Mutant/mutation
The mutant lacks expression of SAS6 (spindle assembly abnormal protein 6).

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 of a mutant lacking expression of SAS6 (RMgm-1109; RMgm-1110) 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.

Analyses of the mutant expressing a cMyc-tagged form of SAS6 showed expression in male gametocytes and gametes (and absence in females).

Additional information
Several attempts at tagging sas6 with GFP were unsuccessful.

Uniform distribution of sas6-myc in male gametocytes changes upon activation: at 10 minutes post-activation (mpa),  distinct punctae of fluorescence were observed varying in number between 4 and 10 per cell.  Absence of detectable protein in asexual stages and aggregation of protein after activation is  consistent with previous electron microscopy data which reported lack of visible centrosome structures  in asexuals and de novo BB formation after male gametocyte activation. At 15 mpa, myc is observed in the  male gametocyte body and at the distal tips of exflagellating gametes. Whilst the maximum number of  punctae does not strictly coincide with the expected maximum number of BB, the location of sas6-myc at the  protruding tips of the male gametes certainly does. The extra-punctae was attributed to putative abnormal BB  segregation caused by the myc tag. Taken together these results indicate that SAS-6 is a male specific protein,  whose distribution is re-organized after male gametocyte activation with a probable BB location.

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 (RMgm-1109, RMgm-1110) 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
Mutant lacking expressing SAS6 (and expressing GFP) (RMgm-1110, RMgm-1109)