Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of SAS6 (spindle assembly abnormal protein 6) and expresses GFP under the constitutive eef1 promoter.
Protein (function)
Each male gametocyte forms 8 male gametes, which are simple flagellate cells. For successful gamete formation, nucleus and cytoplasm of parental gametocytes have to be exquisitely coordinated. In the nuclear compartment, 3 endomitotic divisions produce 8 newly replicated genomes. Simultaneously, in the cytoplasmic compartment, an amorphous microtubule organizing centre develops into two planar tetrads of basal bodies (BB), which separate into 8 individual BBs. Each BB serves as a template for one axoneme and remains connected with the genome trough a nuclear pore. The pairing of a single haploid genome/nucleus with each flagellum is critical for the formation of fully functional male gametes. In a process termed exflagellation, the newly assembled individual haploid flagellate gametes are released, BB first, from the residual gametocyte body.BBs are established platforms for eukaryotic flagella/cilia assembly. Plasmodium genomes contain few conserved BB gene orthologues one of which encodes SAS6 which belongs to an ancestral conserved module of proteins that correlates with presence of centrioles/BBs. SAS-6 family members are required for the earliest steps of centriole formation in a range of organisms and its depletion often results in failure to form centrioles or produces other centriole abnormalities leading to severe flagellar/ciliary anomalies. These anomalies include flagellar absence, loss of flagellar 9-fold symmetry and cilia length reduction.
Phenotype
Phenotype analyses show the absence of formation of motile male gametes (see also 'Additional information' for an explanation). However, a few ookinetes can be formed. These ookinetes form oocysts but these oocysts lack the formation of viable sporozoites, indicating that SAS6 plays also a role during oocyst formation
Additional information
Analysis of a mutant expressing a cMyc-tagged form of SAS6 (RMgm-1111) showed expression in male gametocytes and gametes (and absence in females).
Flagellum formation with an anti a-tubulin antibody was examined and simultaneously nuclear organization by DAPI staining. At 10 min. after activation (mpa), wt and Δsas6 male gametocytes are indistinguishable from each other: tubulin containing microtubule structures are visible in the cytoplasm and DAPI measurements suggest that DNA replication is normal. At 15 mpa, wt parasites undergo exflagellation and tubulin stained wt microgametes can be seen either in the process of release from the gametocyte body or already detached from it. Wt flagella usually exhibit wave-like shapes reflecting motility of male gametes. Δsas6 form tubulin-containing structures that protrude from gametocyte bodies but rarely detach from them. These tubulin structures, which do not appear to move, display abnormal and linear morphology. Malformed microgametes projecting out of Δsas6 male gametocytes rarely contain DNA (3%), as opposed to wt, in which DNA is associated with most flagella detaching or detached from the gametocyte body (92%). The canonical ‘‘9+2’’ microtubule structure of flagella is severely disrupted and basal bodies are rare in Δsas6. Taken together these results indicate that depletion of SAS-6 results in the formation of fewer BBs, which is most certainly responsible for disruption of canonical axonemal structures and lack of axoneme nucleation.
Other mutants
A independent mutant lacking expressing SAS6 (and expressing GFP) (RMgm-1110)
A mutant expressing a cMyc-tagged form of SAS6 (RMgm-1111) |