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

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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.

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.
" data-icon-position data-hide-link-title="0">Fig. 1.
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.

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.
" data-icon-position data-hide-link-title="0">Fig. 2.
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.

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.
" data-icon-position data-hide-link-title="0">Fig. 3.
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).

SPMT Seams Are Oriented Nonrandomly with Respect to the Central Axis of the Parasites.

Each IS is coaxial with the tubulin spirals, leaving a gap spanning a portion of the two protofilaments where the seam occurs. The unique arrangement of IS can also be observed in the averaged density map of SPMT segments computationally extracted from the intact (not detergent-extracted) parasites (Fig. 4A). This can be used to determine the orientation of the microtubule seam. We assigned the 3D SPMT averaged density map back to five reconstructed apical regions of intact parasite cells and retrieved the coordinates applied to align and average the subvolume particles. For each particle, we measured the microtubule seam orientation angle relative to the center of each cell body at cross-section (i.e., the z axis of each SPMT segment). For the five tomograms of intact parasites, half the segments, shown in Fig. 4C in pink, have a mode of their orientation of 10°, while the other half, shown in blue, have a mode in their orientations of 150° relative to the z axis (Fig. 4B). Overall, we find that the seam's orientation changes according to the position of the SPMT around the cell body and preferentially faces the center of the cell body (Fig. 4C). This nonuniform distribution of orientation is likely caused by specimen flattening during the freezing steps.

The seam orientation of SPMT in tomograms of intact cells is nonrandom. (A) In-cell SPMT segment density is oriented with the seam facing down (θ = 180°) or up (θ = 0°). We used the gap in the cryo-EM density map of the SPMT segment as a marker of the microtubule seam. (B) The seam of apical microtubules along their axis is analyzed by measuring its angle relative to the z axis in cell tomograms (n = 5). (C) A schematic representation of SPMT models in the cell body at cross-section, based on the observed cross-section of the cell body in the tomograms. The blue color is used for the microtubules in the "top" half layer of the cell (i.e., toward the electron beam), and the orange color is used for the microtubules in the "bottom" half of the cell. Note that the seam is predominantly oriented toward the center of the somewhat flattened cell body, with orientation angle 90° < θ < 180° for SPMTs in blue or 0° < θ < 90° for SPMTs in orange.
" data-icon-position data-hide-link-title="0">Fig. 4.
Fig. 4.

The seam orientation of SPMT in tomograms of intact cells is nonrandom. (A) In-cell SPMT segment density is oriented with the seam facing down (θ = 180°) or up (θ = 0°). We used the gap in the cryo-EM density map of the SPMT segment as a marker of the microtubule seam. (B) The seam of apical microtubules along their axis is analyzed by measuring its angle relative to the z axis in cell tomograms (n = 5). (C) A schematic representation of SPMT models in the cell body at cross-section, based on the observed cross-section of the cell body in the tomograms. The blue color is used for the microtubules in the "top" half layer of the cell (i.e., toward the electron beam), and the orange color is used for the microtubules in the "bottom" half of the cell. Note that the seam is predominantly oriented toward the center of the somewhat flattened cell body, with orientation angle 90° &lt; θ &lt; 180° for SPMTs in blue or 0° &lt; θ &lt; 90° for SPMTs in orange.

CFs Have&nbsp;an Unusual Tubulin-Based Structure with Additional, Unknown Densities Arranged Between the Protofilaments.

Each CF is tightly associated with its neighbor filament, extending from the top PCR and forming an array of short left-handed spirals. Using detergent extraction to obtain relatively intact cytoskeletons, we were able to refine our subtomogram averaging of the CFs to reveal a periodic density of the CFs (Fig. 2B), which was identified to be 8- and 4-nm layer lines in Fourier space, similar to the typical microtubule tubulin repeats (5). Given previous work showing that tubulins are a main component of the CFs (5, 30), we sought to determine how the tubulins are assembled within these fibrils. To do this, we again used detergent-extracted tachyzoites to obtain their cytoskeleton as described for the SPMT experiments. This extraction preserves the overall helical shape of the CFs (Fig. 2 D–H) while allowing the cell body to be much thinner, improving the signal/noise ratio for tomographic images. The subtomogram averaged map of the CFs was determined at 9.3-Å resolution (SI Appendix, Fig. S6D). The density map accommodates nine columns or protofilaments of 40-Å repeated density that we are able to assign as a tubulin subunit using the homology model, together with uncharacterized associated density (Fig. 5). Although the data were not of sufficient resolution to visualize the difference between α- or β-tubulin, published proteomics analysis has shown that the CFs contain both (14). There are three lines of evidence that suggest the densities in the CF columns are tubulin. First is that the vertical repeat is consistent with that of tubulin dimers (80 Å). Second is that for several of the columns, the side-to-side spacing and angle are consistent with that of microtubules. Third, we have cross-correlated a Protein Data Bank (PDB) model of tubulin (PDB ID: 3jak) with the densities in the columns. The Z-score (31) varies from 2.5 to 14, which is higher than that of a random fit (SI Appendix, Fig. S5). We speculate that the differences in their shapes are due to differences in local resolution and conformational variability due to different interacting partners. The associated components in the center of the CF density map shapes the curvature of nine tubulin columns, with an opening between tubulin column 1 and 9. Three groups of densities are seen within the grooves between columns 3 and 4, 5 to 8, and 8 and 9 (Fig. 5 A and C), and these bind to alternating tubulin units every 80 Å axially (Fig. 5B). Compared to the density volume of tubulin monomer (55 kDa), the molecular weight of each repeating unit can be estimated to be about 30 kDa for each group between columns 3 and 4, 95 kDa for the ones between columns 5 and 8, and 20 kDa for the ones between columns 8 and 9.

