Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Abstract

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample ∼4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the microtubule organizing center (MTOC) and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the MTOC until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an MTOC association during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Expansion microscopy of apicomplexan parasites

“…White scale bars = 2 μm, yellow scale bars = 500 nm. Plasmodium images are derived from the dataset in Liffner et al (2023), Cryptosporidium images are unpublished images derived from the dataset in Guérin et al (2023) and Toxoplasma images are from an unpublished dataset.…”

“…The use of NHS ester, therefore, allows the visualization of a number of protein‐dense structures within the parasite (similar to electron microscopy), without the need for protein‐specific antibodies. Across various stages of the Plasmodium lifecycle, NHS ester has been used to visualize the rhoptries, some cytoskeletal elements, basal complex, cytostomes, apical polar ring (APR) and nuclear microtubule organizing centre (MTOC) (Bertiaux et al, 2021; Liffner & Absalon, 2021; Liffner et al, 2023; Rashpa & Brochet, 2022; Rashpa et al, 2023; Simon et al, 2021; Yang, Cai, et al, 2023); highlighting it is a powerful tool for generally observing changes within the parasite. Following the publication of the first study using U‐ExM in Plasmodium , further significant developments were made when the protocol was optimized for the most common Plasmodium fixative, paraformaldehyde (Simon et al, 2021), followed by the first U‐ExM experiments using P. falciparum (Simon et al, 2021).…”

“…Following the publication of the first study using U‐ExM in Plasmodium , further significant developments were made when the protocol was optimized for the most common Plasmodium fixative, paraformaldehyde (Simon et al, 2021), followed by the first U‐ExM experiments using P. falciparum (Simon et al, 2021). Subsequently, modifications to the U‐ExM protocol were made to streamline the process for harvesting malaria parasites at multiple timepoints/lifecycle stages (Liffner & Absalon, 2021), along with the testing of a wide number of marker antibodies and chemical dyes (Liffner et al, 2023). To date, nearly all lifecycle Plasmodium lifecycle stages have been expanded using U‐ExM (Figure 2).…”

“…Ookinete (replicated with permission from Bertiaux et al, 2021), gametes (replicated with permission from Rashpa & Brochet, 2022) and liver stages are P. berghei (currently unpublished dataset). Asexual blood‐stage (from dataset in Liffner et al, 2023) and gametocyte (currently unpublished dataset) are P. falciparum . Images are not depicted to scale.…”

“…They showed that in each of these stages, along with ookinetes, the subpellicular microtubules (SPMTs), cytosolic microtubules thought to define parasite shape, are stabilized by polyglutamylation and that this polyglutamylation can be recognized using anti‐PolyE antibodies (Bertiaux et al, 2021). The ability to specifically detect SPMTs has subsequently been utilized in other studies to distinguish intranuclear microtubules from SPMTs (Liffner et al, 2023).…”