Plasmodium bergheikinesin-5 associates with the spindle apparatus during cell division and is important for efficient production of infectious sporozoites

Abstract

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>Plasmodium</jats:italic> 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 <jats:italic>Plasmodium berghei</jats:italic> life cycle. Deletion of <jats:italic>kinesin-5</jats:italic> had little visible effect at any proliferative stage. The only significant effect was a 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 exhibited a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging during male gametogony showed that kinesin-5 is closely associated with NDC80, 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 and expressed in proliferating parasite stages, but it is not essential for parasite survival.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>Kinesin-5 is a motor protein with important roles during cell proliferation and therefore considered a strong target for therapeutics development against many diseases. The role of kinesin-5 in <jats:italic>Plasmodium</jats:italic> is unknown, but in this organism there are atypical aspects of cell division that differ from those of model eukaryotes. For example, classical regulators such as polo-like kinase, classical cyclins and cyclin dependent kinases are absent from <jats:italic>Plasmodium</jats:italic>. Here, we show that in <jats:italic>Plasmodium,</jats:italic> unlike in most model eukaryotes, kinesin-5 is dispensable, suggesting that the parasite uses somewhat different machinery for cell proliferation. The study is important because it shows that kinesin-5 is not essential universally for cell division and suggests that there may be other non-canonical mechanisms and regulators. The general concept that kinesin-5 is essential for spindle pole formation and chromosome segregation during cell division is not true for <jats:italic>Plasmodium</jats:italic>, and this is of interest to a broad readership.</jats:p></jats:sec>

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle

“…parasite produced significantly fewer infective salivary gland sporozoites (Fig. 4G) as reported previously (Zeeshan et al, 2020a). However, although several kinesin geneknockout lines exhibited defects in sporozoite production and reduced salivary gland…”

“…In our recent bioinformatic analysis of kinesins in Apicomplexa, we found nine kinesins encoded in the Plasmodium berghei genome, with members of three conserved kinesin subfamilies (kinesin-5, -8B, -8X and -13); kinesin-4, -15 and -20; and two Apicomplexa-specific kinesins: kinesin-X3 and -X4 (Zeeshan et al, 2019b). Surprisingly, kinesin-5 and the two kinesin-8 family members (kinesin-8X and -8B) were found to be not essential for blood stage proliferation (Zeeshan et al, 2020a;Zeeshan et al, 2019a;Zeeshan et al, 2019b). However, deletion of kinesin-5, which showed a clear localisation with the spindle at all proliferative stages, affected the production of infective sporozoites (Zeeshan et al, 2020a).…”

Molecular mechanism of a parasite kinesin motor and implications for its inhibition

“…Indeed, a small molecule screen identified a compound able to inhibit Plasmodium kinesin-5 ATPase activity, but not that of HsK5 21 . P. berghei kinesin-5 localises to mitotic and meiotic spindles in blood and mosquito stages of the parasite life cycle 51 , consistent with a conserved role for this motor in the parasite cell division machinery. Although we know very little else about the function of this motor, we infer from our biochemical and structural data that Plasmodium kinesin-5 is likely to play a MT-organising role within parasite spindles.…”

“…However, knockout of P. berghei kinesin-5 substantially reduces the number of sporozoites in oocysts and mosquito salivary glands. This highlights the operational diversity of replication at different parasite life cycle stages in general, and specifically suggests a key role for kinesin-5 in the multiple rounds of mitosis that occur during sporozoite production in the mosquito host 51 .…”

“…Although we know very little else about the function of this motor, we infer from our biochemical and structural data that Plasmodium kinesin-5 is likely to play a MT-organising role within parasite spindles. Kinesin-5 is not essential during the blood stages of the Plasmodium life cycle 51,52 . However, knockout of P. berghei kinesin-5 substantially reduces the number of sporozoites in oocysts and mosquito salivary glands.…”