasites, but fusion of the two nuclei was impaired in a large proportion of the parasites, and the  DNA content remained at just above 2C, a value consistent with the sum of the two gamete nuclei. In all round cells (i.e. female gametes, zygotes and “failed” ookinetes), the DNA content remained below the 4C value expected in normal ookinetes, indicating that the DNA replication process that precedes meiosis is affected in the mutant parasites.

Analysis of the location of Nek2 in P. falciparum female gametocytes presented in this study indicates that the enzyme localises with what looks like microtubules in female gametocytes. Such a location is consistent with the established role of Neks in the regulation of microtubule dynamics.

See RMgm-59, RMgm-60 for a description of the phenotype of mutants lacking expression of NEK4. In these mutants gamete formation is not affected. Fertilisation occurs, but development is arrested at 2N zygote stage, comparable to the phenotype of the mutant lacking expression of NEK2. Also in the NEK4 mutants the defect is female gamete specific; male gametes are fertile and able to fertilize wild-type female gametes.

The similarity in the phenotypes caused by the loss of Pbnek-2 and Pbnek-4, and the observation that the two enzymes do not trans-phosphorylate or –activate in vitro, suggest that they function in parallel pathways involved in the same cell development process, but are not able to complement for each other.

Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565). After transfection with a KO vector a strong PCR signal diagnostic for gene disruption was observed in transfected populations indicating that this gene is not essential for asexual proliferation. Cloning will however be required to validate this interpretation for this


Other mutants
RMgm-294: Another mutant from the same study lacking expression of Nek2. In addition, this this mutant contains a gfp gene under the control of the eef1a promoter, stably introduced into the genome witthout a drug-selectable marker.