Mutant/mutation

The mutant expresses ARA1 C-terminally tagged with 6HA. In addition, it expresses APR2 tagged with GFP at the C-terminus.

The endogenous ARA1, APRp2 and APRp4 proteins were tagged with a 6HA using the  CRISPR-Cas9 in apr2::gfp background (RMgm-5282) generating 3 single-tagged strains 3 double-tagged strains (apr2::gfp;ara1::6HA, apr2::gfp;aprp2::6HA, and apr2::gfp;aprp4::6HA) (see RMgm-5284, RMgm-5285, RMgm-5286).

Protein (function)
The Plasmodium ookinete, as well as two other invasive “zoite” stages (the salivary gland- and hepatocyte-invading sporozoite and the erythrocyte-invading merozoite), possesses a cortical pellicle unique to apicomplexan organisms. From outside to inside, the pellicle consists of a parasite plasma membrane, a double-membrane organelle inner membrane complex (IMC), and a layer of apically radiating subpellicular microtubules (SPMTs), both of which closely associate with each other and span along the periphery of the parasite. Variable numbers of SPMTs are assembled in different zoite stages, around 60 SPMTs in ookinetes. In Plasmodium, the SPMT arrays play at least two essential roles. One is as a scaffold supporting parasite morphogenesis, maintaining the unusual cell shapes and providing parasite rigidity during gliding and invasion. The other is as a platform for docking the apical secretory organelles, whose protein secretion is through a putative apical gateway for parasite gliding and invasion. Besides SPMTs, the invasive zoites of apicomplexan parasites also possess a highly conserved and specialized structure called the apical polar ring (APR) at the cell apical cortex. In the transmission electron micrograph of the Plasmodium ookinetes, APR has been described as an electron-lucent region beneath an electron-dense layer which is presumably the apical end of IMC. Since all the SPMTs emanate from APR, it has been widely accepted that APR functions as a microtubule-organizing center (MTOC) for nucleating and anchoring SPMTs at the Plasmodium zoites. So far, only two proteins, ARA1 (apical ring associated protein 1) and APR2 have been reported to display an APR-like localization by immunoelectron or fluorescence microscopy in the ookinetes of P. berghei. The orthologues of APR2 are encoded in many apicomplexan parasites, while ARA1 is unique to the Plasmodium parasites. In this study several new APR proteins have been identified including  APRp2 (PY17X_1322400; PF3D7_1454900) and APRp4 (PY17X_1322300; PF3D7_1454800).

Phenotype
The endogenous ARA1, APRp2 and APRp4 proteins were tagged with a 6HA using the  CRISPR-Cas9 in apr2::gfp background (RMgm-5282) generating 3 double-tagged strains (apr2::gfp;ara1::6HA, apr2::gfp;aprp2::6HA, and apr2::gfp;aprp4::6HA) (see RMgm-5284, RMgm-5285, RMgm-5286).

Close association between APR2 and each of ARA1, APRp2, and APRp4 detected at ookinete apical of the double-tagged strains.

Additional information
We demonstrate that APR2 is an APR-residing MT-binding protein that plays an essential role in parasite transmission in mosquitoes. APR2, with its APR-residing partners APRp2 and APRp4 identified by proximity labeling and yeast two-hybrid, coordinately regulate the structural integrity of the APR to ensure apical anchorage and integrity of SPMTs, which serves a critical role in the ookinete development, shape, and motility.

Analyses of mutants lacking expression of APR2 (RMgm-5280) showed the following:
- Measurement of DNA content indicated that female gametes of ∆apr2 could fertilize and further develop from diploid to tetraploid. However, the in vitro assay for zygote to ookinete differentiation revealed that ∆apr2 had a dramatic decrease in mature ookinete formation. The ookinetes from WT parasites were crescent-shaped; however, this shape was lost in all of the ∆apr2 and ∆apr2n ookinetes. Neither Δapr2 nor Δapr2n produced midgut oocysts on day 7 post-infection (pi). No salivary gland sporozoite formation.
The ookinetes from WT and comp parasites were crescent-shaped; however, this shape was lost in all of the Δapr2 and Δapr2n ookinetes with less cell bending, suggesting an alteration in the ookinete cytoskeleton in the absence of APR2. The Δapr2 and Δapr2n ookinetes displayed a significantly reduced gliding speed compared to WT.

