Using dynamic light microscopy and cryo-electron tomography we will investigate the actin cytoskeleton during gliding motility of malaria parasites

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Pfasmodium sporozoites are the forms of malaria parasites transmitted to the mammalian host by an infected mosquito. During a mosquito bite they enter the skin where they migrate to find a blood vessel, which is then invaded to continue the journey to the liver. The ability of the parasites to migrate is essential at many steps during the malaria life cycle. Among many molecular interactions, the polymerization of actin is important in parasite migration. Curiously, polymerized actin filaments in parasites are very short and little is known about where they are formed and how they are oriented. As classical electron microscopy has failed to answer these questions, we are now seeking a new approach, combining light microscopy and cryo-electron tomography. To this end, we want to film hundreds of moving Plasmodium sporozoites on electron microscopy grids using a light microscope. We then rapidly freeze the parasites, by dropping the grid into liquid ethane, which best preserves their native structure. Examination of these grids in a microscope allows the detection of fluorescence at liquid nitrogen temperatures, which will enable us to correlate the position of frozen parasites with those that were prior filmed migrating on the grid. Once motile parasites are identified the grid is transferred into the cryo-electron microscope and a tomographic series of images is taken. The following reconstructions allow us to probe the structure of the dynamically frozen parasites and will hopefully give new insights into their elusive actin cytoskeleton.