Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Abstract

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics are not well understood in the different proliferative stages. Genome-wide homology analysis of Plasmodium spp. revealed the presence of two Kinesin-8 motor proteins (Kinesin-8X and Kinesin-8B). Here we have studied the biochemical properties of Kinesin-8X and its role in parasite proliferation. In vitro, Kinesin-8X showed motile and depolymerization activities like other Kinesin-8 motors. To understand its role in cell division, we have used protein tagging and live cell imaging to define the location of Plasmodium Kinesin-8X during all proliferative stages of the P berghei life cycle. Furthermore, we have used gene targeting to analyse the function of Kinesin-8X. The results reveal a spatio-temporal involvement of Kinesin-8X in spindle dynamics and its association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the Kinesin-8X gene showed that this protein is required for endomitotic division during oocyst development and is therefore necessary for parasite replication within the mosquito gut, and for transmission to the vertebrate host. Consistently, transcriptome analysis of Δkinesin-8X parasites reveals modulated expression of genes involved mainly in microtubulebased processes, chromosome organisation and the regulation of gene expression supporting a role in cell division. 4 Author SummaryKinesins are microtubule-based motors that play key roles in intracellular transport, cell division and motility. Members of the Kinesin-8 family contribute to chromosome alignment during cell division in many eukaryotes. However, the roles of kinesins in the atypical cell division in Plasmodium, the causative agent of malaria, is not known.In contrast to many other eukaryotes, Plasmodium proliferates by endomitosis, in which genome replication and division occur within a nucleus bounded by a persistent nuclear envelope. We show that the Plasmodium genome encodes only nine kinesins and we further investigate the role of Kinesin-8X throughout the Plasmodium life cycle using biochemical and gene targeting approaches. We show that Plasmodium Kinesin-8X has microtubule-based motility and depolymerization activity. We also show that Kinesin-8X is probably localized on putative MTOCs and spindles during cell division in most of the stages of P. berghei life cycle. By gene deletion we demonstrate that Kinesin-8X is essential for normal oocyst development and sporozoite formation. Genome-wide RNA analysis of Δkinesin-8X parasites reveals modulated expression of genes involved in microtubule-based processes. Overall, the data suggest that Kinesin-8X is a molecular ...

Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission

“…This functional discrimination is further supported by a recent study, which shows that deletion of α-1 tubulin does not affect genome replication and nuclear division but results in oocyst in which no MTs are detected (Spreng et al, 2019). There is also evidence that although kinesin-8X of P. berghei is associated with nuclear spindle formation, its deletion does not prevent microgamete formation (Zeeshan et al, 2019 Preprint ). These findings suggest a distinct molecular makeup and functionality of different MT arrays in Plasmodium parasites.…”

“…The metazoan kinesin-8B, Kif19, is involved in ciliary length regulation, but the role of kinesin-8B in axonemal assembly seems to be more profound in Plasmodium than controlling length alone. Kinesin-8B is consistently present in those Apicomplexa with flagellated gametes such as Plasmodium , Toxoplasma , and Eimeria and absent in other genera such as Theileria , Babesia , and Cryptosporidium , which lack flagellated gametes (Zeeshan et al, 2019 Preprint ). The properties of kinesin-8B contrast with those of Plasmodium kinesin-8X, which is located on the mitotic spindle (Zeeshan et al, 2019 Preprint ).…”

“…The Plasmodium genome encodes two kinesin-8s (kinesin-8X and kinesin-8B), along with six or seven other kinesin proteins (Wickstead et al, 2010; Vicente & Wordeman, 2015; Zeeshan et al, 2019 Preprint ). Kinesin-8X is conserved across the Apicomplexa, whereas kinesin-8B is restricted to certain genera such as Toxoplasma , Eimeria , and Plasmodium that have flagellated gametes (Zeeshan et al, 2019 Preprint ). However, the role of kinesin-8s in regulation of Plasmodium flagella is unknown.…”

Protein Phosphatase 1 regulates atypical mitotic and meiotic division inPlasmodiumsexual stages

“…The cell cycle in Plasmodium differs from that of many other eukaryotes with atypical mitotic and meiotic divisions throughout its life cycle 35, 37, 52 . It lacks several classical cell cycle regulators including the protein phosphatases CDC14 and CDC25 23 , and key protein kinases including polo-like kinases 46, 53 .…”

“…To examine PP1-GFP expression during erythrocyte stages, parasites growing in schizont culture medium were used for imaging at different stages (ring, trophozoite, schizont and merozoite) of development. Purified gametocytes were examined for GFP expression and localization at different time points (0, 1-15 min) after activation in ookinete medium 37 . Zygote and ookinete stages were analyzed throughout 24 h of culture.…”