Plasmodium coronin function during malaria parasite migration

冠状蛋白是一个肌动蛋白结合蛋白家族，含有一个或两个含有WD40重复序列的β -螺旋桨，介导与肌动蛋白细丝的结合。它们的c端一半也可以包含一些额外的结构域和区域，这些结构域和区域允许与一系列不同的蛋白质相互作用，包括肌动蛋白、肌动蛋白结合蛋白和微管，并可以介导寡聚化。因此，不同的冠状物通常具有不同的功能。从感染人类的原生动物恶性疟原虫中鉴定出冠状蛋白，并将其定性为肌动蛋白丝状结合蛋白，而在感染啮齿动物的伯氏疟原虫中，冠状蛋白被证明对孢子虫的有效运动很重要，孢子虫是由蚊子传播的高运动形式的寄生虫。我们之前的研究表明，P. berghei冠蛋白功能至少部分定位于WD40结构域。在之前的资助期内，我们在柏氏冠蛋白的c端发现了一个新的位点，该位点在冠蛋白功能中也很重要，可能是由于肌动蛋白丝结合。删除冠蛋白的C端或n端一半进一步显示了以前未观察到的孢子体运动表型，这表明两部分蛋白的合作功能。我们现在的目标是通过(i)在野生型和突变型中表达蛋白质的两个部分以及全长冠蛋白，并研究它们在体外结合肌动蛋白丝的能力，来理解这些表型的机制基础。（ii）研究与荧光蛋白融合的突变蛋白在表达特异性标记肌动蛋白细丝的荧光纳米体的寄生虫中的定位；（iii）利用光学镊子和不同寄生虫品系的表面敏感成像研究力的产生和粘附动力学。

Coronins are a family of actin-binding proteins that harbor one or two WD40 repeat-containing beta-propellers to mediate binding to actin filaments. They can also contain a number of additional domains and regions in their C-terminal half that allow interaction with a range of different proteins including actin, actin-binding proteins and microtubules and can mediate oligomerization. Hence different coronins often mediate distinct functions. Coronin from the protozoan, human-infecting parasite Plasmodium falciparum was identified and characterized as an actin filament-binding protein and coronin in the rodent-infecting parasite P. berghei was shown to be important for effective motility of sporozoites, the highly motile forms of the parasite transmitted by mosquitoes. We previously showed that P. berghei coronin function is at least partly localized in the WD40 domain. During the previous funding period we identified a new site in the C-terminus of P. berghei coronin that is additionally important in coronin function probably due to actin filament-binding. Deleting either the C- or N-terminal half of coronin furthermore showed a previously not observed motility phenotype in sporozoites suggesting a cooperative function of both halves of the protein. We now aim at understanding the mechanistic basis for these phenotypes by (i) expressing both parts of the protein as well as full-length coronin in wild type and mutant forms and investigating their capacity to bind actin filaments in vitro, (ii) investigating the localization of mutant proteins fused with a fluorescent protein in parasites expressing a fluorescent nanobody that labels specifically actin filaments and (iii) investigating force generation and adhesion dynamics using optical tweezers and surface sensitive imaging of the different parasite lines.