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

疟原虫子孢子是通过受感染的蚊子传播给哺乳动物宿主的疟疾寄生虫的形式。在蚊子叮咬期间，它们进入皮肤，在那里它们迁移到找到血管，然后入侵继续到肝脏的旅程。寄生虫的迁移能力在疟疾生命周期的许多步骤中至关重要。在众多的分子相互作用中，肌动蛋白的聚合在寄生虫迁移中起着重要作用。奇怪的是，寄生虫体内聚合的肌动蛋白丝非常短，人们对它们在哪里形成以及如何定向知之甚少。由于经典的电子显微镜未能回答这些问题，我们现在正在寻求一种新的方法，结合光学显微镜和冷冻电子断层扫描。为此，我们想用光学显微镜在电子显微镜网格上拍摄数百个移动的疟原虫子孢子。然后，我们将网格放入液态乙烷中，迅速冷冻寄生虫，这最能保持它们的天然结构。在显微镜下检查这些网格允许在液氮温度下检测荧光，这将使我们能够将冷冻寄生虫的位置与先前在网格上迁移的寄生虫相关联。一旦确定了能动的寄生虫，网格就被转移到低温电子显微镜中，并拍摄一系列断层图像。下面的重建使我们能够探测动态冷冻寄生虫的结构，并有望为他们难以捉摸的肌动蛋白细胞骨架提供新的见解。

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.