Everyone knows the high-pitched buzz of a mosquito in the dark. In many parts of the world, ignoring it can be the opening move in a malaria infection. An infected Anopheles mosquito injects Plasmodium sporozoites into the skin; these slender, slightly curved cells then race between and through cells and possibly along collagen fibers, find a capillary, and ride the bloodstream to the liver. To do this well, they have to move—fast and decisively—through complex environments. If sporozoite motility is blocked in the skin, they do not reach the liver and the infection is stopped before the first symptoms.
Because skin is crowded and heterogeneous, researchers have long complemented in-vivo work with simpler, controllable assays. If you gently rupture an infected salivary gland onto a coverslip and add medium, sporozoites begin to move on glass. The pattern looks peculiar at first: they move in circles, often persistently. In these classic 2D assays (glass or cells with medium on top), the parasites move in mostly counter-clockwise circles.
Such simple assays in combination with the generation of genetically modified parasites allowed scientists to study the machinery driving parasites motility and parasite invasion, which is notoriously difficult to observe. Both are based on complex interactions of myosin motors with short-lived actin filaments, that move adhesins towards the back of the cell, resulting in forward speeds of up to 2 µm/s.
Our story started when we asked how sporozoites move in homogeneous 3D environments, as surrogate for skin, but simple enough to see the parasite intrinsic movement patterns and to quantitatively analyze it with image processing. A previous student noticed that if you place a broken salivary gland onto a very soft polyacrylamide hydrogel and gently press with a glass slide, sporozoites enter the gel right under the gland. That observation gave us a clean way to record 3D trajectories at scale.
Take your right hand, stick out the thumb and turn it down towards the table, envisioning what happens when a right-handed helix hits the table, resulting in a clockwise circle if observed from above, exactly the opposite from the counter-clockwise circles observed on a glass slide before. This initially made no sense and had us scratching our heads. Why would parasites suddenly move differently on glass after migrating through a gel?
These observations also imply that a sporozoite circling on a glass slide in medium, assuming it attempts a right-handed helix, ‘wants to’ move upwards, into the medium, not invade the glass slide (simply use your right hand to follow this argument). To test this unintuitive connection between 2D circling direction and 3D helix, we developed a sandwich assay. Placed between two gels, sporozoites circling counter-clockwise move upwards, while those circling clockwise move downwards.
“It was challenging to get the gels and the sporozoites all together just right, but once we had it, it was stunning how clearly the sporozoites invaded in the respective directions, and for the first time we didn’t need to include the salivary gland to see invasion into the gel!”, Mirko remembers.
From here, the obvious question was to ask how the sporozoite achieves this very particular combination of chiral motion patterns. Biophysically, we considered two routes to chirality:
- Chiral surface flow: the rearward flow of adhesins has an intrinsic twist—like a conveyor belt that’s slightly skewed—due to internal architecture.
- Asymmetric adhesin distribution: adhesins are released/bind unevenly on the cell surface because the apical polar ring (APR) is tilted, making one side effectively “grippier.”
Both mechanisms can generate right-handed helices in 3D in simulations that combine our previous gliding-motility theory to include an adhesion field and torsional deformability (at roughly fixed curvature). However, for 2D substrates the two theories are very different, because they predict opposite stable circling directions. Only the asymmetric adhesion model naturally reproduces CW gliding at the gel bottom and CCW gliding on glass with medium.
“The penny dropped when we asked what each model predicts on a 2D surface embedded in 3D,” Leon recalls. “They make opposite calls—so we suddenly had a decisive, testable difference.”
If adhesins are biased to one side, traction should differ above and below a gliding parasite held in a narrow gap. This essentially called for setting up a two-sided traction force microscopy assay, which was quite challenging; the traction-force gels being stiffer than the ones for the invasion sandwich assay making this setup even less forgiving. But once it worked, the pattern was clear: the side that drives (via the adhesins) versus the side that drags aligns with the predictions of the model for asymmetric adhesin distribution.
But what could be the reason for this uneven distribution of adhesins?
This brought Mirko Singer back to his first weeks working with parasites 15 years ago. At that time, first cryo-electron tomography of sporozoites showed that an ultrastructural feature at the apical tip of these parasites is a strongly tilted apical polar ring. This ring is present in all known apicomplexan parasites but only in Plasmodium sporozoites it seems tilted. To understand its orientation, Mirko originally tried to image sporozoites moving on electron microscopy (EM) grids, then preparing them for EM by removing the medium and then seeing how many of those previously moving sporozoites remained stable at the same position. The goal was to combine the motion information from life cell microscopy with cryo-electron tomography. But this proved to be impossible at that time, leaving the question on how the apical polar ring is tilted in respect to the movement direction of the sporozoite unanswered.
Now, many years later, technology had improved. The old cryo-electron tomography contained a crucial detail to the puzzle: Sporozoites have 16 subpellicular microtubules attaching to the apical polar ring and moving to the center of the parasite directly underlying the plasma membrane. One of these microtubules, however, is special: it always follows the parasite side away from the direction the apical ring tilts towards, while all other 15 microtubules are found on the same side the ring faces.
It was known from previous experiments that all adhesins are secreted through that apical polar ring, and the tilt direction we found was consistent with the side of the sporozoite where our models and experiments suggested the higher density of adhesins. Therefore, we finally concluded that the apical polar ring is the main ultrastructural reason for sporozoites chirality, getting the scientists one step closer to understand why and how parasites show these surprising chiral motion patterns on the cellular scale.