Expansion Microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid

Background

Malaria is caused by unicellular Plasmodium parasites that belong to the phylum Apicomplexa. During their lifecycle, apicomplexan parasites undergo multiple cellular differentiations into morphologically distinct forms capable of sexual and asexual replication, and dissemination via motility, egress and invasion of host cells. Each of these forms relies on microtubule cytoskeletal structures, including a structure unique for Apicomplexans – the apical complex, which is present in ookinete, sporozoites and merozoites. The fine structure and molecular composition of the apical complex differs among apicomplexan parasites, which probably reflects the different mechanical or functional requirements linked to the range of invaded host cells.Plasmodium has been traditionally considered to lack a structure called ‘conoid’ and as such, belongs to the Aconoidasida. To date, our understanding of Plasmodium microtubule structures has heavily relied on electron microscopy (EM). Super- resolution techniques have recently been implemented but various limitations remain. Thus, the structure and molecular composition of the Plasmodium microtubule cytoskeleton remain difficult to interrogate. In this work, Bertiaux and Balestra et al (1) implemented ultrastructure expansion microscopy (U-ExM) to expand various Plasmodium stages, and successfully resolved the structure of the axonemes, the mitotic hemispindles as well as the subpellicular microtubules. Moreover, the authors were able to study the conoid in Plasmodium ookinetes.

Key findings and developments

The authors began by testing the potential of U-ExM (2) to successfully expand and image Plasmodium, by focusing on tubulin structures. Imaging of the development of P. berghei microgametocytes into microgametes was successful, and enabled a 4.2-fold increase in size in the linear dimension. Imaging of the ookinete was also successful, and allowed the clear resolution of individual subpellicular microtubules that radiate from the apical polar ring, and also enabled a 4.2-fold increase in size in the linear dimension. Finally, expansion and staining of late schizonts highlighted previously described mitotic hemispindles (3), and achieved a 4.2-fold increase in size without major morphological distortions.

Having shown that sample expansion and visualization was successful in various stages, the authors went on to analyze axonemal assembly (labeling for a/b tubulin) in cellulo in expanded microgametocytes, as the exact mode of assembly remains unknown. They observed snapshots of axonemal assembly, and a wide range of patterns ranging from incomplete axoneme formation to fully assembled axonemes. Detailed analysis of whole-cell projections revealed that only 4% of activated gametocytes showed fully-formed axonemes, while the rest presented three main and non-exclusive intermediate states: a) less than 8 axonemes; b) within single gametocytes, incomplete and fully assembled axonemes coexisting within a shared cytoplasm; and c) comprising 44% of observations, axonemes displaying free singlets or doublets microtubules. Converse to the frequent observation of mis-assembled axonemes in developing gametocytes, the axoneme of free microgametes did not show such apparent defects. The authors then stained the gametocytes for polyglutamylated tubulin (PolyE), a post-translational modification that stabilizes microtubules with various roles in regulating flagellar motility. Polyglutamylation was observed on both assembling and full-length axonemes. Altogether, the authors prove that U-ExM allows visualization of axonemal microtubules and post-translational modifications in fully reconstructed gametocytes.

The authors then analyzed expanded late P. falciparum schizonts stained for a/b tubulin, and observed two distinct microtubule structures by U-ExM: the mitotic hemispindles and the subpellicular microtubules.

Finally, despite previous observations of the apical complex by ultrastructural studies, there is relatively limited knowledge of its molecular composition and variation across the different zoite stages in Plasmodium. To analyze the apical complex in ookinetes, the authors stained them for a/b tubulin and polyglutamylated tubulin. U-ExM allowed visualization of a large number of sub-pellicular microtubules radiating from the apical polar ring, covering most of the ookinete body. Importantly, the improved resolution of U-ExM in ookinetes allowed observing a ring of tubulin above the apical polar ring, which had never before been described, but was reminiscent of a conoid. While the conoid is thought to be absent in the Plasmodiumlineage, some proteins associated to the conoid in Toxoplasma are also conserved in Plasmodium (as recently shown (4)). The authors visualized various conoid proteins. Moreover, using U-ExM combined with genetic modification of parasites, they showed that the position of the apical tubulin ring relative to the apical polar ring, depends on the activation of secretion and motility in ookinetes. Altogether, the authors use U-ExM to reveal that a divergent and reduced form of the conoid is actually conserved in the Plasmodium genus.

What I like about this preprint

I have a great interest in expansion microscopy, and have done some work with it for some time. I find it exciting as a method that can be used to answer many biological questions in parasitology. I think in this work, it was put to excellent use, and has opened various interesting questions for the future.

References

1. Bertiaux E, Balestra AC, et al, Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid. bioRxiv (2020).
2. Gambarotto, D. et al. Imaging cellular ultrastructures using expansion microscopy (U- ExM). Nature Methods (2019).
3. Mehnert, A.-K., et al. Immunofluorescence staining protocol for STED nanoscopy of Plasmodium-infected red blood cells. Molecular and Biochemical (2019).
4. Koreny et al. Conservation of the Toxoplasma conoid proteome in Plasmodium reveals a cryptic conoid feature that differentiates between blood- and vector-stage zoites, bioRxiv (2020).

 

doi: https://doi.org/10.1242/prelights.23206

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