A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers

Abstract

Background: TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella.

Results: We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a "C"-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or "C"-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite.

Conclusions: These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Fig. 1

Conoid architecture and endogenous TgDCX localization. a Diagrams of the T. gondii cytoskeleton [modified from [6]], in which several tubulin containing structures (22 cortical microtubules, 2 intra-conoid microtubules, and 14 conoid fibers) are highlighted in red. EM images of a cross-section of each of those polymers [5] are also shown. Shown in brown are several rings mentioned later in the text. The apical polar ring is the anchoring structure for the 22 cortical microtubules. A complex structure (the preconoidal rings, unlabeled), rich in intricate detail, lying at the apical end of the conoid, is portrayed in this cartoon as two featureless rings. IMC: Inner Membrane Complex. A replicating parasite is also shown, with daughter parasites being built inside the mother. The cortical microtubules of the adult are omitted for clarity. On the right, a cartoon shows how the conoid responds to increasing [Ca²⁺] by extending and changing its shape. (b-d) Z-projections of SIM images of mCherryFP-TgDCX (red, “K-in mCh-TgDCX”) knock-in parasites [6] expressing mNeonGreenFP-β1-tubulin (green, mNe-TgTub). b Two interphase adult parasites. One adult is outlined with a dashed white border. The arrowhead indicates the apical complex of one parasite, shown 2x enlarged and contrast enhanced in the inset. Tubulin and TgDCX are co-localized in the conoid, appearing as an annulus with a ~ 0.2 μm central opening. c Two dividing parasites at an early stage of daughter formation, with two daughters in each adult. One of the developing daughter’s apical complex is indicated by the arrowhead, and enlarged 1.5x in the inset. d Parasites at a later stage of daughter formation. The daughter apical complexes (white arrowhead) are nearly mature, and daughter cortical microtubules have grown to ~ 1/3 of their length in the adult. e-g Electron microscope (EM) images of the conoid region of negatively stained whole-mount mCherryFP-TgDCX knock-in (e, “K-in mCh-TgDCX”), TgDCX knockout (f, “ΔTgDCX”) parasites (two images), and a complemented line generated by transfecting the TgDCX knockout parasite with a plasmid driving expression of TgDCX-EGFP (g, “Comp”). The conoids are shorter, distorted, and disordered in the TgDCX knockout parasites (f) compared to their parental strain in (e), but supplying TgDCX completely restores conoid structure (g)

Fig. 2

Conoid diagram and EM images of conoids isolated from wild-type, mCherryFP-TgDCX knock-in and TgDCX knockout parasites. a CryoEM image of disassembled apical complexes from wild-type (“WT”) parasites. Several groups of conoid fibers (“CF”, arrowheads) and fragments of cortical microtubules (“MT”, arrows) are seen. Note that the cortical microtubules are straight, whereas the conoid fibers are uniformly curved. The conoid fibers appear to become wider along their length, and their protofilaments become clearer, indicating a twist in the fibers, as diagrammed in the cartoon. The cartoon represents the cluster of fibers in the upper right of the cryoEM image. The hollow arrow in the cartoon shows the direction of view in the EM image, and the boxes contain cross-sections of the fiber at the indicated locations. Near the apical end of the fibers (towards the bottom in the cartoon and the image), the direction of view yields a narrow fiber with protofilaments obscured by superposition. As the fiber twists along its length, its profile becomes wider, and there is decreasing superposition of protofilaments, giving the splayed appearance at the basal region of the fibers. b End on (left) and side views (right) of negatively stained isolated conoids from mCherryFP-TgDCX knock-in parasites (“K-in mCh-TgDCX”). In the left image, the apical polar ring with stumps of broken cortical microtubules encircles the conoid. A second conoid, almost completely disassembled, is also seen. In the right image, the two intra-conoid microtubules are seen projecting through the conoid, which is detached from the apical polar ring. c Three examples of disassembled conoids isolated from mCherryFP-TgDCX knock-in parasites (“K-in mCh-TgDCX”). All 14 of the fibers that formed each conoid are seen. Arrows: preconoidal rings, which often remain attached to the apical ends of the fibers. d End on views of conoids isolated from TgDCX knockout parasites (“ΔTgDCX”). The conoids are encircled by the apical polar ring with attached fragments of cortical microtubules. Isolated conoid fibers or conoids detached from the apical polar ring were never observed in preparations from the TgDCX knockout parasite. e Diagram illustrating the changing geometry of the TgDCX-containing fibers of the conoid. Extension of the conoid through the apical polar ring, which occurs as the parasites reactivate motility and exit their lysed host cell, is accompanied by a change in conoid shape from more conical to more cylindrical. The structural implications, for the conoid fibers, that follow from this change in overall shape are described in the Discussion. For clarity, the change in fiber orientation has been exaggerated in the diagram. Note also that the diagrams here are oversimplified for clarity: in an untilted sagittal section as diagrammed, the fiber profiles cannot be clearly seen. In order to make the profiles visible, the section must be tilted in the microscope by plus (to see the profiles on one side) or minus (to see the profiles on the other side) the pitch angle of the fibers. See Fig. 6 in [5] for a demonstration

