Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Benjamin Liffner, Ana Karla Cepeda Diaz, James Blauwkamp, David Anaguano, Sonja Frölich, Vasant Muralidharan, Danny W. Wilson, Jeffrey Dvorin, Sabrina Absalon

Posted on: 13 June 2023 , updated on: 3 July 2023

Preprint posted on 15 May 2023

Divide et Impera. Thanks to U-ExM, cheap and high resolution imaging maps the asexual reproduction cycle of malaria parasites in red blood cells

Selected by Nadja Hümpfer

Categories: biochemistry, cell biology, developmental biology, microbiology

Background

Malaria is a fatal disease transmitted by parasites of the species Plasmodium. Their life cycle includes replication of the organism inside red blood cells of infected vertebrates. Five species are known to infect humans, but Plasmodium falciparum accounts for the majority of severe cases in human. Plasmodium have a complex life cycle with different stages. Their sexual reproduction takes place in insect (mostly mosquito) vectors, after which parasites at the sporozoite stage are transmitted to the human host during blood meals. Inside the human host, they first infect hepatocytes (liver stages) and then erythrocytes (blood stages). At these life stages, they reproduce asexually by duplication of genetic and cellular material to produce several merozoites inside the infected host cell. Merozoites are released and the erythrocyte is destroyed in the process, which causes the infected host to feel the typical malaria symptoms such as fever and malaise. The merozoites can then infect the next pool of erythrocytes. Depending on the duration of the replication cycle, the malaria symptoms can occur in turns of two to three days. A fraction of the parasites turns into gametocytes (sexual stage), which are ingested by the mosquito to enable transmission between the host and vector.

In this preprint, the authors use ultrastructure expansion microscopy (U-ExM) [1] to investigate the cellular organization of P. falciparum asexual reproduction in erythrocytes. This technique allows magnification by physically expanding the sample. Therefore, the resolution can be increased by the factor of expansion, 4.5-fold in the case of U-ExM. In addition, the U-ExM variety of ExM [2] is optimally suited for the staining of denatured proteins after expansion. This makes tightly packed structures accessible. In their work, Liffner & Cepeda Diaz et al. combine antibody-based staining for 13 different organelles and markers with a pan-staining [3] for cellular context. The pan-protein staining was achieved by labelling the entire protein content with NHS-ester coupled dyes. They also visualized the membranes by employing BODIPY TRc. The EM-like context together with specific information from antibodies allowed the authors to collect a full ‘atlas’ of P. falciparum development in erythrocytes.

Key findings

Overall, the study provides a comprehensive overview of the P. falciparum developmental stages inside red blood cells. As the authors frame it, they put together an ‘atlas’ of unprecedented detail that shows how the parasite transforms from the ‘ring state’, right after the infection, into a trophozoite and finally into the schizont to produce the merozoites that are the infectious particles. The improved resolution of U-ExM, together with the decrowding effect and the cellular context, allows the authors to describe the Plasmodium life cycle in great detail and to support common hypotheses in the field with their data.

The emphasis of the study is the biological side; the method was merely altered compared to the original U-ExM protocol. In this respect, the preprint is of particular interest to Plasmodium biologists, who are at ease with the specific Plasmodium terminology of organelles and developmental stages. For an overview of the intraerythrocytic cycle of P. falciparum, check out this schematic as published in [4].

Glossary

Apicoplast	A plastid found in most Apicomplexa. It is non-photosynthetic but indispensable for the invasion of host cells.
Basal Complex	A ring-shaped protein complex that segments the multinucleated schizont into individual merozoites during cytokinesis.
Cytostome	Endocytic compartment that allows the parasite to feed of the host red blood cell’s hemoglobin.
Merozoite	Infectious unit of Plasmodium that is produced after the segmentation of the schizont into multiple of merozoites.
Rhoptry	Secretory organelle inside the merozoites. It secretes proteins that are involved in host cell invasion
Ring	The first stage after Plasmodium infects the red blood cell.
Schizogony	The process of multiple rounds of mitosis and nuclear fission, followed by cytokinesis; which allows the Plasmodium to replicate asexually.
Schizont	Multinucleated stage of Plasmodium inside the red blood cell.
Trophozoite	Feeding stage of Plasmodium inside the red blood cell.

Asexual developmental stages of *Plasmodium falciparum* in erythrocytes. Liffner & Cepeda Diaz et al. use U-ExM together with pan-staining for the total protein content (NHS) and membranes (BODIPY), SYTOX DNA stain and a marker for the cytoplasm (aldolase) to show ultrastructural details of *Plasmodium* schizogony. Image taken from the supplementary material (S1) of Liffner & Cepeda Diaz *et al*., 2023, bioRxiv.

