Simone Lepper

Positioning of large organelles by a membrane- associated cytoskeleton in Plasmodium sporozoites

Cellular organelles are usually linked to the cytoskeleton, which often provides a scaffold for organelle function. In malaria parasites, no link between the cytoskeleton and the major organelles is known. Here we show that during fast, stop‐and‐go motion of Plasmodium sporozoites, all organelles stay largely fixed in respect to the moving parasite. Cryogenic electron tomography reveals that the nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. These tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nm repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane‐associated filaments. While the spatial organization of the large parasite organelles appears dependent on their linkage to the cortex, the specialized secretory vesicles are mostly not linked to microtubules or other cellular structures that could provide support for movement.

Geometric constrains for detecting short actin filaments by cryogenic electron tomography

Polymerization of actin into filaments can push membranes forming extensions like filopodia or lamellipodia, which are important during processes such as cell motility and phagocytosis. Similarly, small organelles or pathogens can be moved by actin polymerization. Such actin filaments can be arranged in different patterns and are usually hundreds of nanometers in length as revealed by various electron microscopy approaches. Much shorter actin filaments are involved in the motility of apicomplexan parasites. However, these short filaments have to date not been visualized in intact cells. Here, we investigated Plasmodium sporozoites, the motile forms of the malaria parasite that are transmitted by the mosquito, using cryogenic electron tomography. We detected filopodia-like extensions of the plasma membrane and observed filamentous structures in the supra-alveolar space underneath the plasma membrane. However, these filaments could not be unambiguously assigned as actin filaments. In silico simulations of EM data collection and tomographic reconstruction identify the limits in revealing the filaments due to their length, concentration and orientation. PACS Codes: 87.64.Ee

Structural Differences Explain Diverse Functions of Plasmodium Actins

Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.

Rapid quantification of the effects of blotting for correlation of light and cryo-light microscopy images

Recent technical developments allowed the accurate correlation of fluorescently labelled organelles in living cells to cryo‐electron micrographs. We aimed at expanding this approach to Plasmodium berghei sporozoites, the motile forms of a rodent malaria parasite, which can be imaged by cryo‐electron tomography in toto without the need for sectioning. Sporozoites are crescent shaped eukaryotic cells that move on flat supports including EM grids in a circular, unidirectional manner. While sporozoites can be visualized with fluorescent light and cryo‐light microscopy prior to tomography, few motile sporozoites remained on the grid after blotting excess liquid impairing a complete correlation from light microscopy to cryo‐electron tomography. Comparison with cells showing different adhesion strengths demonstrated that the ratio of cells remaining on the grid can be rapidly determined, but that the integrity of the cells has to be carefully monitored as the blotting applies high physical stress to the cells. We demonstrate a quick technique to assess not only feasibility of direct correlation without fixation but also the damage caused by blotting.

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites.

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.

Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments

Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using Plasmodium, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of Plasmodium actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit–subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.

Spotlight on pathogens: 'Imaging Host-pathogen Interactions'.

The self-evidence of pictures has nowadays taken a back seat, as pictures are abundantly present in our world. Only proverbs like ‘seeing is believing’ still acknowledge the predominance of the picture over the written word. While the social sciences are still struggling who should deal with the analysis of the influence of all the pictures on society (Boehm, 1994), the natural sciences already accepted the high impact of pictures on their hypotheses and are waiting for the impact films will have on science. Despite this, we will give a written overview of a symposium held in Heidelberg, which tried to connect the rather new field of cellular microbiology (Cossart et al., 1996) with the latest advances in imaging techniques. Several meetings were already dedicated to this subject over the last years (Lehmann and Frischknecht, 2006; Celli and Knodler, 2008). These clearly showed that pathogens represent not only interesting microscopic objects themselves, but can also be used as probes to decipher complex cell biological processes as they turn host cell functions for their own benefit (Lehmann and Frischknecht, 2006). To successfully image the interactions between pathogens and their host cells an interdisciplinary approach between imaging scientists and microbiologists is required. The symposium in Heidelberg put a strong emphasis on different infection paradigms and on dynamic imaging of infections, as real-time imaging in multiple dimensions is getting widely accessible. Lectures on in silico modelling, image rendering and object tracking completed the programme. Freddy Frischknecht and Maik Lehmann (both from the University of Heidelberg, Germany) welcomed the congress in a charming villa in the old city centre of Heidelberg. The probes – viruses, fungi, parasites, bacteria and prions – seemed almost like a diverse collection out of Pandora’s box. But unlike Pandora, the masters of these beasts found the matching imaging techniques to observe the probes’ fate in detail. Like in a vitreous Pandora’s box, the audience could watch the destructive pathogens unfold their virulence. In a short retrospective on the history of microscopy, Freddy Frischknecht pointed out that the foundation of modern physical optics was actually laid in a book written by the Arab Ibn al-Haytham. Being under house arrest in Cairo from 1011 until 1021, he wrote ‘The Book of Optics’, which influenced the development of that field and especially the emerging understanding of light and vision. About 650 years later the Dutch tradesman Antoni van Leeuwenhoek built many single-lens microscopes and noticed, for example, bacteria on rotten teeth and red blood cells in the vasculature of living fish. Observing for the first time single cells and pathogens with a magnification of more than 200 times, he thus can be considered as the founder of cell and pathogen imaging despite the fact that a formal acceptance of microorganisms as causative agents of disease had to wait another 200 years.

Correlated Microscopy: From Dynamics to Structure

It has always been the aim of functional studies in biology to establish structurefunction relationships of the molecular machineries involved in the process of interest. A powerful tool within this field has always been electron microscopy continuously adapting to answer latest questions [1, 2] and therefore, principally shaping our view on cell architecture. Correlative approaches now allow to bridge the gap of information by combining dynamic data obtained by fluorescent light microscopy (LM) with the structural information of cryo-electron microscopy (CEM) or cryo-electron tomography (CET) and vice versa. Plasmodium berghei sporozoites are an ideal system for such studies. They are the reactive agents transmitting malaria from mosquitoes to rodents, and due to their close relation to the human pathogen Plasmodium falciparum they are widely used as a model organism. Since they are only 1 ?m thick they can be viewed in toto with ET. The sporozoites represent both, a motile, highly polarized eukaryotic cell and a devastating pathogen.