Fig. 3

FP-tagged TgDCX generates curved microtubules in a heterologous system, Xenopus laevis S3 cells. a Deconvolved wide-field images of Xenopus S3 cells expressing EGFP-α-tubulin (green) and TgDCX-mCherryFP (red), showing that TgDCX-mCherryFP is localized to EGFP-α-tubulin containing fibers. TgDCX-mCherryFP expression drives the formation of curved fibers, many of which are short and share similar curvatures (also see Additional file 3: Movie S1). The areas within the dashed outlines are enlarged 1.5x in the small white boxes. b Deconvolved wide-field images of Xenopus S3 cells expressing EGFP-α-tubulin (green) and mCherryFP-TgDCX (red), showing that, similar to TgDCX-mCherryFP, mCherryFP-TgDCX expression drives the formation of curved fibers (also see Additional file 4: Movie S2). Arrows indicate microtubule bundles coated with mCherryFP-TgDCX. Note the fibers containing EGFP-tubulin only in the neighboring untransfected cell, which follow the meandering, gently curved, paths of typical cytoplasmic microtubules. Insets: 1.5x. c Histogram of radii of curvature of microtubules in Xenopus S3 cells, untransfected (green), or transfected with mCherryFP tagged TgDCX (red). Data include 844 microtubule segments (total length 7488 μm) for the untransfected cells and 599 segments (total length 2621 μm) for the transfected cells. All measurable microtubule segments in 8 representative untransfected cells and 7 transfected cells were included in the two histograms. Insets: fluorescent images of subregions of an untransfected (top) and a transfected cell (bottom). d Distribution of photon counts in images of Sindbis virus containing a capsid protein tagged with mCherryFP to be used as the fluorescence calibration standard for molecule counting. Y-axis: number of virions. X-axis: 10⁴ photons/sec/virion. The images commonly include both single virus particles and a variable proportion of unresolved pairs, and occasionally a small number of larger aggregates. The single and double particles can be distinguished as two peaks in the histogram of measured intensities. The smooth blue and green curves overlying the histogram show the least squares fit to a Gaussian distribution with two components. The empirical means (sem) for the two components derived from this fitting are 38,318 (494) and 77,406 (683) photons/sec/virion. Inset: fluorescent image of a field of mCherryFP tagged Sindbis virus particles. e Photon count distribution in images of mCherryFP tagged TgDCX associated with “flexible” microtubules (green) or short curved arcs (red). Arrow indicates the expected photon count for FP tagged TgDCX molecules to decorate a single microtubule in a 1:1 TgDCX to tubulin dimer ratio. Y-axis: % of total. X-axis: 10⁵ photons/sec/μm. Insets: fluorescent images containing “flexible” microtubules (bottom) or short curved arcs (top)

