Variable microtubule architecture in the malaria parasite

Abstract

Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. This arrangement is considered the canonical form and is adopted by most organisms, with rare exceptions. Here, we use in situ electron cryo-tomography and subvolume averaging to analyse the changing microtubule cytoskeleton of Plasmodium falciparum, the causative agent of malaria, throughout its life cycle. Unexpectedly, different parasite forms have distinct microtubule structures coordinated by unique organising centres. In merozoites, the most widely studied form, we observe canonical microtubules. In migrating mosquito forms, the 13 protofilament structure is further reinforced by interrupted luminal helices. Surprisingly, gametocytes contain a wide distribution of microtubule structures ranging from 13 to 18 protofilaments, doublets and triplets. Such a diversity of microtubule structures has not been observed in any other organism to date and is likely evidence of a distinct role in each life cycle form. This data provides a unique view into an unusual microtubule cytoskeleton of a relevant human pathogen.

Fig. 1. Imaging parasites across the Plasmodium life cycle: from live parasites to high-resolution 3D volumes.

a Simplified Plasmodium life cycle of parasite forms studied here. Sporozoites are injected into the host. After differentiating in the liver, merozoites are released into the blood. The majority enter an asexual replication cycle (merozoites) and a small percentage commit to becoming gametocytes. Gametocytes are taken up by a mosquito and after fusion of male and female gametes in the mosquito gut, zygotes transform into ookinetes. Ookinetes cross the mosquito midgut, develop into oocysts and form thousands of sporozoites, which migrate to the salivary glands. b Schematic representation of our workflow: (i) live parasites are vitrified on EM grids. (ii) cells are thinned into lamella and then (iii) imaged by electron cryo-tomography (cryo-ET). Tilt-series are collected and computationally reconstructed into 3D volumes. c Columns 1–4: representative images of parasites at different workflow steps. 1: compositions of fluorescence images of cells highlighting overall parasite shape. Inset: cartoon representation of each stage. 2: Scanning Electron Microscopy (SEM) micrographs showing Plasmodium parasites (some false-coloured in yellow and green) surrounded by host cells (green asterisks). 3: micrographs showing overviews of lamellae. 4: slices through example tomograms.

Fig. 2. Sporozoite subpellicular microtubules (SPMTs) contain a periodic luminal density inside 13 protofilament microtubules.

a Slice through a tomogram illustrating the overall architecture of the pellicle within a cell. One SPMT is seen weaving in and out of the slicing plane. Insets: slices through EM map (shown in B, C, D) placed back into the tomogram at positions determined by SVA. b Orthogonal slices through the EM map. c Isosurface representation of the EM map with pseudoatomic model (7MIZ)  fitted into the EM density. p1-p13 = protofilament numbers, dotted line = seam position. d Top: section through the EM map showing the position of an α/β tubulin dimer relative to TrxL1. Bottom: radial projection with one period of the ILH highlighted in purple. e The apical pole of a P. falciparum sporozoite with a full set of microtubules (13 blue + 1 green). The APR is represented by an isosurface, the SPMTs as pin models, where the pinhead marks the centre and the line is oriented towards the seam. f Segmented sporozoite apical pole. The tubulin density (13 protofilaments) of two SPMTs was hidden to reveal the ILH. Unannotated slice through tomogram shown in Fig. S3a and the full volume in supplemental movie S1.

Fig. 3. Ookinete SPMTs contain interrupted luminal helices and are organised in a complex apical pole.

a Tomogram slice through the apical end of an ookinete with SPMTs oriented along the apico-basal axis. A cartoon of an ookinete on the right shows approximate centre positions of lamella in a, b and d. b Slice through an ookinete apex proximal region with SPMTs cut transversally, the annotation colours are the same as in a. SPMTs get closer to the IMC as inner apical collar tapers off (from left to right). The rotational orientation of SPMTs (centre to seam) is indicated with arrows. c Orthogonal sections through EM map determined by SVA of ookinete SPMTs. d Segmentation of an apex proximal region of an ookinete with transversally cut SPMTs, highlighting the two apical collar layers between the SPMTs and IMC. The conoid is shown in green. e Segmentation of the apical pole of an ookinete. The tubulin density of one SPMT was hidden to reveal the ILH. Inset shows a slice through an average volume of the conoid periodic structure. Unannotated slice through tomogram shown in Fig. S3b and full volume in supplemental movie S2.

