Additional remarks phenotype | Mutant/mutation
In the 'promoter swap mutant': the promoter of nek1 gene has been replaced with the asexual blood stage promoter clag2 (PBANKA_1400600) that is not active in gametocytes. In addition, it expresses GFP under control of the constitutive eef1a promoter.
Protein (function)
Mitosis has a key role in the eukaryotic cell cycle when the cell divides and produces daughter cells. During mitosis, centrosomes act as microtubule organising centres (MTOCs) coordinating spindle dynamics with chromosome congression and segregation. They also act as signalling hubs for regulators of mitosis, including cyclin-dependent kinases (CDKs), and Aurora, Polo and NIMA-related (NEK) kinases.
Budding and fission yeast express a single NEK kinase, but the family is expanded to 11 members in mammals, which may reflect the increased complexity of microtubule-dependent processes. Several NEKs are essential to form functional cilia and flagella in mammals and other organisms, including Chlamydomonas, Trypanosoma and Tetrahymena. Other NEKs are more directly implicated in mitotic cell division, with defects in NEK expression likely to contribute to aberrant chromosome segregation in cancer cells. NEKs are located at MTOCs in many organisms, including Aspergillus, yeast and human cells. Of the eleven mammalian NEKs, NEK2, NEK6, NEK7 and NEK9 localise to centrosomes, playing roles in mitotic spindle assembly.
Plasmodium contains a bipartite MTOC, that consists of a cytoplasmic part (outer MTOC) and a nuclear component (inner MTOC). Four NIMA-like kinases were identified in Plasmodium, and all are expressed most highly in gametocytes. These kinases have been named NEK1 to NEK4, but they are not directly analogous to mammalian NEK1 to NEK4.
Previous gene knockout studies showed that Plasmodium NEK2 and NEK4 are required for zygote differentiation and meiosis, but not for mitotic division of parasite cells in mammalian red blood cells, whereas NEK1 is (likely) essential for asexual blood stage schizogony and proliferation.
Phenotype
A Pclagnek1 (NEK1clag) parasite line was generated for transcript depletion by double homologous recombination to insert the cytoadherence-linked asexual protein (CLAG – PBANKA_1400600) promoter upstream of nek1, in a parasite line that constitutively expresses GFP, to take advantage of stage-specific expression of clag that is transcribed in blood stages but not in gametocytes.
Quantitative real time PCR (qRT-PCR) confirmed that nek1 mRNA is downregulated by approximately 90% in these gametocytes.
Male gamete formation (exflagellation) was severely affected in NEK1clag compared to GFP (wild type) parasites, and very few or no ookinetes (differentiated zygotes after fertilisation) were formed. Female gametogenesis was not affected as observed by using an antibody to a surface marker (P28) that expresses highly in female gametes. In mosquitoes fed with NEK1clag parasites we observed no oocysts in the midguts, while hundreds of oocysts were present in comparable mosquitoes fed GFP wild type parasites. To further confirm the absence of any oocyst or sporozoite, infected mosquitoes were allowed to feed on naïve mice. Mosquitoes infected with NEK1clag parasites failed to transmit this parasite to susceptible mice, whilst mosquitoes were able to transmit the WT-GFP parasite at the same time, and blood stage infection was observed in naïve mice 4 days later.
See RMgm-5533 for a mutant expressing NEK1-GFP. Analysis of these parasites showed that NEK1-GFP was undetectable in ring stages but was visible with a diffuse cytoplasmic location during later stages (trophozoites). At the start of schizogony, NEK1-GFP re-located In the cytoplasm initially to single focal points near individual nuclei, which later divided into two tightly associated punctae. NEK1-GFP signal disappeared at the end of nuclear division and was undetectable in free merozoites.The location of NEK1-GFP was also studied during other asexual stages like liver schizogony and mosquito gut sporogony. The live cell images of the proliferative liver schizogony and sporogony stages showed similar patterns of NEK1-GFP foci formation during proliferative stages.
