Abstract
Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus 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 in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Delta kinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.
Author summary Kinesins 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 of 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 up to nine kinesins and we further investigate the role of kinesin-8X throughout the Plasmodium berghei life cycle using biochemical, cellular 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 located on putative microtubule organizing centres (MTOCs) and spindles during cell division in most stages of the life cycle. By gene deletion we demonstrate that kinesin-8X is essential for normal oocyst development and sporozoite formation. Genome-wide RNA analysis of Delta kinesin-8X gametocytes revealed modulated expression of genes involved in microtubule-based processes. Overall, the data suggest that kinesin-8X is a molecular motor playing an essential role during endomitosis in oocyst development in the mosquito and parasite transmission.