Fig. 4. Merozoites have canonical subpellicular (SPMTs) and nuclear spindle microtubules.

a Slice through a tomogram of an apical pole. Inset: slice through the microtubule subvolume average showing 13 protofilaments. b Segmentation of a nucleus in a dividing schizont with a partial spindle body. All four nuclear pore complexes observed in this section of the nucleus are in proximity of the spindle. Inset: slice through an isosurface of the SPMT EM map. c Segmentation of a single merozoite within a fully segmented schizont. Note: for simplification the IMC is segmented as a continuous double membrane although multiple discontinuities were observed. Unannotated slice through tomogram shown in Fig. S3c and full volume in supplemental movie S3.

Fig. 5. Gametocyte subpellicular microtubules (SPMTs) have a wide range of protofilament numbers with random polarity.

a Micrograph showing a row of singlet and doublet SPMT cross sections and highlighting the range of sizes. Protofilament numbers are indicated (for doublets of the A tubule). Micrographs at two different tilt angles were stitched to show SPMT transversal views. b Segmentation of a stage III gametocyte nucleus with a spindle pole body at a nuclear pore complex. Microtubule colours correspond to protofilament numbers as shown in c, d. c. Bar chart of the distribution of different protofilament numbers in SPMTs (blue) N = 155 and nuclear spindles (lilac) N = 31. Distributions are significantly different (p = 5 × 10⁻⁸, chi-squared). d Isosurfaces of microtubules from subvolume averaging with protofilament numbers from 13 to 18. e Schematic representation of the differences in microtubule diameter. f Segmentation of a stage III gametocyte with transversely sectioned SPMTs. Microtubule colours correspond to protofilament numbers as shown in c, d. ‘+/−’ indicates the polarity of each microtubule. Unannotated slice through tomogram shown in Fig. S3d and full volume in supplemental movie S4.

Fig. 6. Microtubule distance from the IMC is consistent in all forms, but the interrupted luminal helix may be needed to uniquely determine their radial orientation.

Panels a–c left: scatter plots of individual SPMT distance (dIMC) and radial orientation (φIMC) with respect to the IMC. Each point represents the median along a single SPMT. Ookinete and sporozoite SPMTs have a defined polarity whereas gametocyte orientations are random (see Fig. S7 for 2D histogram representation of these data). Parasite form cartoons indicate subcellular locations where data points were sampled: at the cell body or the apex. Right: average volumes of SPMTs sampled at the indicated subcellular locations. Although the subvolume averaging was focused on SPMTs, IMC and APR components can be seen in the EM maps after extracting large subvolumes (200 nm edges). This is a consequence of the consistent SPMT-IMC distance and orientation. Cartoons below average volumes are models of the pellicle architecture at each location. Note that since the IMC wraps around the torus-shaped sporozoite APR, it was not possible to measure a single apical dIMC (apical φIMC angles are shown in Fig. S6b). Gametocyte panel (c) shows sections through individual SPMTs rather than an average, to indicate their inconsistent orientations and different number of protofilaments. *See supplementary materials for details and assumptions made in gametocyte orientation assignment. d Violin plot comparing dIMC between forms, widths are scaled according to the amount of data.

Fig. 7. Structural diversity of Plasmodium microtubules across its life cycle.

a Cartoon representations of the four Plasmodium forms analysed. b Table summarising the main architectural differences in the four forms. Solid outlines indicate properties that are likely correlated: the presence of an apical polar ring sets the SPMT polarity and ILH is directly linked to setting the seam orientation with respect to the IMC.