Cryo‐Electron Tomography of Toxoplasma gondii Indicates That the Conoid Fiber May Be Derived from Microtubules

Abstract

Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis and can infect numerous warm‐blooded animals. An improved understanding of the fine structure of this parasite can help elucidate its replication mechanism. Previous studies have resolved the ultrastructure of the cytoskeleton using purified samples, which eliminates their cellular context. Here the application of cryo‐electron tomography to visualize T. gondii tachyzoites in their native state is reported. The fine structure and cellular distribution of the cytoskeleton are resolved and analyzed at nanometer resolution. Additionally, the tachyzoite structural characteristics are annotated during its endodyogeny for the first time. By comparing the structural features in mature tachyzoites and their daughter buds, it is proposed that the conoid fiber of the Apicomplexa originates from microtubules. This work represents the detailed molecular anatomy of T. gondii, particularly during the budding replication stage of tachyzoite, and provides a reference for further studies of this fascinating organism.

The initiation and early development of the tubulin-containing cytoskeleton in the human parasiteToxoplasma gondii

“…Twenty eight had the grouping pattern of 4, 4, 4, 4, and 6 MTs, and 15 had a pattern that could be easily derived from that grouping (e.g., 4, 4, 6, 2, 6). Of note, a 4, 4, 4, 4, 4, 2 grouping was reported in a previous cryo electrontomography study (Li et al, 2023). We thus propose that the grouping into 4, 4, 4, 4, and 6 MTs is the foundational pattern defined by the location of the nucleation sites.…”

“…Genetic manipulations that disrupt the integrity of the apical polar ring result in abnormalities in the MT array, failed mosquito transmission for Plasmodium ookinete (Qian et al ., 2022) and highly impaired lytic cycle for the Toxoplasma tachyzoite (Leung et al ., 2017; Tosetti et al ., 2020; Back et al ., 2021). In Toxoplasma , the same tubulin subunits that polymerize into the cortical MTs are also the building blocks for the conoid, which is formed of 14 ribbon-like, novel tubulin polymers organized into a left-handed spiral (Hu et al ., 2002b; Sun et al ., 2022; Gui et al ., 2023; Li et al ., 2023) (Figure 1). Like a piston, the conoid can protrude and retract in a calcium-dependent manner, passing through the apical polar ring (Hu et al ., 2002b; Hu et al ., 2006).…”

“…We previously showed that the cortical MTs grow at their distal ends (Hu et al ., 2002b), suggesting that their minus ends are anchored at the apical polar ring as proposed earlier (Russell and Burns, 1984). This polarity arrangement was later confirmed by cryo-electron microscopy studies (Wang et al ., 2021; Sun et al ., 2022; Li et al ., 2023). During cell division, while the mitotic spindle is assembled in the nucleus, the cortical microtubules of the developing daughter polymerize in the cytosol (Liu et al ., 2015).…”

Expansion microscopy of apicomplexan parasites

“…The past few years have seen tremendous efforts to define the apical end of Toxoplasma tachyzoites in incredible detail, using a variety of imaging modalities (Li et al, 2023; Mageswaran et al, 2021; Segev‐Zarko et al, 2022; Sun et al, 2022). At the apical end of a tachyzoite is the conoid, a barrel‐shaped structure comprised angled tubulin fibres and containing a further two intraconoidal microtubules (Dos Santos Pacheco et al, 2020).…”

The Molecular Architecture of the Nuclear Basket

“…To collect tachyzoites, trypsin-treated, parasite-infected HFFs were mechanically disrupted using a 27-gauge syringe, and the mixture was filtered to separate tachyzoites from HFF debris. The tachyzoites were then centrifuged, resuspended in DMEM with 30% FBS and 10% DMSO, and deposited on EM grids for vitrification, as described previously (84). Excess media was manually blotted from the back (opposite to the carbon film and seeded cells).…”