Cryo-ET of Toxoplasma parasites gives subnanometer insight into tubulin-based structures

Abstract

Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite’s long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.

Form follows function: Variable microtubule architecture in the malaria parasite

“…2B-D, 3C). They were similar to the recent T. gondii SPMT structures determined in detergent solubilised cells (Sun et al, 2022; Wang et al, 2021). T. gondii ILH consists of thioredoxin-like proteins 1 and 2 (TrxL1, TrxL2) and subpellicular microtubule protein 1 (SPM1).…”

“…3E). The average volume cross section is consistent with the L-shaped tubulin-based conoid described in T. gondii (Sun et al, 2022). The ookinete conoid measured 70 nm in height and 300 nm in diameter, resembling a retracted state.…”

“…Our observations contribute to a growing list of organisms that make use of an ILH in their microtubule cytoskeleton, which now includes the SPMTs of apicomplexans (Sun et al, 2022; Wang et al, 2021), and the cilia and flagella of mammals (Leung et al, 2021; Zabeo et al, 2018). With our observations it seems likely that ILH consisting of TrxL1 and SPM1 are conserved across the Apicomplexa .…”

“…2A, 3) (Cyrklaff et al, 2007). Analogous structures, named Interrupted Luminal Helices (ILH) were first observed in flagellar ends of human spermatozoa (Zabeo et al, 2018), and more recently in equine and porcine spermatozoa (Leung et al, 2021), and tachyzoites of Toxoplasma gondii (Sun et al, 2022;Wang et al, 2021). We adopted the nomenclature of Zabeo et al To generate an unbiased EM density map, 422 sporozoite and 177 ookinete SPMTs were analysed by SVA without applying any (pseudo-) symmetry.…”

“…APRs form a prominent part of the apical assembly which defines the parasite polarity. A feature unique to apicomplexan tubulin is its formation of L-shaped “semi tubules” which, with accessory proteins, form a helical structure termed the conoid (Hu et al, 2002; Sun et al, 2022), located within APRs. The conoid is a specialised structure involved in invasion.…”