Fig. 4

Curved fibers coated with mCherryFP-TgDCX are stable. Deconvolved wide-field images of Xenopus S3 cells expressing EGFP-α-tubulin (green) and mCherryFP-TgDCX (red) before (a) and after (b) treatment with 16 μM nocodazole for 65 min. Note that the curved fibers coated with mCherryFP-TgDCX are resistant to depolymerization by nocodazole. Arrows indicate untransfected cells, expressing EGFP-α-tubulin only, in which virtually all microtubules have depolymerized

Fig. 5

TgDCX bridges TgCPH1 to tubulin. a Deconvolved wide-field images of Xenopus S3 cells expressing mCerulean3FP-TgCPH1, mCherryFP-TgDCX and EGFP-α-tubulin. Inset: a control Xenopus S3 cell that expresses mCerulean3FP-TgCPH1 but not TgDCX (also see Additional file 2: Figure S1). CPH1 on its own does not bind to microtubules in Xenopus cells, but is recruited to the microtubules by TgDCX. b RHΔku80Δhx (“WT”) and TgDCX knockout (ΔDCX) parasites expressing mCherryFP tagged TgCPH1. CPH1 localizes to daughters (arrows) and adult conoids (arrowheads) regardless of the presence of TgDCX

Fig. 6

The DCX domain alone does not support stable microtubule binding in Xenopus cells, or conoid targeting in Toxoplasma. a-b Deconvolved wide-field images of Xenopus S3 cells expressing EGFP-tubulin (green) and either mCherryFP-TgDCX148–243 (a, red) or mCherryFP-TgDCX71–243 (b, red). The boxed insets in b are 1.5x enlarged and contrast-enhanced views of small regions over the nucleus that include the slices from the 3D stacks in which these individual arcs are clearly seen (also see Additional file 5: Movie S3). The DCX domain alone (TgDCX148–243) is not sufficient for microtubule binding, but P25α + DCX domain together (TgDCX71–243) cause binding to microtubules and generation of short arcs. c-d Deconvolved wide-field images of the parental RHΔku80Δhx (“WT”) and TgDCX knockout (“ΔTgDCX”) parasites expressing either mCherryFP-TgDCX148–243 (c), or mCherryFP-TgDCX71–243 (d), two examples are shown for ΔTgDCX). Arrowheads in c indicate the nucleus. Arrows in d point to the conoid; arrowhead in d points to a daughter conoid. e-h EM images of the conoid region of negatively stained T. gondii. Parental RHΔku80Δhx (e, “WT”); TgDCX knockout (f, “ΔTgDCX”); knockout parasites transfected with a plasmid expressing either EGFP tagged full-length TgDCX (g, “ΔTgDCX/TgDCX”), or mCherryFP-TgDCX71–243 (h, “ΔTgDCX/TgDCX71–243”), both expressed under control of the T. gondii α-tubulin promoter (constitutive, See Fig. 9d). i Plaque assays (see Methods) of the parasite strains used for e-h; the parental T. gondii, TgDCX-knockout parasites, and knockout parasites complemented with full-length TgDCX or the fragment containing only the partial P25α domain and the DCX domain, TgDCX71–243. Annotations are the same as e-h. j Domain structure of TgDCX in which the amino acid boundaries of the partial P25α domain and the DCX domain are numbered

