Mutant/mutation
The mutant expresses a C-terminal GFP tagged version of CDPK4

To examine the spatiotemporal expression of the three protein kinases CDPK4 (PBANKA_0615200), SRPK1 (PBANKA_0401100) and MAP2 (PBANKA_0933700), transgenic P. berghei parasite lines were generated expressing the genes modified to code for a C-terminal  GFP tag (RMgm-5602, RMgm-5603, RMgm-5604). An in-frame gfp coding sequence was inserted at the 3′ end of the endogenous gene locus using single crossover homologous recombination.

Protein (function)
CDPK4 belongs to an expanded family of Ca²⁺ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. In plants, CDPKs translate Ca²⁺ signals generated by external stimuli into cellular responses, thereby regulating cell division and differentiation, the development of tolerance to stress stimuli and the specific defense responses to pathogens.
CDPK4 has a role in male gamete formation: in male gametocytes axoneme formation, formation of mitotic spindles and DNA synthesis is inhibited/blocked,.

In the rodent malaria parasite Plasmodium berghei (Pb), several studies have highlighted three male specific PKs, which are defined as essential during male gametogenesis but not at the asexual blood stage. These are calcium-dependent protein kinase-4 (CDPK4), a serine/arginine-rich protein kinase (SRPK1) and mitogen-activated protein kinase-2. The deletion of each ablates exflagellation (male gamete release) and completely blocks sexual reproduction and parasite transmission through the mosquito. However, the deletion of each PK has little to no effect on the development of the parasites during the asexual blood stage. Each PK has been postulated to act at a distinct point: CDPK4 is considered to be a master regulator, initiating assembly of the pre-replicative complex and formation of the first mitotic spindle within the first 18 seconds following gametocyte activation; SRPK1 is for subsequent DNA replication, and MAP2 is required for cytokinesis and axoneme motility.

Phenotype
Western blot analysis of schizont (for CDPK4 and SRPK1) and gametocyte (for MAP2 as MAP2 seems to be barely expressed in asexual blood stages) protein extracts, using an anti-GFP antibody, revealed major bands at 90 kDa for CDPK4-GFP, 183 kDa for SRPK1-GFP, 90 kDa for MAP2-GFP, and 29 kDa for unfused GFP (WT-GFP); the expected sizes for each. Each GFP-tagged PK was also observed by fluorescence in activated gametocytes.
Live-cell fluorescence imaging of P. berghei asexual blood stages revealed a diffuse cytoplasmic distribution of CDPK4-GFP in both the schizonts and merozoites. In contrast SRPK1-GFP showed weak diffuse cytoplasmic expression with a strong focus at the location of the nuclear pole and towards the apex of the forming or mature merozoites. In contrast, no MAP2-GFP fluorescence was observed in asexual development. During male gametogenesis, CDPK4-GFP had a diffuse location in both cytoplasm and nucleus, along with a distinct focus in the cytoplasm six min post-activation. At the early stage of male gametogenesis (30 s to 6 min post activation), up to two foci of CDPK4-GFP were observed, whereas at the late stage (>6 min post activation, more than three foci were sometimes observed. SRPK1-GFP was located in the cytoplasm, concentrically surrounding the nucleus, MAP2-GFP had a nuclear location in activated male gametocytes.
Because these PKs are essential for axoneme development and are located at distinct regions of the parasite cell (in particular the distinct CDPK4-GFP focus in male gametocytes), we examined their co-localisation with markers of MTOC biology, including chromosome segregation (kinetochore protein, NDC80) and basal body formation (cytoplasmic axonemal protein, kinesin-8B, which were C-terminally tagged with mCherry (mCh). No co-localisation was observed between CDPK4-GFP, SRPK1-GFP or MAP2-GFP and NDC80-mCh during male gametogenesis, or between SRPK1-GFP and NDC80-mCh during schizogony, suggesting that these PKs do not directly interact with kinetochores during chromosome condensation. However, kinesin-8B-mCh appeared as a ring around SRPK1-GFP, suggesting that SRPK1 may be interacting with axoneme proteins or proteins involved in their assembly. CDPK4-GFP did not co-localise with kinesin-8B-mCh at any point.

Evidence is presented in the paper for the following:
- CDPK4, SRPK1 and MAP2 interact with each other and key regulators of male gametogenesis (by immunoprecipitation analyses with anti-GFP antibody CDPK4-GFP, SRPK1-GFP, MAP2-GFP and WT-GFP (as a control) from lysates of cells, 6 min post-activation of gametocytes).
- A role of reversible protein phosphorylation as a key driver of male gametogenesis
- Ultrastructure analysis reveals defects in MTOC formation, kinetochore dynamics, and axoneme formation in (previously published) CDPK4, SRPK1 and MAP2 deletion mutants

Additional information
From the Abstract:
'We localise each protein kinase (PK) during rapid male gamete formation using live-cell imaging, identify their putative substrates by immunoprecipitation, and determine the morphological consequences of their absence using ultrastructure expansion and transmission electron microscopy. Each PK has a distinct location in either the nuclear or cytoplasmic compartment. Protein interaction studies revealed that CDPK4 and MAP2 interact with key drivers of rapid DNA replication, while SRPK1 is involved in RNA translation. The absence of each PK results in severe defects in either microtubule organising centre (MTOC) organisation, kinetochore segregation or axoneme formation.

The following dual-tagged parasite lines were generated by crossing the GFP-tagged CDPK4, SRPK1 and MAP2 parasite lines with mCherry-tagged lines of the kinetochore marker NDC80 (PBANKA_1115700; RMgm-4844) and axoneme marker kinesin-8B (PBANKA_0202700; RMgm-5324)

The mutant RMgm-5605 expresses a C-terminal GFP-tagged version of CDPK4  and an mCherry-tagged version of Kinesin-8b. The mutant has been obtained by crossing CDPK4-GFP (RMgm-5602) with Kinesin-8b::mCherry (RMgm-5324).
The mutant RMgm-5606 expresses a C-terminal GFP-tagged version of SRPK1  and an mCherry-tagged version of Kinesin-8b. The mutant has been obtained by crossing SRPK1-GFP (RMgm-5603) with Kinesin-8b::mCherry (RMgm-5324)
The mutant RMgm-5607 expresses a C-terminal GFP-tagged version of MAP2  and an mCherry-tagged version of Kinesin-8b. The mutant has been obtained by crossing MAP2-GFP (RMgm-5604) with Kinesin-8b::mCherry (RMgm-5324)

The mutant RMgm-5608 expresses a C-terminal GFP-tagged version of CDPK4  and an mCherry-tagged version of NDC80. The mutant has been obtained by crossing CDPK4-GFP (RMgm-5602) with NDC80-mCherry (RMgm-4844).
The mutant RMgm-5609 expresses a C-terminal GFP-tagged version of SRPK1  and an mCherry-tagged version of NDC80. The mutant has been obtained by crossing SRPK1-GFP (RMgm-5603) NDC80-mCherry (RMgm-4844).
The mutant RMgm-5609 expresses a C-terminal GFP-tagged version of MAP2  and an mCherry-tagged version of NDC80. The mutant has been obtained by crossing MAP2-GFP (RMgm-5604) NDC80-mCherry (RMgm-4844).

Other mutants