Imaging Motile Malaria Parasites

How an Ancient Eukaryote can Serve as a Motile Model Cell

  • Fig. 1: (A) Cartoon of the malaria parasite life cycle. The parasite causes disease in the blood, where it replicates in erythrocytes. Invasion of erythrocytes depends on an actin-myosin motor of the parasite. Sexual stages can be transmitted to the mosquito during a bite and after fertilization a motile egg (the ookinete) is generated which again relies on an actin myosin motor to penetrate the gut of the mosquito. Within cysts hundreds of Plasmodium sporozoites develop that ultimately invade salivary glands of the mosquito. From there they can be injected into the host, where they use the actin-myosin motor to move in the skin, enter and exit blood vessels and invade liver cells. Sporozoites also migrate through cells in the skin and liver. (B) Simplified tree of life modified after Simpson and Roger, Current Biology 2004. Animals and fungi belong to the Opisthokonta, Plasmodia to the Chromalveolata.Fig. 1: (A) Cartoon of the malaria parasite life cycle. The parasite causes disease in the blood, where it replicates in erythrocytes. Invasion of erythrocytes depends on an actin-myosin motor of the parasite. Sexual stages can be transmitted to the mosquito during a bite and after fertilization a motile egg (the ookinete) is generated which again relies on an actin myosin motor to penetrate the gut of the mosquito. Within cysts hundreds of Plasmodium sporozoites develop that ultimately invade salivary glands of the mosquito. From there they can be injected into the host, where they use the actin-myosin motor to move in the skin, enter and exit blood vessels and invade liver cells. Sporozoites also migrate through cells in the skin and liver. (B) Simplified tree of life modified after Simpson and Roger, Current Biology 2004. Animals and fungi belong to the Opisthokonta, Plasmodia to the Chromalveolata.
  • Fig. 1: (A) Cartoon of the malaria parasite life cycle. The parasite causes disease in the blood, where it replicates in erythrocytes. Invasion of erythrocytes depends on an actin-myosin motor of the parasite. Sexual stages can be transmitted to the mosquito during a bite and after fertilization a motile egg (the ookinete) is generated which again relies on an actin myosin motor to penetrate the gut of the mosquito. Within cysts hundreds of Plasmodium sporozoites develop that ultimately invade salivary glands of the mosquito. From there they can be injected into the host, where they use the actin-myosin motor to move in the skin, enter and exit blood vessels and invade liver cells. Sporozoites also migrate through cells in the skin and liver. (B) Simplified tree of life modified after Simpson and Roger, Current Biology 2004. Animals and fungi belong to the Opisthokonta, Plasmodia to the Chromalveolata.
  • Fig. 2: (A) Cartoon (top) and rendered tomogram (right) of the Plasmodium sporozoite [10]. Note the microtubule basket (green) at the front end of the sporozoite that encircles specialized secretory vesicles (magenta and cyan), the nucleus at the rear and the chloroplast-like apicoplast (yellow). A further blow-up (grey) shows a schematized drawing of selected proteins that play a role in sporozoite motility, the plasma membrane proteins TRAP, TLP, S6, the cytoplasmic aldolase and actin as well as the myosin, which is anchored to the inner membrane complex (IMC, shown in yellow in the rendered tomogram), a specialized organelle subtending the entire plasma membrane. (B) DIC images of the rodent malaria model organism Plasmodium berghei moving in circles on glass. Time is indicated in seconds. Scale bar: 10 µm. (C) Fluorescent images of motile P. berghei sporozoites expressing the green fluorescent protein in their cytoplasm. Three subsequent images are colored red, green and blue to highlight different movements (insets) [1].
  • Fig. 3: (A) Reflection interference contrast microscopy of migrating sporozoites reveals individual adhesion sites (dark spots) and their dynamics [5]. Numbers indicate time in seconds. (B) Sporozoites move rapidly (blue) in a stop-and-go fashion that corresponds to the formation (stop) and rupture (go) of substrate adhesions [5].
  • Fig. 4: Trajectories of sporozoites moving (A) in two different sites of the skin of a mouse after transmission during a mosquito bite and (B) in patterned PDMS-obstacle arrays (top: DIC top views of obstacles spaced 3 and 5 µm apart; bottom: sporozoite tracks in green and obstacle outlines in red) [8]. (C) Comparison of mean square displacements over time from sporozoites in A and B [8].

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.

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 [4]. 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 [5] and never assume productive motility [6]. They also fail to enter mosquito salivary glands, as the TRAP protein is necessary for binding to their surface [6]. 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 [5]. 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 [5]. 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 [8].

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.

Acknowledgements
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.

References
[1] Hegge S. et al.: Biotechnol. J. 4, 903-13 (2009)
[2] Hegge S. et al.: FASEB J. 24, 5003-12 (2010)
[3] Hellmann J.K. et al.: PLoS One 5, e8682 (2010)
[4] Hegge S. et al.: FASEB J. 24, 2222-34 (2010)
[5] Münter S. et al.: Cell Host Microbe 6, 551-62 (2009)
[6] Sultan et al.: Cell, 90, 511-22 (1997)
[7] Steinbüchel et al.: Cell. Microbiol. 11, 279-88 (2009)
[8] Hellmann J.K. et al.: PLoS Pathogens 7, e1002080 (2011)
[9] Amino R. et al.: Nat. Med. 12, 220-4 (2006)
[10] Kudryashev M. et al.: Cell. Microbiol. 12, 362-71 (2010)

Authors
Dr. Friedrich Frischknecht
Janina Kristin Hellmann
Mirko Singe
r
University of Heidelberg Medical School
Parasitology, Department of Infectious Diseases
Heidelberg, Germany
www.klinikum.uni-heidelberg.de

 

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