Cell motility is an essential process for most uni- and multi-cellular forms of life. The study of evolutionary ancient and thus divergent cells can yield interesting insights into our very basic understanding of molecular processes conferring cellular motility. The Plasmodium sporozoite represents a unique cell that migrates in vitro in near perfect circles due to its crescent shape. This allows the combination of high throughput imaging with reverse genetics to dissect how these cells move. As Plasmodium sporozoites are the stage of the malaria parasite transmitted by mosquitoes, understanding their movement might in addition give us a new handle for stopping these deadly parasites before they cause havoc.
Motility of Malaria Parasites
Malaria is transmitted by the bite of a mosquito, when Plasmodium sporozoites are deposited in the skin of an animal, be it house sparrow, lizard or man. Sporozoites of species infecting mammals migrate through the skin to enter blood vessels, attach to the endothelium of liver sinusoids and migrate further to ultimately invade a liver cell. Like in other stages of the malaria parasite's complex life cycle (fig. 1A), motility is driven by an actin-myosin motor located underneath the plasma membrane. The generated force is transduced to the substrate via adhesion mediating proteins spanning the plasma membrane of the parasite. The basic motor machinery is conserved among all apicomplexa, which belong to the chromalveolata, one of the six known eukaryotic groups (fig. 1B). It is for this reason that we think Plasmodium deserves attention not just as a medically important parasite, but as an interesting unicellular model organism that can reveal more insights into how life can work.
A Rodent Parasite as Model Cell
Importantly, the rodent malaria model species Plasmodium berghei can be readily genetically manipulated and imaged throughout its life cycle. Plasmodium also only encodes a limited set of actin binding proteins, which are homologous to known actin binding proteins from metazoans or yeast and thus likely play a role in forming filaments.
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Due to their divergence we might, however, also expect to find some unique proteins. Sporozoites are particularly interesting from a cell biological viewpoint, as they constitute highly polarized cells that move, usually with their front end leading, in a macroscopic simple circular fashion (fig. 2A,B). Sporozoites expressing the green fluorescent protein can be imaged by the hundreds in a standard wide field fluorescent microscope and their movement (fig. 2C) and potential morphological changes can be quantitatively analyzed [1-3]. This should in principle allow the identification of the necessary components of the motility machinery and the dissection of the molecular events leading to motility.
Membrane Proteins with Different Functions
A first quantitative study of the adhesion process prior to motility showed that the parasite follows a step-by-step sequence before it assumes motility . This suggests that different surface proteins of the parasite might mediate distinct adhesion steps. Indeed, a number of proteins are specifically expressed on the sporozoite surface that could fulfill such functions including the TRAP family members TRAP, TLP and S6. These proteins show a conserved cytoplasmic tail and extracellular adhesion domains (fig. 2A). Parasites lacking TRAP can only undergo one adhesion step, seem to be blocked from further adhesion  and never assume productive motility . They also fail to enter mosquito salivary glands, as the TRAP protein is necessary for binding to their surface . Sporozoites lacking S6 show a similar albeit weaker loss of their adhesive property as those lacking TRAP but some can still enter salivary glands and commence motility [4,7]. Parasites lacking TLP show only a weak phenotype that could only be clarified using detailed image analysis [4,8]. It appears that these parasites lack the capacity to adhere strongly to the substrate and thus detach more frequently during motility compared to wild type parasites. This can be compensated by the application of flow, which presumably presses the motile sporozoites to the substrate and thus compensates for the lack of adhesion.
Adhesion Turnover of Sporozoites
Sporozoites are just over 10 micrometer in length and it was thus assumed that they could form distinct adhesion sites. To investigate these, reflection interference contrast microscopy was used . This showed individual attachment zones interspersed with regions where the parasite appeared further away from the substrate surface (fig. 3). Curiously the attachment zones did not behave like classic adhesion sites of motile cells: the parasite does not establish an adhesion at the front, then moves over that adhesion while establishing new ones at the front and ultimately detaches from them at the rear end. Rather the sporozoites showed two types of adhesions: one type (at the front and rear ends) appeared to stay at the same spot relatively to the parasite during movement. The other type appeared to progress rearwards faster than the parasite translocated forward. During movement the parasite continuously detaches from the substrate at the front and rear and undergoes cycles of rapid acceleration and slow-down. Whenever an adhesion of the first type is lost, the parasite speeds up and when it reattached to the substrate, it slows down . This was named stick-slip motility. Importantly, similar stop-and-go movements were also observed with sporozoites moving on cells or in their natural tissues.
Migration without Chemotaxis
Within tissues, such as the skin, sporozoites do not move in circles but move on apparently random paths [8, 9]. Curiously, sporozoites move in different patterns in different skin environments (fig. 4A). This raised the question if sporozoites follow chemotactic cues or whether they simply squeeze through the tissue in a random manner. To probe these micro-patterned PDMS substrates were used as obstacle arrays (fig. 4B). This showed that sporozoites placed between the obstacles could, at certain obstacle densities, migrate with similar patterns and mean square displacements (fig. 4C) as those migrating in vivo .
Taken together, we postulate that gliding motility of Plasmodium sporozoites is a form of cellular movement that is specialized for rapid crossing of tissue barriers inside its host. We hope that further studies will reveal general insights into the basic concepts of coupling actin-myosin motors to the substrate.
We thank Stephan Hegge, Misha Kudryashev and Sylvia Münter for figures, Leandro Lemgruber for comments, the German Federal Ministry for Education and Science (BioFuture and NGFN), the German Science Foundation (SFB 544, SPP1464), the University of Heidelberg Frontier Program and the Chica and Heinz Schaller Foundation for funding.
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