Mutant/mutation
The mutant expresses a C-terminal-tagged version of SUN1

Protein (function)
SUN1 is a protein with a carboxy-terminal SUN (Sad1p/UNC-84) domain. SUN domain proteins were present in the first eukaryote and typically span the inner nuclear envelope (NE), where they form part of the LINC (linker of nucleoskeleton and cytoskeleton) complex that anchors the nucleus by connecting the nuclear lamina to the cytosolic cytoskeleton. Apicomplexa lack the nuclear lamins and canonical LINC complex components. SUN1 interacts with a novel Allantoicase-like protein (termed ALLAN). Functional disruption of either SUN1 or ALLAN impairs cytoplasmic basal bodies (BB) segregation, disrupts kinetochore attachment, and results in defective flagellum assembly.
 
Phenotype
No expression in asexual blood stages.
Expression in both male and female gametocytes following activation. In activated male gametocytes undergoing mitotic division, SUN1-GFP displayed a dynamic localization around the nuclear DNA (Hoechst-stained), associated with loops and folds forming in the nuclear envelope (NE). In activated female gametocytes within one-minute post-activation, SUN1-GFP was observed to form a half-circle around the nucleus, eventually encompassing the entire nucleus by six to eight minutes. In contrast to SUN1-GFP in male gametocytes, SUN1-GFP in female gametocytes was more uniformly distributed around the nucleus without apparent loops or folds in the NE.
In zygotes/ookinetes/oocysts SUN1-GFP had a spherical distribution around nuclear DNA at two hours post-fertilization, but by 24 hours post-fertilization, as the zygote developed into a banana-shaped ookinete, the SUN1-GFP-marked NE became elongated or oval, possibly due to spatial constraints within the cytoplasm.

Additional information
Analysis of a mutant lacking expression of SUN1 (RMgm-5636) showed the following:
Normal asexual development/growth. Normal gametocyte production. Reduced male gametogenesis, as shown by a significant decrease in gamete formation (exflagellation). Reduced ookinete formation. Reduced oocyst formation. At day 7, oocysts were comparable in size to wild type, but failed to grow further, and they had degenerated by day 21. Mosquitoes infected with Δsun1 parasites were unable to transmit the parasite to naïve mice.

Evidence is presented that: 
- Transcriptomic analysis of Δsun1 gametocytes shows modulation in lipid metabolism and microtubule organization
- Disruption of SUN1 Perturbs Lipid Homeostasis and Membrane Biogenesis in Gametocytes
- Ultrastructural microscopy reveals defects in spindle formation, Basal Body segregation and nuclear attachment to axonemes in Δsun1 gametocytes
- SUN1 Interactome Reveals Associations with Nuclear Envelope, ER and Chromatin components. PbSUN1 interacted with proteins harbouring a divergent carbohydrate binding domain, such as the allantoicase-like protein ALCC1, referred to as ALLAN (PBANKA_1144200)(see RMgm-5638 and RMgm-5639).
ALLAN is an uncharacterized protein with sequence homology to allantoicase, an enzyme of purine metabolism
- ALLAN is located at nuclear poles (NP) and influences Basal Body/NP segregation in male gametogenesis

Analysis of a mutant expressing a C-terminal GFP-tagged version of SUN1 (RMgm-5637) showed the following:
- No expression in asexual blood stages.
- Expression in both male and female gametocytes following activation
- In activated male gametocytes undergoing mitotic division, SUN1-GFP displayed a dynamic localization around the nuclear DNA (Hoechst-stained), associated with loops and folds forming in the nuclear envelope (NE)
- The parasite line expressing SUN1-GFP (green) was crossed with lines expressing mCherry (red)-tagged kinesin-8B (PBANKA_0202700; PF3D7_0111000), EB1 (PBANKA_0405600; PF3D7_0307300) or NDC80 (PBANKA_1115700; PF3D7_0616200). Results of analysis of these double-tagged parasites indicated suggest that although SUN1-GFP is partially colocalized with spindle poles as indicated by EB1, it shows no overlap with kinetochores (NDC80) or BB/axonemes (kinesin-8B).
- In activated female gametocytes within one-minute post-activation, SUN1-GFP was observed to form a half-circle around the nucleus, eventually encompassing the entire nucleus by six to eight minutes. In contrast to SUN1-GFP in male gametocytes, SUN1-GFP in female gametocytes was more uniformly distributed around the nucleus without apparent loops or folds in the NE
- In zygotes/ookinetes/oocysts SUN1-GFP had a spherical distribution around nuclear DNA at two hours post-fertilization, but by 24 hours post-fertilization, as the zygote developed into a banana-shaped ookinete, the SUN1-GFP-marked NE became elongated or oval, possibly due to spatial constraints within the cytoplasm.

