Significance
Tubulin polymers are essential for a variety of cellular functions. Using cryo-ET, we reveal the 3D organization of the apical complex in Toxoplasma gondii, an intracellular eukaryote with tubulin-based structures, including an apical "conoid" involved in host cell invasion. Our development of an advanced subtomogram averaging protocol for filamentous structures enabled us to accurately assign tubulins in cellular context. At the subnanometer resolution achieved, tubulins were confirmed to assemble into two major forms: canonical subpellicular microtubules (SPMTs) and noncanonical conoid fibrils (CFs). The data further revealed associated proteins in both structures, a dominant orientation of SPMTs, and a unique patterning of the CFs. This work demonstrates an approach that can be used to determine cellular filamentous structures at multiscale resolutions.
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.
Toxoplasma gondii belongs to the phylum Apicomplexa, which consists of a diverse group of obligate intracellular parasites. This single-celled eukaryote possesses a broad host range, capable of infecting almost any nucleated cell in any warm-blooded animal (1). Symptoms associated with Toxoplasma infection are diverse, ranging from mild to fatal, especially in the developing fetus or immunocompromised patients (e.g., those with AIDS, heart transplants, etc.). Because of its prevalence worldwide, Toxoplasma presents a global health problem.
Upon infection, tachyzoites, which are the rapidly growing, asexual forms of Toxoplasma, proliferate within the host cell and eventually cause host cell lysis. Released free tachyzoites are highly motile, actively invading more host cells. To accomplish this, they deploy a sophisticated and complex strategy involving a specialized, apical architecture and tightly choreographed secretion from two distinct secretory organelles: micronemes and rhoptries (2⇓–4). Among the events associated with invasion is rearrangement of the cytoskeleton at the apical end, including protrusion of the conoid, a prominent bundle of spirally organized fibers (5⇓⇓–8).
The tubulin-based cytoskeleton that forms the pellicle and apical complex of Toxoplasma is not only responsible for cellular structural integrity but also provides a scaffold for cell motility and a polarized discharge of key invasion factors (9, 10). These crucial roles make these tubulin-based assemblies, including their interacting nontubulin partners, attractive drug targets for treating infection (11, 12). Proteomic analysis of a detergent-insoluble tachyzoite cytoskeleton fraction indicates that α- and β-tubulin proteins are expressed in Toxoplasma (13, 14), and a novel tubulin polymer in the conoid has been observed in detergent-extracted parasites (5). The precise molecular organization of tubulins in the apical complex and pellicle remained unclear, making it difficult to study how they interact and function during infection.
Cryogenic electron tomography (cryo-ET) is a powerful tool to study molecular structure in situ (15⇓⇓–18). This method can define the spatial context of cellular architecture without using chemical fixatives or metal stains. Toxoplasma tachyzoites are crescent shaped and ∼2 by 6 μm. Although these cells are generally too thick for the entire intact parasite to be studied by cryo-ET, our interest is in the tapered apical region, which is about 400 nm thick, thin enough for a 200- to 300-kV electron beam to form images. In this study, we focused on detailed image analysis of the subpellicular microtubules (SPMTs) and conoid fibrils (CFs) in intact parasites. In addition, we used detergent to remove the membranes and soluble components, leaving the insoluble cytoskeleton. This has the effect of making the specimen thinner overall and removing other obscuring cellular components. Subtomogram averaging was subsequently used for cytoskeleton components in both intact and detergent-extracted cells. Our analysis revealed unexpected structural differences in the tubulin protofilament organization of SPMTs and CFs at subnanometer resolution and enabled detection of their specific association with additional, yet-to-be-identified molecular components. Dissection of the components in apical complexes containing CFs and pellicles containing SPMTs provides a detailed understanding of how different tubulin-based macromolecular complexes are assembled in a cellular context and provides clues to their crucial roles in parasite biology.
Results
Cryo-ET Reveals Structural Details in the Apical Region of Toxoplasma.
