The threePlasmodium falciparumAurora-related kinases display distinct temporal and spatial associations with mitotic structures in asexual blood stage parasites and gametocytes

Abstract

Aurora kinases are crucial regulators of mitotic cell cycle progression in eukaryotes. The protozoan malaria parasite Plasmodium falciparum replicates via schizogony, a specialised mode of cell division characterized by consecutive asynchronous rounds of nuclear division by closed mitosis followed by a single cytokinesis event producing dozens of daughter cells. P. falciparum encodes three Aurora-related kinases (PfARKs) that have been reported essential for parasite proliferation, but their roles in regulating schizogony have not yet been explored in great detail. Here, we engineered transgenic parasite lines expressing GFP-tagged PfARK1-3 to provide a systematic analysis of their expression timing and subcellular localization throughout schizogony as well as in the non-dividing gametocyte stages, which are essential for malaria transmission. We demonstrate that all three PfARKs display distinct and highly specific and exclusive spatiotemporal associations with the mitotic machinery. In gametocytes, PfARK3 is undetectable and PfARK1 and PfARK2 show male-specific expression in late stage gametocytes, consistent with their requirement for endomitosis during male gametogenesis in the mosquito vector. Our combined data suggest that PfARK1 and PfARK2 have non-overlapping roles in centriolar plaque maturation, assembly of the mitotic spindle, kinetochore-spindle attachment and chromosome segregation, while PfARK3 seems to be exquisitely involved in daughter cell cytoskeleton assembly and cytokinesis. These important new insights provide a reliable foundation for future research aiming at the functional investigation of these divergent and possibly drug targetable Aurora-related kinases in mitotic cell division of Plasmodium falciparum and related apicomplexan parasites.

ThePlasmodium falciparumhistone methyltransferase PfSET10 is dispensable for the regulation of antigenic variation and gene expression in blood stage parasites

“…5B). To provide an unbiased quantification, we integrated the signal intensities originating from PfSET10-mNG and Pfg377 labeling using ImageJ as described previously 56 . This analysis revealed that PfSET10-mNG was only expressed in gametocytes that also displayed strong Pfg377 signal intensities, indicating that in late stage gametocytes PfSET10 is indeed only expressed in females (Fig.…”

“…To assess the sex-specific expression of PfSET10-mNG in stage V gametocytes (day 13), IFAs α-mNeonGreen and α-Pfg377 antibodies and DAPI were performed on methanol/acetone-fixed thin blood smears. Image acquisition with the 40x objective was followed by automated quantification of the fluorophore signal intensities emitted from the secondary antibodies (Alexa Fluor 488-conjugated α-mouse IgG, Alexa Fluor 568-conjugated α-rabbit IgG) and DAPI using a Fiji macro (ImageJ version 1.52n) as described previously 56 . In brief, images were segmented based on the Pfg377 signal using the auto-threshold “Triangle”, which identified potential male and female gametocytes.…”