Plasmodium berghei Kinesin-5 Associates With the Spindle Apparatus During Cell Division and Is Important for Efficient Production of Infectious Sporozoites

Abstract

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-color live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites

“…We further clarify microtubule dynamics, which underlie their heterogeneous organization, previously described in fixed cells (Read et al, 1993;Fennell et al, 2008;Arnot et al, 2011), or using the microtubule-associated protein Kinesin-5 in live-cell imaging (Zeeshan et al, 2020a). Hemispindles are already present early in mononucleated cells indicating that they are not per se postanaphase remnants as suggested earlier (Canning & Sinden, 1973).…”

“…In early transmission EM studies, mainly carried out in oocysts in the mosquito midgut, centriolar plaques appear as electron-dense areas that neither show centrioles nor any other distinct structures (Aikawa et al, 1967;Terzakis et al, 1967;Aikawa & Beaudoin, 1968;Howells & Davies, 1971;Canning & Sinden, 1973;Sinden et al, 1976;Schrevel et al, 1977). Centriolar plaques seem partially embedded in the nuclear membrane, but their positioning relative to the nuclear pore-like "fenestra" remains unclear (Aikawa & Beaudoin, 1968;Wall et al, 2018;Zeeshan et al, 2020a). Generally, the amorphous appearance of centriolar plaques in EM has precluded a detailed analysis of their organization so far.…”

“…In another study, hemispindles observed in oocysts were interpreted as postanaphase spindles (Canning & Sinden, 1973). More recent fluorescence live-cell imaging data of the microtubule-associated protein Kinesin-5 in asexual and sexual stages demonstrated the dynamic elongation of the mitotic spindle, while hemispindles were not resolved (Zeeshan et al, 2020a). These data provide a controversial view on the occurrence, dynamics, and function of hemispindles, which needs to be clarified.…”

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly

“…Observations by electron and fluorescence microscopy gave general insights into the dynamics of cellular structures during microgametogenesis [ 4 , 11 – 13 ] ( S1 Fig ). The mature microgametocyte shows two electron-dense structures that have been linked with the formation of the mitotic spindles and the axonemes, respectively.…”

The Modular Circuitry of Apicomplexan Cell Division Plasticity

“…The next step is the separation of the chromosome sets. Kinesin motors have been characterized in Plasmodium berghei: kinesin-5 (Eg5 ortholog) and kinesin-8X associate with the spindle across various developmental stages, including schizogony, but surprisingly, were only essential during the mosquito stages (Zeeshan et al, 2019;Zeeshan et al, 2020a). Phenomenologically, completion of mitosis is not necessarily concluded by karyokinesis, which is skipped in some apicomplexan division modes.…”