Variable resolvability of tubulin in a CF segment of cryo-EM density by subtomogram averaging from detergent-extracted parasite. (A) CFs are unusual tubulin-based fibrils. Each fibril contains nine columns of tubulin (blue and cyan), associated with CF-associated densities within the concave inner face of the CF (yellow) and on the outer convex face (magenta, green, orange). This is shown axially from various views rotated around the long axis. (B) Zoomed-in cryo-EM density of two tubulin components in each of columns 1 to 9. Tubulin columns were fitted to the conoid average densities. A total of nine columns were segmented, shown axially with alternating light and dark blue colors (top) from various views. (C) Nontubulin groove densities appear every 80 Å, shown between columns 3 and 4 (magenta), on the outside of columns 5 to 8 (orange and brown), and between columns 8 and 9 (green). The plus (+) and minus (−) ends of the microtubule are as indicated in A and the first column of B.
" data-icon-position data-hide-link-title="0">Fig. 5.
Fig. 5.

Variable resolvability of tubulin in a CF segment of cryo-EM density by subtomogram averaging from detergent-extracted parasite. (A) CFs are unusual tubulin-based fibrils. Each fibril contains nine columns of tubulin (blue and cyan), associated with CF-associated densities within the concave inner face of the CF (yellow) and on the outer convex face (magenta, green, orange). This is shown axially from various views rotated around the long axis. (B) Zoomed-in cryo-EM density of two tubulin components in each of columns 1 to 9. Tubulin columns were fitted to the conoid average densities. A total of nine columns were segmented, shown axially with alternating light and dark blue colors (top) from various views. (C) Nontubulin groove densities appear every 80 Å, shown between columns 3 and 4 (magenta), on the outside of columns 5 to 8 (orange and brown), and between columns 8 and 9 (green). The plus (+) and minus (−) ends of the microtubule are as indicated in A and the first column of B.

The Plus End of CFs Is Oriented Toward the Top of the Conoid.

With the successful composition assignment of tubulin in the CFs, we were able to determine the structural polarity of the conoid. Fig. 5B shows that the plus ends of all nine protofilaments are directed toward the bottom of that figure, based on the fit of tubulin subunits modeled in the density map and by analogy to the plus end of canonical microtubules. This plus direction is toward the top (anterior end) of the conoid. This polarity direction suggests that the conoid is assembled from its base toward what will ultimately be its anterior end. Within the density of extracted CF segments, the nine tubulin columns are organized into a comma shape in cross-section (Fig. 6A). The interprotofilament angles of CFs are much more variable and often much greater than the relatively constant ∼27°of the interprotofilaments in SPMTs (Fig. 6 E–G). Interestingly, there is a very pronounced kink between protofilaments 3 and 4, possibly as a result of the observed density between these two protofilaments (magenta in Fig. 6).

CFs are oriented in parallel with a plus-like end at the anterior. (A) Reconstruction of a CF segment from protruded conoids from detergent-extracted cells, generated by subtomogram averaging and shown in cross-section, viewed from the minus-like end. The cartoon (on the Bottom) illustrates the structural arrangement of the nine tubulin columns that form the comma-like architecture. (B) Low-resolution reconstruction of a CF segment from conoid-protruded intact cells, generated by subtomogram averaging. Above this is shown the higher-resolution structure of A inserted into the low-resolution structure from intact cells. (C) The CFs in a protruded conoid are assembled into spiral filaments with a "parallel" orientation and the opening of the comma shape facing the interior of the conoid. (D) The spiral CFs viewed from the conoid surface. (E) Zoomed-in model of tubulins within a CF segment. An example of how interprotofilament angles were determined is shown for the angle between a line connecting the centers of protofilaments 1 and 2 relative to such a line for protofilaments 2 and 3; this was defined as α2 and was determined to be 37°. (F) The interprotofilament angles were measured for each protofilament relative to the ones on either side. (G) As for F, except the interprotofilament angles were measured for the SPMT protofilaments. (H) Comparison of the protofilament arrangement in the SPMTs and CFs with the tubulin model fitted into the cryo-ET density map. Note the marked kink at protofilaments 3 and 4 in the CF. (I) Closest spacing distance between neighboring CFs showing the increased spacing in protruded CFs (blue) relative to retracted ones (orange). Data are from analysis of three protruded and four retracted conoids of intact cells. (J) The turn/rise distance ratio was measured every 1 nm along the CF axis, showing that the CFs in retracted (orange) conoids are higher than in protruded conoids (blue).
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Fig. 6.