Fig. 7

Sequence and structure conservation among DCX domains of TgDCX orthologues. a Sequences of DCX domains from Toxoplasma gondii, Vitrella brassicaformis, Chromera velia, Trichoplax adhaerens, N- and C-terminal domains of humans doublecortin, and a consensus DCX domain from the NCBI Conserved Domain Database (CD01617), were aligned using the MUSCLE program accessed through JalView (V2.10.5, http://www.jalview.org) with default parameters and displayed colored by polarity. Yellow: non-polar (G, A, V, L, I, F, W, M, P); Green: polar, uncharged (S, T, C, Y, N, Q); Red: polar, acidic (D, E); Blue: polar, basic (K, R, H). Secondary structure elements in TgDCX (beta sheet, “β”; alpha helix, “α”; turns, “T”; and a short stretch of 3₁₀ helix), derived from our X-ray crystal structure (PDB 6B4A) of TgDCX148–243, are indicated above the alignment. Black arrowheads indicate residues discussed in the text (TgDCX R152 and HsDCX-N K53; TgDCX D201). CvDCX1 (EupathDB ID: Cvel_6797), CvDCX2 (EupathDB ID: Cvel_18664), CvDCX3 (EupathDB ID: Cvel_28653), VbDCX1 (EupathDB ID: Vbra_15441), VbDCX2 (EupathDB ID: Vbra_12284), VbDCX3 (EupathDB ID: Vbra21191), PfDCX (EupathDB ID: PF3D7_0517800), TaDCX (Uniprot ID: B3RTF1) and HsDCX (NG_011750). b Superposition of backbone ribbon traces of TgDCX148–243 (dark gray-green; X-ray, 6B4A) on the N-terminal DCX domain of human doublecortin (yellow-orange; solution NMR, 1MJD) docked with the structure of αβ-tubulin (α-tubulin blue, β-tubulin cyan; electron crystallography, 1JFF) onto the cryoEM map of human doublecortin bound to microtubules (4ATU). The DCX domain binds in the groove between protofilaments, making contacts with two αβ-dimers. Side-chains are shown for some of the surface-exposed residues. The view is from the outside of the microtubule, corresponding to Fig. 2c of [9]. The (+)-end of the microtubule is towards the top. c Backbone ribbon threading of apicortin orthologue sequences onto the superimposed experimentally-determined structures of human DCX-N docked on microtubules (4ATU) and TgDCX148–243 (6B4A). The view is from the outside of the microtubule. The structures are rotated 135 degrees clockwise relative to the orientation shown in b. The microtubule (+)-end points toward the bottom right corner of the diagram. Only the portion of one β-tubulin close to the DCX domain is included. Side chains are shown for the electronegative patch on β-tubulin, close to DCX, identified as contact region #3 on β-tubulin by [9]. Side-chains of the residues discussed in the text, K53 on HsDCX-N and R152 on TgDCX are also shown, pointing away from tubulin in these structures

Fig. 8

Microtubule binding in Xenopus S3 cells by TgDCX and its orthologues. a-i Deconvolved wide-field images of Xenopus S3 cells expressing both EGFP-tubulin and mCherryFP tagged DCX orthologues. The grayscale images show the fluorescence from the mCherryFP channel only. For a subarea, both channels are displayed to show both the EGFP tagged microtubules (green) and mCherry tagged orthologues (red). In a, b, c, and g, the orthologues are associated with microtubules, whereas in d, e, f, h, and i they are distributed diffusely throughout the cytoplasm and nucleus

Fig. 9

Localization of TgDCX and its orthologues in Toxoplasma. a-b Deconvolved wide-field images of dividing TgDCX knockout (a, “ΔTgDCX”, two examples) and RHΔku80Δhx (b, “WT”) parasites transiently expressing TgDCX-eGFP driven by the T. gondii α-tubulin promoter. TgDCX-eGFP is highly enriched in the mother conoid (green arrowhead) and daughter conoids (green arrows) and is absent from the cortical microtubules of mother parasites. However, in contrast to expression regulated by the endogenous promoter, when expression is driven by this nearly constitutive (see d) α1-tubulin promoter, in some cases TgDCX-eGFP signal is also detected on the daughter cortical microtubules, centrosomes (cyan arrowheads), and basal complexes (cyan arrows). Dashed cyan lines in a outline two of four parasites in the same parasitophorous vacuole. Insets: 1.5x. The lower panels show merged DIC and fluorescence (in red) images. c Deconvolved wide-field images of RHΔku80Δhx (WT) parasites expressing FP tagged DCX orthologues. Two examples are shown for CvDCX1. In the left example, dashed blue lines outline 4 of the 8 parasites in the vacuole. In the right example, the dashed blue oval outlines two nearly mature daughters, shown enlarged 1.5x in the oval inset with white outline. Note that among the eight orthologues, only CvDCX1 closely mimics the pattern of localization shown by TgDCX (when expressed under this T. gondii α1-tubulin promoter). Green arrows: daughter conoids. Green arrowheads: mother conoids. Cyan arrowhead: centrosome. d Time course of RNA expression levels [21] in Toxoplasma gondii for α1-tubulin (green) and TgDCX (red). Tubulin expression is nearly constitutive, whereas TgDCX varies by more than 30-fold across the cell-cycle