CFs and SPMTs are two types of tubulin-based cytoskeletal elements that maintain the shape and polarity of the apical complex and pellicle, which are key parts of the invasion machinery in Toxoplasma. Under calcium flux, a chemical stimulus critical to the invasion process in this phylum of parasites, the CFs protrude from the surrounding SPMTs (19), making the apical end of the parasites fall well within the thickness limitation of cryo-ET imaging (∼500 nm). Using phase optical microscopy, we assessed the percentage of extracellular parasites exhibiting CF protrusion following calcium ionophore (A23187) treatment. Examination of tachyzoites before loading them on the EM grid showed ∼87% presented a protruded conoid, ensuring a large number of parasites suitable for imaging. To maximize the recognition of distinct subcellular components in this structurally complex area of the cell, we used a Volta phase plate (VPP) located in the focal plane of the objective lens of the electron microscope (20). The resulting enhancement of image contrast facilitates the use of convolutional neural networks (15) for annotation of distinct secretory organelles and cytoskeleton elements in the apical region. Fig. 1 and SI Appendix, Movie S1 show data for a representative tomogram generated in this way. Among the features revealed were the tubulin-based CFs and intraconoidal microtubules, bounded by two distinct preconoidal rings (PCRs) on top and the apical polar ring (APR) on the bottom, with micronemes, rhoptries, and other organelles filling the conoid's interior space (Fig. 1 B and E). The SPMTs were aligned to the inner surface of the pellicle along the entire extent of the cell body apparent in our field of view. For tachyzoites presenting intermediate or completely retracted conoids, we can capture the retracted conoid shielded by the ring of SPMTs.
Fig. 1. Three-dimensional organization of the apical complex in Toxoplasma tachyzoites reveals the subpellicular microtubule and conoid architecture. (A) Cartoon of the Toxoplasma tachyzoite, the life stage of which has been imaged by cryo-ET in B and annotated in E. (B) Tomographic slice of a representative apical complex of an intact tachyzoite recorded with VPP optics in a 200-kV electron microscope. The dotted and solid rectangles show the regions of the apical complex represented in greater detail in C and D using different z slices (scale bar, 100 nm). (C) A zoomed-in view of a tomographic slice in the dashed black rectangle of B, showing two CFs in the xy plane, their anterior ends extending toward a PCR (white bracket) with filamentous density (a white arrowhead) between them (scale bar, 25 nm). (D) Portion of a tomographic slice in the black rectangle of B, showing the SPMTs in the xy plane, and their association with the APR (white bracket). Note columnar densities (white arrows) emerging from the APR and positioned between the SPMTs (scale bar, 25 nm). (E) Three-dimensional segmentation of the tomogram shown in B including the PCR (red), CFs (cyan), APRs (golden), SPMTs (blue), intraconoidal microtubules (red), micronemes (pink), rhoptries (yellow), and plasma membrane (pale pink) (scale bar, 100 nm). A movie with the complete tomogram is available in SI Appendix, Movie S1.
Transverse slices of the CF from the reconstructed tomograms exhibit a comma shape, consistent with previous negative staining electron microscopy studies (5). Close examination of the apical end of the CF revealed filamentous densities extending toward the PCR both in intact and detergent-extracted parasites, showing a close connection between the conoid and the PCR (Fig. 1C; SI Appendix, Fig. S1 A–D). These feature discoveries were enabled by the use of VPP imaging. We aligned and averaged 50 subvolumes of the region that includes the PCR pairing units, the anterior end of the CF, and the filamentous densities from six cellular tomograms. The averaged map shows that one CF spans about four ring-pairing units of the PCR (SI Appendix, Fig. S1 E and F). The filaments seen in Fig. 1C and SI Appendix, Fig. S1 A–D are not visible after averaging, probably because they are not always oriented in the same way. We annotated the reconstructed tomogram to directly examine the number of assembled CFs per apical complex, but the complexity and image limitations hindered our ability to trace them reliably. To reliably count the number of CFs in each conoid, we annotated the individual CF in detergent-extracted parasites, whose conoid is thin enough to be fully annotated; the results showed that each conoid consists of 14 (n = 6) or 15 (n = 4) CFs (SI Appendix, Fig. S2 A–D).
The SPMTs span most of the cell body length, contributing to the elongated shape of Toxoplasma tachyzoites because of their tight connection to the inner membrane complex (21). The SPMTs are defined as polar structures, emanating from the APR. Examination of the intact cell tomograms in the area of the APR revealed microtubules that are regularly and radially spaced around the ring, with additional discrete density appearing between neighboring SPMTs (white arrows in Fig. 1D). Annotation of the 3D volume allows us to count the number of SPMTs per cell that otherwise overlap or would be completely missed in a 2D slice view. To avoid potential uncertainty from intraconoidal or broken microtubules, we counted only the SPMTs emanating from the APR of intact parasites. The results showed that 15 of our tomograms displayed the previously reported number of 22 SPMTs, while 10, 6, and 1 tomograms showed 21, 23, and 24 SPMTs per cell, respectively (SI Appendix, Fig. S2 E and F).