CFs are oriented in parallel with a plus-like end at the anterior. (A) Reconstruction of a CF segment from protruded conoids from detergent-extracted cells, generated by subtomogram averaging and shown in cross-section, viewed from the minus-like end. The cartoon (on the Bottom) illustrates the structural arrangement of the nine tubulin columns that form the comma-like architecture. (B) Low-resolution reconstruction of a CF segment from conoid-protruded intact cells, generated by subtomogram averaging. Above this is shown the higher-resolution structure of A inserted into the low-resolution structure from intact cells. (C) The CFs in a protruded conoid are assembled into spiral filaments with a "parallel" orientation and the opening of the comma shape facing the interior of the conoid. (D) The spiral CFs viewed from the conoid surface. (E) Zoomed-in model of tubulins within a CF segment. An example of how interprotofilament angles were determined is shown for the angle between a line connecting the centers of protofilaments 1 and 2 relative to such a line for protofilaments 2 and 3; this was defined as α2 and was determined to be 37°. (F) The interprotofilament angles were measured for each protofilament relative to the ones on either side. (G) As for F, except the interprotofilament angles were measured for the SPMT protofilaments. (H) Comparison of the protofilament arrangement in the SPMTs and CFs with the tubulin model fitted into the cryo-ET density map. Note the marked kink at protofilaments 3 and 4 in the CF. (I) Closest spacing distance between neighboring CFs showing the increased spacing in protruded CFs (blue) relative to retracted ones (orange). Data are from analysis of three protruded and four retracted conoids of intact cells. (J) The turn/rise distance ratio was measured every 1 nm along the CF axis, showing that the CFs in retracted (orange) conoids are higher than in protruded conoids (blue).

Extensive density can be resolved within the structure of the CF segments averaged from intact cells, but to a lower resolution than described above for the detergent-extracted parasites. Nevertheless, we were able to readily fit the higher-resolution CF structure into the structure obtained from intact cells with protruded conoids. The results showed considerable extra density within the CFs of intact cells, arrayed along the outside of the tubulin-based protofilaments and associated densities seen for the detergent-extracted parasites (Fig. 6B). Presumably, this extra material dissociates upon detergent extraction. To determine the polarity orientation of the CFs within the cell, their in situ density was mapped back to the cell tomogram based on the coordinates of each extracted particle. The CF segment density revealed the orientation of the CFs to be in "parallel" with respect to each other, with the opening of the cross-sectional comma shape being toward the center of the conoid (Fig. 6 C and D). The patterning of retracted and protruded conoids in calcium ionophore–stimulated parasites was further investigated by measuring the spacing of neighboring fibrils and the ratio between turn and rise distance along the axis under these two conditions (Fig. 6 I and J). The results indicated that relative to retracted conoids, the distance between neighboring fibrils is slightly increased when the conoid is protruded, with a median spacing of 38.4 nm in the protruded conoid versus 32.6 nm in the retracted structures. In addition, the turn/rise ratio was markedly lower in the protruded versus retracted conoids (medians of 1.48 and 2.55, respectively).

Discussion

Single-particle cryo-EM of thin or purified samples is ideal to determine near atomic resolution structures of biomolecules in solution; however, this usually comes at the expense of studying the cellular context of their native environment. Here, we developed a protocol for image acquisition and processing by cryo-ET to investigate the cytoskeleton-based apical regions of Toxoplasma, examining the cellular machinery in context both in intact and detergent-extracted parasites and extending to the level of single macromolecules in detergent-extracted parasites at subnanometer resolution. Cryo-ET of intact parasites revealed the spatial organization of distinct components in the highly crowded apical region. By annotating the tubulin-based SPMTs and CFs, we conclude that their numbers are not fixed, implying that their assembly process is regulated but not rigid (SI Appendix, Fig. S2 D and F). Interestingly, lower expression rates of α-tubulin in Plasmodium berghei resulted in fewer microtubules (32). Further investigation into the relationship between SPMT and CF numbers and tubulin expression in Toxoplasma will be interesting to study.

The SPMT minus end emanates from the APR, which has been suggested to function like a microtubule organizing center, i.e., as an anchoring site for the SPMTs (23). Previous studies revealed that even in the absence of fully formed APR, there remains some organization of the SPMT array (33). This argued for additional elements that support and stabilize the arrangement of the SPMT. The pillar densities we observe between neighboring SPMTs (Figs. 1D and 2A) where they attach to the APR could be involved in such organization. The identities of these short densities are not yet known, but they may be related to previously described structure found between the SPMTs (23) and could possibly be composed of the recently discovered AC9 and/or AC10 proteins, which by light microsc...