Real time live-cell imaging of PbARK2 reveals spindle association and kinetochore dynamics during male gametogony. The upper schematic shows the major stages of male gametogony with subcellular structures identified. (A) Imaging of ARK2-GFP (green) during male gametogony reveals an initial location at the putative microtubule organizing centre (MTOC) just after activation (1 minute post activation; mpa), and at the spindles and spindle poles in later stages. The protein accumulates diffusely in the residual nuclear body after gamete formation and is not present in the flagellate gametes (15 mpa). Scale bar = 5 μm. (B) Still images (5 sec intervals) showing development of an ARK2-GFP bridge from one focal point followed by further division into two halves within 1 to 2 mpa. Scale bar = 5 μm. (C) The location of ARK2-mCherry (red) relative to kinetochore marker, NDC80-GFP (green). Scale bar = 5 μm. (D) Still images (5 s intervals) of the dynamic location of ARK2-mCherry and NDC80-GFP between 1 and 2 min of activation. Scale bar = 5 μm. (E) The relative location of ARK2-GFP (green) and the basal body and axoneme marker, kinesin-8B-mCherry (red). Scale bar = 5 μm. (F) Still images (5 s intervals) of the dynamic location ARK2-GFP and kinesin-8B-mCherry between 2 and 3 min of activation. Scale bar = 5 μm. (G) Indirect immunofluorescence followed by STED confocal microscopy showing co-localization of ARK2 (purple) and α-tubulin (green) at spindle but not at cytoplasmic microtubules at 1 mpa. Scale bar = 1 μm

Zeeshan M, Rea E, Abel S, Vukušić K, Markus R, Brady D, Eze A, Raspa R, Balestra A, Bottrill AR, Brochet M, Guttery DS, Tolić IM, Holder AA, Roch KGL, Tromer EC, Tewari R. Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages. bioRxiv [Preprint]. 2023 PMID: 36778504;