Additional information
Since NEK1 is expressed in male gametocytes and parasite development is affected after fertilisation, we investigated whether the defect is inherited from the male gamete. We performed genetic crosses between NEK1clag parasites and other mutants deficient in production of either male (Δhap2) or female (Δdozi and Δnek4) gametocytes. Crosses of NEK1clag with Δdozi and Δnek4 mutants produced some mature banana shaped
ookinetes, showing a partial rescue of the NEK1clag phenotype. In contrast, crosses between NEK1clag and Δhap2 did not rescue the NEK1clag phenotype. These results reveal that a functional nek1 gene from a male gamete is required for subsequent zygote formation and ookinete development.
Transmission electron microscopy (TEM) analysis of NEK1clag and GFP-expressing wild type (WT-GFP) gametocytes at eight- and fifteen minutes post-activation provided evidence that NEK1 downregulation affected MTOC organisation, chromosome segregation and kinetochore attachment.
The following mutants were generated to analyse the location and function of NEK1 in more detail:
- A mutant expressing a C-terminal 3xHA tagged version of NEK1 (RMgm-5534)
- (Conditional) knockdown mutants of NEK1: i) a mutant expressing a AID/HA tagged version of NEK1 to degrade NEK1 in gametocytes in the presence of auxin and ii) a mutant in which the promoter region of NEK1 was replaced with the promoter of clag, a gene that is specifically expressed in asexual blood stages (and not in gametocytes) (RMgm-5535).
By (mosquito-)crossing NEK1-GFP parasites (RMgm-5533) with other (existing) transgenic lines expressing mCherry tagged proteins (NDC80 RMgm-4844; EB1 RMgm-5329; ARK2 RMgm-5322; kinesin-8b RMgm-5324) 'dual tagged' parasites were generated that expresses:
- GFP-tagged NEK1 and mCherry tagged kinetochore marker NDC80
- GFP-tagged NEK1 and mCherry tagged spindle microtubule plus-end binding protein EB1
- GFP-tagged NEK1 and mCherry tagged spindle-associated aurora kinase 2 ARK2
- GFP-tagged NEK1 and mCherry tagged cytoplasmic axonemal protein kinesin 8B
Generation of dual tagged parasite lines
The NEK1-GFP parasites were mixed with either NDC80-cherry or EB1-mCherry or ARK2-mCherry or kinesin-8B-mCherry parasites in equal numbers and injected into mice. Mosquitoes were fed on mice 4 to 5 days after infection when gametocyte parasitaemia was high. These mosquitoes were checked for oocyst development and sporozoite formation at day 14 and day 21 after feeding. Infected mosquitoes were then allowed to feed on naïve mice and after 4 - 746 5 days, and the mice were examined for blood stage parasitaemia by microscopy with Giemsa-stained blood smears. In this way, some parasites expressed both NEK1-GFP and NDC80-mCherry; or EB1-mCherry; or ARK2-mCherry; or kinesin-8B-mCherry in the resultant gametocytes, and these were purified, and fluorescence microscopy images were collected as described above.
The analyses of these mutants revealed the following (from the Abstract):
'We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organizing centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation and kinetochore attachment. These data suggest that Plasmodium NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation'
NEK1 is (likely) essential for asexual blood stage schizogony and proliferation.
Evidence is presented that:
- Plasmodium NEK1 has a punctate location, partially overlapping with the kinetochore and MTOC during blood stage schizogony
- NEK1-GFP associates with MTOC formation and spindle dynamics during male gamete formation
- The spatiotemporal location of NEK1 with respect to kinetochore, spindle and axoneme markers during male gametogenesis
- NEK1-GFP interacting proteins are components of the axoneme and flagellum
- Conditional knockdown/depletion of NEK1 reveals an essential role during male gametogenesis
- Abnormal MTOC organization and kinetochore attachment following of conditional knockdown of NEK1 level
Other mutants |