The MTOC has an important part in scaffolding organelles during segmentation

A big section of the study focuses on the role of the microtubule organizing center (MTOC) in mitosis and cytokinesis. The MTOC could be visualized by NHS-ester staining. Imaging suggested that the MTOC anchors the mitotic nuclei to the plasma membrane in the trophozoite. The MTOC cytoplasmic extensions remained in contact with different organelles during cytokinesis, such as the Golgi, the rhoptries, and the basal complex. The MTOC also associated with the mitochondrion and the apicoplast at the end of segmentation, anchoring them to a common point inside the cell. Therefore, the authors assign the MTOC the important role of controlling the apico-basal polarity during schizogony.

Organelle segmentation follows the branching point fission model

An interesting section of the study investigates the fission of two organelles: the mitochondrion and the apicoplast. An early trophozoite has one of each organelle, but during schizogony, both need to increase their size and volume and be split into smaller bits to be inherited by the developing merozoites upon segmentation. Staining of mitochondrion and apicoplast markers, together with the basal complex that is responsible for the segmentation of the schizont into smaller individual units (i.e. the merozoites), argues for a branching point fission model: That is, first, the organelles grow; second, they are segmented at a few points. Finally, they undergo another round of fission to produce the accurate number of organelles mirroring the number of merozoites.

NHS-ester staining provides contrast to depict the cytostome of P. falciparum

The pan-protein staining with NHS-ester coupled dyes highlights several protein-dense structures in the Plasmodium cell. Some of them are clearly identifiable by their morphology (such as the rhoptry bulbs, which are discussed below). The authors noticed a small ring-like structure at the interface of the parasite membrane and the parasitophorous vacuole that harbors Plasmodium inside the erythrocyte. They speculated this to reflect the collar of the cytostome, an endocytic structure that allows the parasite to feed on hemoglobin from the erythrocyte cytoplasm. They used Kelch13 (K13), a recently described marker for the endocytic micropore in Toxoplasma (Plasmodium and Toxoplasma are both members of the phylum Apicomplexa of parasites) to detect the collar in Plasmodium. K13 localized to the protein-dense rings and was confirmed as a marker for the cytostome. Altering the fixation protocol even allowed the authors to visualize the content of the endocytic pore.

Asymmetric rhoptry biogenesis

Rhoptries are organelles that merozoites need to invade erythrocytes. Like other organelles, they need to be multiplied and segregated equally between the developing merozoites. The authors observed two interesting aspects of rhoptry biogenesis. First, they noticed a tight association between the rhoptries and the microtubule organizing center (MTOC) throughout schizogony. Second, they observed different densities in terms of protein content in the rhoptries. Surprisingly, the differences in protein density did not reflect the maturity of the rhoptry. At least the localization of a specific marker of mature rhoptries, RON4, to the less dense rhoptries suggested that density is not positively correlated with rhoptry age.

What I like about this preprint

This work shows the application of an upcoming imaging technology. Expansion microscopy circumvents the diffraction limit of fluorescent microcopy by physical expansion of the sample. Since its first publication in 2015 [2], many methodological papers have been published, focusing on the improvement of the expansion factor or the combination of expansion microscopy with other super-resolution imaging techniques. As is often the case with the advent of a new technique, it takes time until the method can be used to answer biological questions. In their study, Liffner & Cepeda Diaz et al. prove the value of ExM to biology and generate an impressive amount of data, which will be made available to the community so people can browse the atlas and answer additional questions. They achieved this by taking advantage of the traits of U-ExM. It is cost effective, easy to implement with already established staining protocols, increases the spatial resolution and provides ultrastructural context. Therefore, it could serve as a bridging technology between super-resolution fluorescence imaging and electron microscopy.

I especially like how biologists working with diverse model systems adopt ExM. They apply the general protocol to different organisms, such as Plasmodia [5], Chlamydia [6], Trypanosoma [7] and Toxoplasma [8]. ExM was even explored on the pilot expedition of EMBL’s Traversing European Coastlines project to study plankton.

The authors also point out structures that could not be resolved with their protocol. They find that the hemozoin crystal that fills the inside of the schizont could not be detected. This is likely due to its composition. Hemozoin is produced by the parasite during digestion of hemoglobin. It leaves the heme groups and turns them into a crystalline form, which likely cannot be expanded. The different layers of parasite and cellular membranes were too dense to be distinguished with the improved resolution, unfortunately.

Nevertheless, this Plasmodium atlas could become a powerful resource for researchers in the field, especially once the imaging data will be publicly available.

The preprint is published in eLife together with reviewers’ comments.

References

[1] Gambarotto, D., et al. (2019). “Imaging cellular ultrastructures using expansion microscopy (U-ExM).” Nat Methods 16(1): 71-74.

[2] Chen, F., et al. (2015). “Optical imaging. Expansion microscopy.” Science 347(6221): 543-548.