Analyses of mutants containing tagged APR2 (and other mutants) revealed the following:

- APR2 localizes at apical polar ring (APR) throughout zygote to ookinete development.
To investigate the expression and localization of APR2 during ookinete development, we tagged the APR2 with a 6HA at either the N- or C-terminus in the 17XNL parasite using the CRISPR-Cas9 method, generating 6HA::apr2 and apr2::6HA clones (see RMgm-5281). APR2 expression was detected in other parasite stages, including asexual blood stage, gametocyte, midgut oocyst, and salivary gland sporozoite.  Apical localization of APR2 was also observed in merozoites and sporozoites.
We generated another two independent clones gfp::apr2 and apr2::gfp (see RMgm-5282) with endogenous APR2 tagged with GFP at the N- and C-terminus.
To compare the APR2 localization to proteins localized at the apical tubulin ring (ATR), a structure at the apical extremity of ookinetes, we genetically tagged the ATR-localized proteins Myosin-B (see RMgm-5290) and SAS6L (see RMgm-5291) respectively with a quadruple Myc epitope (4Myc) from the apr2::6HA parasite. Under the confocal microscope, Myosin-B and SAS6L marked the cellular localization of ATR, and APR2 was only detected at APR but not at ATR.
- APR2 associates with apical subpellicular microtubules (SPMTs)
Co-immunostaining of APR2 (HA epitope) and SPMT (alpha- and beta-Tubulin) in the apr2::6HA parasites detected apical co-localization of APR2 with SPMTs throughout ookinete development subpellicular microtubules (SPMTs)
- APR2 N-terminal region shows microtubules (MT)-binding and -stabilization properties
- APR2 C-terminal region could localize at APR.
Whether APR2-N (1–600 aa) with MT-binding activity is sufficient to target APR2 to the APR remains unknown. To test this, we deleted the coding region of 2–600 aa of endogenous APR2 in the apr2::gfp parasite, generating a modified clone APR2-ΔN. (see RMgm-5283).Surprisingly, the C-terminal part (601–1394 aa) of APR2 in the APR2-ΔN ookinetes still showed APR localization, although its fluorescent signal at APR was greatly decreased compared with that of the full-length APR2 in the apr2::gfp ookinetes.
- APR2 is required for integrity and apical anchorage of APR-SPMT.
- Cortical arrangement of SPMT is impaired in the APR2-null ookinetes.
- Three proteins were identified with a close interaction with APR2, ARA1 (PY17X_1412750; PF3D7_1312450), APRp2 (PY17X_1322400; PF3D7_1454900) and APRp4 (PY17X_1322300; PF3D7_1454800). 
The endogenous ARA1, APRp2 and APRp4 proteins were tagged with a 6HA using the  CRISPR-Cas9 in both WT and apr2::gfp background (seeRMgm- 5284, RMgm-5285, RMgm-5286) generating 3 single-tagged strains (ara1::6HA, aprp2::6HA, and aprp4::6HA) and 3 double-tagged strains (apr2::gfp;ara1::6HA, apr2::gfp;aprp2::6HA, and apr2::gfp;aprp4::6HA). The close association between APR2 and each of ARA1, APRp2, and APRp4 was further confirmed using the PLA assay, in which the signals were detected at ookinete apical of the double-tagged strains.
- APR2-APRp2-APRp4 module regulates apical anchorage of APR-SPMT.
To explore the functional relevance of ARA1, APRp2, and APRp4 in controlling the integrity and apical anchorage of APR-SPMT, we disrupted each of these genes in WT parasite using the CRISPR-Cas9, obtaining 3 mutant strains Δara1, Δaprp2, and Δaprp4 (see RMgm-5287, RMgm-5288, RMgm-5289) 
We deleted the endogenous apr2 gene in each of aprp2::6HA and aprp4::6HA strains, obtaining aprp2::6HA;Δapr2 and aprp4::6HA;Δapr2mutants. 

Other mutants