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

Abstract

<jats:title>Abstract</jats:title><jats:p>Malaria is caused by unicellular <jats:italic>Plasmodium</jats:italic> parasites. <jats:italic>Plasmodium</jats:italic> relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication or dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy. Here, we demonstrate that the nanoscale cytoskeleton organization is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows to monitor the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the subpellicular microtubule arrays that confer cell rigidity. In ookinete, we additionally identify an apical tubulin ring above the subpellicular microtubules that colocalises with markers of the conoid in related Apicomplexa parasites. This microtubule structure was presumed to be lost in <jats:italic>Plasmodium</jats:italic> despite its crucial role in both motility and invasion in most apicomplexans. Here, U-ExM reveals that a divergent and reduced form of the conoid is actually conserved in the <jats:italic>Plasmodium</jats:italic> genus.</jats:p>

Conservation of theToxoplasmaconoid complex proteome reveals a cryptic conoid inPlasmodiumthat differentiates between blood- and vector-stage zoites

“…This presence of tubulin in a Plasmodium conoid complex provides additional support to our data showing the conservation of numerous conoid-associated proteins in all Plasmodium zoite forms. We have previously shown that SAS6L and Myosin B locate to an apical ring in Plasmodium also [42,63], and these co-locate with the tubulin ring in Plasmodium ookinetes [62].…”

“…With any further reduction in the tubulin component in Plasmodium spp., or other Aconoidasida, detection of conoid tubulin by ultrastructural approaches would be even more challenged. However, the very recent application of ultrastructure expansion microscopy (U-ExM), in combination with anti-tubulin staining, has revealed that a ring of tubulin is present in P. berghei ookinetes at the very apex of the cell, beyond the apical termini of the subpellicular microtubules at APR2 [62]. This position is consistent with the location of the three conoidal rings we observe by TEM (Fig 8B-D) and the thicker posterior conoidal ring is the most likely location of this tubulin given its presence here in Haemoproteus and Leucocytozoon.…”

The Riveting Cellular Structures of Apicomplexan Parasites

“…This suggests that this curious polarity must offer some advantage over a uniform distribution of microtubules [64]. The polar rings are arranged perpendicularly to the microtubules in T. gondii tachyzoites and Plasmodium ookinetes, yet are tilted towards the ventral side in sporozoites [64,69] (Figure 1B). This could direct secretion of microneme-based adhesins specifically to the ventral side, allowing a better maintenance of substrate contact and hence more persistent gliding.…”

“…One of the stumbling blocks in understanding the roles of the alveolins was imaging the small, densely packed structures. However recent advances in expansion microscopy has allowed unprecedented localization of individual alveolins and microtubules in the cell [69,72,89]. Determining their precise localization will allow functions for individual proteins to be suggested, informing future research.…”

The Dynamic Roles of the Inner Membrane Complex in the Multiple Stages of the Malaria Parasite

“…The round zygotes undergo huge and rapid morphological changes, gaining a protrusion, elongating and maturing to finally form an elongated, crescent-shaped ookinete ( Guttery et al., 2015 ; Bennink et al., 2016 ). The ookinete’s apical rings are formed at the same time and organize the SPM of > 40 microtubules ( Bertiaux et al., 2020 ) under the IMC. These are initially seen at the apical protrusion of the zygote but extend during zygote elongation into a “dome-like” structure under the IMC ( Wang et al., 2020 ).…”