SPMTs Contain Intralumenal Spirals (ISs) of Unknown Components, with Unique Organization at the Seam.
Intact parasites are too thick to observe structures in most of the cell body at high resolution. To investigate the structural details of the SPMTs, therefore, detergent-extracted cells (Materials and Methods) were vitrified, imaged, and examined as done previously (5). The extracted cells are free of loosely bound cellular contents, thereby maximizing the signal/noise ratio of the remaining highly organized cytoskeleton components, especially the interior of the microtubules. The imaging magnification was chosen to optimally cover the microtubules as they emerge from the APR. The SPMTs of these lysed cells remain connected to the APR, together with interspersed pillar densities that were detected in the tomograms of intact parasites (Figs. 1D and 2A). These preserved details indicate that lysed cells have a similar cytoskeletal organization as intact cells. To obtain an intermediate resolution structure of these SPMTs, we performed subvolume alignment and averaged with 8-nm periodicity, based on the expected repeat for tubulin heterodimers and as also observed in the IS density repeats in Fig. 2A and previous reports (22). It has been shown previously that SPMTs have a standard microtubule arrangement (5, 23); our subvolume averaging assumed the tubulin protomers followed the microtubule-specific pseudohelical symmetry (24), in which each microtubule consists of 13 protofilaments of alternating α- and β-tubulin, with a unique junction called the "seam," where α- and β-tubulin interact laterally. This interaction is different from the other interactions between the protofilaments. Beginning and ending at the seam, there is a 12-nm axial spacing of a single spiral of α- or β-subunits. Our subvolume averaging resulted in a 6.7-Å resolution map for an SPMT segment over a length of 24 nm (Fig. 2 C and E; SI Appendix, Fig. S6C). Visualizing the density, we were able to identify 5- to 7-Å–wide rod-shaped features, characteristic of α-helices, corroborating the subnanometer resolution estimate (Fig. 3 A–C). The dispositions of these helices are consistent with the atomic structure of the tubulin molecules (25). However, the smoothness of the helix density suggests that the resolution appears to correspond to structural features expected in 7- to 8-Å–resolution cryogenic electron microscopy (cryo-EM) maps of single particles (26). Though our high-resolution results of individual monomers of tubulin or IS are derived from this map with the microtubule pseudohelical symmetry imposed, we also created a subtomogram average without imposing any symmetry (c1) using a search template having a well-defined seam, here called "symmetry-released" (Materials and Methods). While the symmetry-released map does not have sufficient resolution to distinguish α- and β-tubulin, the secondary structural elements of tubulin were apparent. Both maps were used for the interpretation of the topological organization of the SPMT and its associated IS.
Fig. 2. Determination of the 3D structures of apical SPMT (blue) and CF (cyan) segments from detergent-extracted Toxoplasma with tilt series recorded in a 300-kV electron microscope. (A) Portion of a tomographic slice showing the SPMTs with internal striation density (white arrows) and short interspersed pillars (white arrowheads; scale bar, 50 nm). (B) Portion of a tomographic slice showing individual CFs (red stars; scale bar, 50 nm). (C, E) Reconstruction of a representative SPMT segment viewed from top and a tilted angle. (D, F) The top view and tilted view of a reconstructed CF segment showing an asymmetric semicircular profile. (G) A representative tomographic slice at the apical end of a detergent-extracted cell, containing individual SPMTs (white arrows) and CFs (red stars; scale bar, 100 nm). (H) The 3D organization of SPMTs and CFs in the local region of the reconstructed tomogram shown in G based on the refined coordinates of individual particles.
Fig. 3. Subtomogram average of SPMT tubulins and their associated lumen complex from detergent-extracted parasites. (A) The arrangement of α- (cyan) and β- (blue) tubulin subunits in the averaged density map of the SPMT segment, viewed from the seam and showing the arrangement of the 13 protofilaments. The plus (+) and minus (−) ends of the microtubule are as indicated. The minus (up) end is toward the APR. (B) Density for an αβ-tubulin dimer as represented by the white boxed area of A, viewed from within the SPMT lumen. Note the S loop (green loop indicated by black arrows) in α-tubulin has extended density, but not in β-tubulin; this is validated by C, an αβ-tubulin dimer segmented from a porcine microtubule (EMD6349) showing the S loop density of differences displayed at 8 Å, also viewed from the lumen side. (D) Nontubulin densities inside the tubulin spiral, viewed within a cross-section of SPMT, are colored. (E) Stacked spiral architecture of the IS complex with four stacked spirals shown in pink, magenta, yellow, and orange. (F) One stack of the IS in yellow viewed from the seam at different angles. (G) Unwrapping the SPMT between protofilaments 4 and 5, indicated by an arrowhead in D, shows the SPMT assembly from the inside, with an IS density axially arrayed along the tubulin lattice with apparent repeating units.
