Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of of EB1 

Protein (function)
End binding proteins (EBs) are the conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. It is not expressed in asexual blood stages but is expressed in the nucleus of gametocytes.

Phenotype
EB1-GFP showed during male gamete formation a spatiotemporal distribution with distinct foci and elongated spindle ‘bridges’ at certain time points after gametocyte activation (see also information below).
Live cell imaging of EB1-GFP during early meiosis located the protein on spindles and spindle poles, but it then disappeared as the ookinete matured. There was also an accumulation of EB1-GFP at the nascent apical end of the developing ookinete.
In later stages of ookinete differentiation, it was distributed around the periphery of the growing protuberance, potentially associated with sub-pellicular MTs but had disappeared in mature ookinetes.

Analysis of a mutant lacking expression of EB1 (RMgm-5318) showed the following: Significantly reduced oocyst numbers on day-10 post-infection of mosquitoes. The oocysts that were present were smaller than those of WT parasites, and by day-21 no oocysts were detectable.

Additional information
Real time live cell imaging of parasites expressing EB1-GFP reveals its association with the spindle and kinetochore throughout male gamete formation.

Chromatin immunoprecipitation with parallel sequencing (ChIP-Seq) was used to determine the DNA binding sites of EB1, and indicated its co-location with the outer kinetochore marker NDC80 centromeric chromatin marker. These results were corroborated by live-cell imaging of EB1-GFP/NDC80-mCherry dual reporter lines. An additional cross to produce EB1-GFP/ARK2-mCherry parasites showed overlap of fluorescence signals at 1 to 2 min post-activation of gametocytes, confirming their co- location and interaction with spindles and the kinetochore. Finally, parasite lines expressing EB1-mCherry and the basal body marker SAS4-GFP showed EB1’s association with the formation of basal bodies that serves as the MT organising centre for axonemes.

To further resolve the location of EB1 with respect to the kinetochore and basal body at higher resolution, 3D-SIM was performed on EB1-GFP/NDC80-mCherry, EB1-GFP/ARK2-mCherry and EB1-mCherry/SAS4-GFP fixed gametocytes. The 3D-SIM images of gametocytes expressing EB1-GFP/NDC80-mCherry showed EB1 bridge(s) across the nucleus with NDC80 distributed like beads on the bridge, each bead representing a kinetochore. The 3D-SIM images of gametocytes expressing EB1-GFP/ARK2-mCherry showed EB1 bridge(s) across the nucleus with ARK2, overlapping each other. The bridged pattern of spindles for EB1 were restricted to the nucleus as shown by 3D-SIM images of gametocytes expressing EB1-mCherry/SAS4- GFP; whereby SAS4 was located in the cytoplasm but aligned with the EB1 bridge in the nucleus. We also performed STED microscopy on fixed EB1-GFP gametocytes stained with anti-GFP and anti-tubulin antibodies, which confirmed EB1’s location on the spindle: the images showed EB1 distribution on the nuclear spindle MTs with a distribution like that of ARK2. These real time imaging data, together with the interactome data, confirm that ARK2 and EB1 form a functional/structural axis associated with the spindle and the acentriolar MTOC, and are associated with kinetochore dynamics.

In the paper evidence is presented for a unique scaffold of an aurora-related kinase (ARK2; PBANKA_0407400; PF3D7_0309200; serine/threonine protein kinase ARK2, putative) at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission.

Other mutants