[3] M’Saad, O. and J. Bewersdorf (2020). “Light microscopy of proteins in their ultrastructural context.” Nat Commun 11(1): 3850.

[4] Tilley, L., et al. (2011). “The Plasmodium falciparum-infected red blood cell.” Int J Biochem Cell Biol 43(6): 839-842.

[5] Bertiaux, E., et al. (2021). “Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.” Plos Biology 19(3).

[6] Kunz, T. C., et al. (2019). “Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy.” Front Cell Infect Microbiol 9: 276.

[7] Kalichava, A. and T. Ochsenreiter (2021). “Ultrastructure expansion microscopy in Trypanosoma brucei.” Open Biol 11(10): 210132.

[8] Oliveira Souza, R. O., et al. (2022). “IMC10 and LMF1 mediate mitochondrial morphology through mitochondrion-pellicle contact sites in Toxoplasma gondii.” J Cell Sci 135(22).

Tags: parasitology

doi: https://doi.org/10.1242/prelights.34838

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Author's response

Ben, Ana Karla, James & Sabrina shared

Firstly, a big thank you to Nadja for writing this PreLights article, we greatly appreciate your efforts! While we obviously work on malaria parasites, we were hopeful that this study could be useful to the broader cell biology community, and your article provides great accessibility to the findings of our study for that audience.

Prior to the publication of this PreLights article, Nadja reached out to us and during our correspondence proposed a few questions. Below, we will state and answer these questions.

What were some of the particular challenges you had to overcome to get U-ExM working in your labs?

As is the case for any protocol that occurs over multiple days, with dozens of steps, going from written protocol to working experiment is never easy. Speaking on behalf of the Absalon Lab, we first tried U-ExM in 2020 without a lot of success. After 3 or 4 attempts that were unsuccessful for various reasons, we got in contact with Julien Guizetti’s lab (Heidelberg University Hospital) who had U-ExM up and running in the lab. They generously gave their feedback on what could’ve been causing our problems, and with their help we were able establish U-ExM in the Absalon Lab.

From there, working out how best to image and analyse our samples became the major hurdle. U-ExM increases your sample volumetrically approximately 90-fold (4.5³), which makes for a drastically increased imaging area, imaging time and file size. Finding the best balance between taking high quality images, taking as many images as possible, and minimising sample bleaching took many months to get right. On top of that, many of our images were coming out at >10 Gb, generating file and dataset sizes that we weren’t used to handling or storing. These were issues that were really overcome through experience, trying little tweaks, seeing what works and what doesn’t.

Finally, once U-ExM and our imaging and analysis pipelines were established, we got to optimizing the protocol for our samples. In this study we looked at a whole range of parasite lifecycle stages. To do this, we needed to synchronise parasites and then harvest them at designated times. In a practical sense, this clashed with the original U-ExM protocol, because the first day was over 10 hours long. Considering this, harvesting parasite lifecycle stages that may be separated by many hours (like pre and post segmentation for example) created for an impractically long day in the lab. To overcome this, we optimized the protocol to have overnight anchoring and primary antibody steps. In practice, this meant the day where parasites were harvested only required about 30 minutes of work and was therefore conducive to harvesting sequential lifecycle stages. With experience we have also optimized the protocol in other ways that have helped with out productivity and efficiency, but this timing change made the most significant difference.

Do you have any advice for people who want to try U-ExM on Plasmodium or other small organisms?

Give it a go with an open mind and follow up anything you find interesting. While U-ExM can take some time to establish in a lab, once it is routine, it is not significantly more difficult than conventional immunofluorescence microscopy. We started using expansion in our labs for specific projects that already existed. This study, however, was born out of all the incredibly interesting but incidental findings we made whenever we would take our expanded parasites to the microscope. On any organism or structure that is particularly small, it can be difficult to infer exactly what you’re looking at by conventional light microscopy. For us, we found that U-ExM really disambiguates the parasite and you can almost always infer exactly where you are within the cell.

Are there plans to analyse the data further? Especially seeing as these are all 3D images.

At the moment, we haven’t started analysing the data further than what is already in the paper but we hope that others do! We are in the process of making all the ~700 images from this study available through the Image Data Repository (IDR, idr.openmicroscopy.org) for anybody to browse and analyse themselves. With all the markers and lifecycle stages we imaged, we are certain that this dataset contains hints to questions other groups are interested in answering.

We hope this has been some help, and we are always happy to answer more questions for anybody who is interested!

Regards,

Ben, Ana Karla, James & Sabrina.

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Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Background

Key findings

Glossary

The MTOC has an important part in scaffolding organelles during segmentation

Organelle segmentation follows the branching point fission model

NHS-ester staining provides contrast to depict the cytostome of P. falciparum

Asymmetric rhoptry biogenesis

What I like about this preprint

References

Share this:

Have your say Cancel reply

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