Because the defect in Δsun1 parasites led to a transmission block, we investigated whether the defect was rescued by restoring sun1 into Δsun1 parasites, using the Δnek4 parasite that produces normal male gametocytes but is deficient in production of female gametocytes. We performed a genetic cross of Δsun1 and Δnek4 parasites, which produced some normal-sized oocysts that were able to sporulate, showing a partial rescue of the Δsun1 phenotype. As controls, Δsun1 and Δnek4 parasites alone were examined and no oocysts/sporozoites were detected. These results indicate that a functional sun1 gene is required from a male gamete for subsequent oocyst development.

The SUN1 Interactome Reveals Associations with Nuclear Envelope, ER and Chromatin components. PbSUN1 interacted with proteins harbouring a divergent carbohydrate binding domain, such as the allantoicase-like protein ALCC1, referred to as ALLAN (PBANKA_1144200)(see RMgm-5638 and RMgm-5639).
ALLAN is an uncharacterized protein with sequence homology to allantoicase, an enzyme of purine metabolism. ALLAN is located at nuclear poles (NP) and influences Basal Body/NP segregation in male gametogenesis.

Analysis of a mutant expressing a C-terminal GFP-tagged version of ALLAN (see RMgm-5639) showed the following:
-  During asexual blood stages, ALLAN-GFP exhibited a diffuse nucleoplasmic signal in trophozoites and schizonts with distinct focal points adjacent to dividing DNA, consistent with a role in mitotic regulation. By late schizogony, the ALLAN signal had diminished
- In male and female gametocytes and during zygote to ookinete transformation, ALLAN-GFP showed a spherical distribution around Hoechst-stained nuclear DNA and was enriched to form distinct focal points.
During oocyst development and liver schizogony, ALLAN-GFP also exhibited distinct focal points adjacent to dividing DNA suggesting a role in mitotic regulation, like in asexual blood stages.
- In activated male gametocytes, ALLAN-GFP rapidly localized within a minute post-activation to the NE, forming strong focal points that correlated with spindle poles or MTOCs. This localization persisted as ploidy increased from 1N to 8N in successive rounds of genome replication during male gametogenesis
- The location of ALLAN-GFP showed no overlap with that of kinesin-8B, a BB marker, and that of kinetochore marker NDC80
- Crosses with EB1-mCherry, a spindle marker, revealed an overlap at spindle poles suggesting a role for ALLAN in nuclear spindle pole organization. Thus, the spatial relationship of SUN1 and ALLAN suggests coordinated roles in nuclear architecture and division of labour. SUN1 likely spans the NE, linking nuclear and cytoplasmic compartments, while ALLAN localizes more specifically to nuclear MTOCs.

Analysis of a mutant lacking expression of ALLAN (see RMgm-5638) showed the following:
- Normal asexual development/growth.
- Normal gametocyte production. Slightly reduced male gametogenesis, as shown by a a decrease in gamete formation (exflagellation).
- Reduced ookinete formation.
- Significant reduced oocyst formation, with diminished oocyst size and survival at 14- and 21-days post-infection.
- Δallan parasites were successfully transmitted to mice in bite-back experiments, showing that some viable sporozoites were produced

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