In order to determine the relationship between the microtubule and the IS, we needed to distinguish between the α- and β-tubulin. It is known that Toxoplasma's α- and β-tubulin are slightly different in their structure, with α-tubulin having a long S loop compared to β-tubulin, and that the sequences of both tubulins are ∼85% identical to their mammalian counterparts (13). Based on that, we generated a homology model of Toxoplasma tubulins to distinguish the α- from the β-tubulin isoforms in the averaged density map. While the Toxoplasma genome carries three α-tubulin genes, we used the protein sequence of α-1 (TGME49_316400) for the homology model as it is the only one of the three that is appreciably expressed in tachyzoites, both at the RNA and protein level (14, 27), and, as therefore expected, it is also the only one of the three that appears essential for tachyzoite viability (28). The Toxoplasma genome also carries three β-tubulin genes (TGME49_266960, 221620, and 212240), with all isoforms reportedly detected in one proteomics study (14), although transcriptomic studies indicated the β-3 isoform (TGME49_212240) is essentially not expressed in tachyzoites (https://toxodb.org/toxo/app) and appears dispensable for tachyzoite growth (28); the other two genes are abundantly expressed in tachyzoites (https://toxodb.org/toxo/app ) and appear essential (28). By amino acid sequence, β-1 (TGME49_266960) and β-2 (TGME49_221620) share 96.9% identity and 98.9% similarity; given this near complete identity, we chose the β-1 isotype at random for modeling purposes. The α-1 tubulin model displays the longer S loop compared to β-1, corresponding to residues 359 to 372 in the α-1 protein and similar to the resolved structure of the porcine microtubule. Our subtomogram average resulted in a map in which the α-tubulin is readily differentiated from the β-tubulin unit because of this difference in the S loop (Fig. 3 B and C). In summary, based on the correlation and visual analysis between the segmented tubulin subunit density in the subtomogram average and the map corresponding to ∼8-Å resolution computed from the porcine α- and β-tubulin model (EMD-6439), we were able to distinguish these two subunits in our density map (Fig. 3A).
The IS is clearly visible in the cross-sectional view, lining the luminal surface of the microtubule protofilaments (Fig. 3D). Viewing from the longitudinal axis of the SPMT segment (Fig. 3E), this IS complex follows a left-handed and discontinuous helical pattern, built up stack by stack, similar to a series of split-ring washers (Fig. 3 E and F). The axial repeat of the washers is 8 nm along the microtubule axis, and the two split ends of each washer are about 12 nm apart (Fig. 3F), similar to the spirals of α- or β-tubulin. Based on the start and stop ends of each washer, we segmented 13 structurally similar repeating units X1 to X13 in the symmetrized map, each estimated to be ∼25 kDa based on the volume density (SI Appendix, Fig. S3) (29). In examining the density features of the IS units, each appears to have rod-shaped (α-helical) features, although the helices cannot be connected due to limited resolution (SI Appendix, Fig. S3D). Each IS stack is accompanied by a spiral of 13 rod-shaped densities (Rn) in green (SI Appendix, Fig. S3F), positioned between the IS components and the tubulin proteins and connecting between each spiral axially in the symmetrized average map (Fig. 3G; SI Appendix, Fig. S3F). Unwrapping the SPMT density map arbitrarily between protofilaments 4 and 5 further revealed the positioning of IS units with respect to the tubulin lattice, viewed from the luminal side (Fig. 3G). The organization of IS units and rod-shaped densities were also verified in symmetry-released maps (SI Appendix, Fig. S3F). However, the low visibility of unit X1 in the symmetry-released map suggests that this unit located over the seam is not actually present but is an artifact of symmetry imposition (SI Appendix, Fig. S3F). Each putative monomeric unit of an IS spans between tubulin heterodimers in adjacent protofilaments and is positioned every 80 Å at the interface between an α-tubulin and the β-tubulin above it (SI Appendix, Fig. S4). The IS units marked X2 to X13 are close to β-tubulin at residues 34 to 42, and the IS units marked R2 to R13 are close to α-tubulin at two loops, residues 30 to 43 and 359 to 372 (the second is the S loop, which is longer in α- compared to β-tubulin).
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