Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages

Abstract

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, omics and live-cell fluorescence imaging, we set out to investigate the contribution of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus associated with kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1 and lacking some other conserved molecules. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.

A non-canonical Arp2/3 complex is essential forPlasmodiumDNA segregation and transmission of malaria

“…In contrast, deletion of EB1 in P. falciparum and P. yoelli leads to the formation of gametes that were entirely deficient of DNA and incapable of fertilising females, highlighting interesting species-specific differences in DNA segregation in male gametocytes (32,47). A paternal phenotype followed by an early oocyst arrest has also been described for other Plasmodium parasite lines lacking proteins such as the formin-like protein MISFIT (49), the microtubule motor kinesin-8X (50), the aurora kinase Ark2 (48), and for a parasite line that expresses actin 1 in place of actin 2 (44). While their precise connection to the Arp2/3 complex or Arp2/3mediated actin polymerization remains to be determined, a unifying feature of these paternal defects is that they do not affect ookinete development but arrest parasites only in the oocyst stage.…”

“…A core protein connecting the kinetochores to the spindle during male gametogenesis is end-binding protein 1 (EB1) ( 32 , 47 , 48 ). Deletion of EB1 in P. berghei resembles the phenotype of ARPC1(-), with an arrest in a delayed-death like manner in early oocysts ( 48 ).…”

“…A core protein connecting the kinetochores to the spindle during male gametogenesis is end-binding protein 1 (EB1) ( 32 , 47 , 48 ). Deletion of EB1 in P. berghei resembles the phenotype of ARPC1(-), with an arrest in a delayed-death like manner in early oocysts ( 48 ). In contrast, deletion of EB1 in P. falciparum and P. yoelli leads to the formation of gametes that were entirely deficient of DNA and incapable of fertilising females, highlighting interesting species-specific differences in DNA segregation in male gametocytes ( 32 , 47 ).…”

“…While their precise connection to the Arp2/3 complex or Arp2/3- mediated actin polymerization remains to be determined, a unifying feature of these paternal defects is that they do not affect ookinete development but arrest parasites only in the oocyst stage. Interestingly, immunoprecipitation of Ark2 and EB1 revealed many interacting proteins including Akit7 but not a single subunit of the Arp2/3 complex ( 48 ), suggesting a dynamic interplay between the kinetochores and the Arp2/3 complex.…”

PlasmodiumNEK1 coordinates MTOC organisation and kinetochore attachment during rapid mitosis in male gamete formation

“…During male gametogenesis, this pattern was even clearer: the number and location of the NEK1 foci correlated with the ploidy of the parasite, and the splitting of the MTOC was observed associated with NEK1 foci at the different mitotic stages during genome replication from 1N to 8N (Fig 2A). The localisation of NEK1 in real time relative to a number of markers for kinetochores (NDC80) (Zeeshan et al, 2020), microtubules (EB1), spindle kinase (ARK2)(Zeeshan et al, 2023), and axoneme marker (Kinesin 8B) (Zeeshan et al, 2019a), showed that NEK1 is part of the outer MTOC in the cytoplasmic compartment. This was confirmed by expansion microscopy and super resolution microscopy using various centrosome and spindle markers, including centrin, γ-tubulin and α-tubulin.…”