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

Eloïse Bertiaux, Aurélia C Balestra, Lorène Bournonville, Mathieu Brochet, Paul Guichard, Virginie Hamel

Preprint posted on 8 July 2020

Article now published in PLOS Biology at

Expansion microscopy: a new toolkit for parasitology - investigating the conoid.

Selected by Mariana De Niz

Categories: cell biology


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.

Figure 1. Identification and characterisation of a conoid-like structure in P. berghei ookinetes. Highlight of 3 subregions: (left) distal, (middle) centre and (right) apical regions.

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.


  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).


Posted on: 19 July 2020


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Author's response

Virginie Hamel, Mathieu Brochet, Paul Guichard, Eloise Bertiaux shared

Open questions

1.Were there any specific limitations you found when using U-ExM in red blood cells and Plasmodium?

We did not encounter any specific limitations when using U-ExM in the studied Plasmodium stages. We did not have either to optimize the classical U-ExM protocol used for human cells as it worked readily. The only limitation was the relatively long acquisition time at the confocal microscope for such expanded samples! However, we noticed that some of the antibodies we tested did not work in U-ExM notably the ones specific for membrane or membrane-associated proteins (p21, AMA1, and MSP1). It is likely that some optimization of the protocol (antibodies, fixation, denaturation time, etc) would be needed to alleviate this issue.

2.You mention early in the manuscript, previous work that has been done using SIM and STED. U-ExM is an alternative to super-resolution microscopy. What limitations did you observe in terms of resolution and otherwise, compared to using SIM or STED?

We did not use SIM ourselves on Plasmodium samples. However, when we tried STED on gametocytes, we encountered the same problems that other groups previously described, i.e., the rapid cell disintegration when hemozoin crystals from the parasites are illuminated with the high-power STED laser. U-ExM alleviates such issues and allows imaging the microtubule structures within gametocytes with unprecedented details. However, so far, the expansion factor is limited to 4.5, so the resolution may not still be sufficient to discriminate very tiny structures such as the ones found in the schizonts for example.

3.Did you notice any artefacts that could be introduced during expansion of the sample? If so, how often did this happen, and should users of the method be aware of specific things to look for?

We did not notice any obvious deformation of the sample that could have been introduced during the expansion procedure. To ensure that the expansion is isotropic, the users of the method should always measure the expansion factor of the gel itself as well as the expansion of the biological sample. Both expansion factors should be identical.

4.Out of curiosity, did you try the method and visualization in P. falciparum and P. berghei for all the questions? If not, why did you chose specific Plasmodium strains for the different questions? If yes, did you see any difference among them?

When we initially discussed testing U-ExM in Plasmodium, we first thought about imaging axonemes and/or centrioles as these structures represented the obvious shared interest between our two groups. Could we use U-ExM to study the mysterious biology of these structures in Plasmodium? When we saw the first images of P. berghei gametocytes, we immediately realised that U-ExM had a potential to change our vision of other Plasmodium stages. So, we decided to look at our other favourite stages we study in the lab, i.e. motile P. berghei ookinetes and P. falciparum schizonts, which all express distinct microtubule structures. We believe our analysis only represents the tip of the iceberg and that many groups will now implement U-ExM and develop new protocols to specifically address their research projects in many other developmental stages.

5.What do you hypothesize is the relevance of the conoid in Plasmodium zoites, and to what extent are those functions conserved relative to other organisms (parasitic or otherwise)?

Good question! Together with the work from Ross Waller and Rita Tewari, we show that the conoid is a more conserved feature in the phylum Apicomplexa than previously expected. We also confirm that not only the conoid itself significantly varies across apicomplexans but also the apical complex as a whole. It was suggested that the conoid plays a mechanical role in the invasion or traversal of the host cell by parasites. The variations in both the structure and composition of the apical complex likely reflect adaptations to the different host cells or environment encountered by apicomplexan zoites.

So far, it was difficult to tackle the role of the apical complex in Plasmodium without knowing its composition and without an accessible approach to visualise it. Now that some of the components of the Plasmodium apical complex have been identified by the groups of Ross Waller and Rita Tewari, it will be possible to investigate their function in Plasmodium and we are certain that U-ExM will be instrumental to do so!

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