Fig. 10

CvDCX1 cannot rescue the structural and lytic cycle defects of the TgDCX knockout parasite. a-c EM images of the conoid region of negatively stained T. gondii. Parental RHΔku80Δhx (a, “WT”), TgDCX knockout (b, “ΔTgDCX”, two examples); a clone of TgDCX knockout parasite stably expressing CvDCX1-mNeonGreenFP under control of the T. gondii α-tubulin promoter (c, “ΔTgDCX/CvDCX1”, three examples). Compare with Fig. 6g and h. d Plaque assay (see Methods). Knockout parasites complemented with TgDCX-eGFP expressed under control of the T. gondii α-tubulin promoter (“ΔTgDCX/TgDCX”). Other annotations same as in a-c. Compare with Fig. 6i

Fig. 11

Correlative light and electron microscopy analysis of microtubules in untransfected CvDCX1-expressing Xenopus cells. a EM images of microtubules in sections of an untransfected Xenopus cell. In all cases where they are countable, 13 protofilaments (pf) are present. No microtubules with other than 13 pf were seen in untransfected cells. b Fluorescence and DIC light microscope images of the cell sectioned in a. The cell is from a line expressing EGFP-α-tubulin. The plane of sectioning in the EM images is shown by the white bar. c EM images of microtubules in sections of a Xenopus cell transfected with mCherryFP-CvDCX1. All microtubules have 13 pf. The tannic-acid-enhancement of microtubule staining (see Methods) is more effective when the microtubules are heavily decorated, which makes the protofilaments more obvious and more easily countable, but the diameter of the microtubules is approximately the same as in untransfected cells. d Fluorescence and DIC light microscopy images of the cell sectioned in c. mCherryFP-CvDCX1 is shown in red, EGFP-α-tubulin in green. The plane of section is shown by the white bar. The magnification is the same as for b. Note that the elongated narrow extension of the transfected cell lies on top of another untransfected cell, running over the edge of the latter’s nucleus. e Low magnification EM images of a cross-section of the cell shown in d. The thin extension of the transfected cell is seen crossing over the underlying untransfected cell. The region within the white box, shown enlarged on the right, contains > 100 parallel microtubules viewed in cross-section, appearing as tiny black doughnuts at this magnification

Fig. 12

Correlative light and electron microscopy analysis of microtubules in Xenopus cells expressing TgDCX. a Montage of images of microtubule rafts viewed in cross-section by EM, from a Xenopus cell expressing TgDCX-mCherryFP. Often the microtubules on one edge of a raft are incomplete tubes (white arrows), as are some of the single microtubules in these cells. The light micrographs at the bottom show fluorescence and DIC images of the sectioned cell, which is also expressing EGFP-α-tubulin. The plane of sectioning is shown by the white bar. TgDCX-mCherryFP is shown in red, EGFP-α-tubulin in green. b Light and electron microscope images of a Xenopus cell expressing mCherryFP-TgDCX71–243 and EGFP-α-tubulin. mCherryFP-TgDCX71–243 is shown in red, EGFP-α-tubulin in green. The plane of section is indicated by the white bar. The EM images show microtubule cross-sections in these cells, which are similar to those in cells expressing full-length TgDCX, quite different from microtubules in untransfected cells (c.f. Fig. 11). c-f Comparison of microtubule shapes and sizes. c&d: a single microtubule with more than 13 pf, and a cluster of three microtubules from TgDCX71–243 transfected cells. Note that the width of the incomplete microtubules is often larger than the complete tubes, suggesting that the gap results from a tube expanding in diameter and splitting open, rather than from loss of protofilaments. e: a 13 pf microtubule from a cell expressing CvDCX1. f: a 13 pf microtubule from an untransfected cell