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

Abstract

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.

Plasmodium SAS4: basal body component of male cell which is dispensable for parasite transmission

“…Whereas kinesin-8B is part of the axoneme assembly, SAS4 is limited to the BB. This is consistent with previous results showing SAS4 in cytoplasmic compartment associated with the dense BB ( Rashpa & Brochet, 2022 ). We show that SAS4 duplication is synchronized with the accumulation of NDC80 and spindle formation during successive rounds of genome replication during mitosis.…”

“…In contrast to Plasmodium SAS6 that was shown to be important for male gamete formation ( Marques et al, 2015 ), we found that SAS4 is not essential for exflagellation as there was no significant change in flagellated gamete formation. Our findings are corroborated with a recent study showing a non-essential role of SAS4 in parasite transmission, although it described a significant decrease in male gamete formation ( Rashpa & Brochet, 2022 ). At other stages of parasite development, including zygote formation, ookinete and sporozoite development, and parasite transmission and infectivity, no significant differences from the WT-GFP parasite were observed.…”

“…It will be interesting in the future to analyse in depth the 3D structure of Plasmodium BBs and their components, for example, by using these BB and mitotic spindle markers and correlative light and electron microscopy (CLEM) as described recently for the bryophyte Physcomitrium ( Pereira et al, 2021 ). In a recent study, Raspa and Brochet have used expansion microscopy to study the MTOC structures showing its bipartite architecture in Plasmodium ( Rashpa & Brochet, 2022 ), complementing our findings by live cell imaging, super resolution, and EM. The protein interactome obtained using these lines and others will provide tools to identify BB components and understand their evolution in this divergent organism, which assembles the complete BB complement de novo in 8 min.…”

Form follows function: Variable microtubule architecture in the malaria parasite

“…2-4). Although recent expansion microscopy data hints at a presence of ring-like structures at gametocyte extremities (Rashpa and Brochet, 2022), there was no evidence of an APR-like structure that could act as a MTOC at gametocyte poles. SPMTs originated at apparently uncoordinated positions and there was no discernible pattern in the distribution of SPMTs along the IMC with respect to the number of protofilaments or secondary tubules.…”

“…Recently, expansion microscopy has provided overviews of the SPMT scaffold in some Plasmodium species and forms (Bertiaux et al, 2021; Rashpa and Brochet, 2022) and results from the first purification of tubulin from asexual blood forms were published (Hirst et al, 2022). Even with these advances, our understanding of P. falciparum’s microtubules remains incomplete, particularly in less-studied forms such as the mosquito forms and “pre-sexual” gametocytes.…”