Exit of Plasmodium gametes from red blood cells

导致疟疾的寄生虫以疟原虫配子体的形式传播给蚊子。在蚊子体内，这些性细胞离开受感染的红细胞，并迅速分化为配子，配子融合形成受精卵。特别引人注目的是雄配子体分化成8个配子，这些配子构成非常简单的精子细胞，只包含一个轴丝和一个被质膜包围的核。雄配子的形成和它们的出口是紧密耦合的，但还不是很清楚。在这个项目中，我们打算通过基因修饰、生化实验和3D电子断层扫描来阐明这一耦合过程的分子机制。在细胞内基因组倍增后，配子需要拉出含有一组染色体的核的一部分，这一过程似乎很容易出错，因为只有大约50%的雄配子含有核。连接细胞质轴丝和核纺锤体的两部分微管组织中心(MTOC)可能促进了形成配子的核吸收。通过类比弓形虫的子细胞形成和卵囊中疟原虫子孢子的形成，我们假设横纹根纤维在两个MTOCs之间提供了联系。为了排出寄生虫周围的膜，需要裂解寄生虫的液泡和红细胞质膜。有几种蛋白质已被鉴定为配子形成和膜破坏所必需的。然而，我们仍然对这些蛋白质的功能只有有限的机械性了解，因为很少有蛋白质-蛋白质相互作用被建立。我们以前已经证明，在感染伯氏疟原虫的啮齿动物中，一种名为MTRAP的膜跨越黏附蛋白对于配子从红细胞中流出是重要的。为了进一步了解MTRAP的功能，我们将采取两种主要方法：对于MTRAP的功能研究，我们将从基因上删除MTRAP的关键结构域进行功能研究，并利用MTRAP与生物素化酶的融合来寻找相互作用伙伴。我们在第一个资助期为这两种方法产生了转基因寄生虫系，现在的目标是彻底分析这些系。最后，我们将研究条纹纤维集合体的功能，这是细根纤维的可能构建块，与基因缺失，并使用3D电子断层扫描来阐明这些配子出口的耦合事件。

Malaria-causing parasites are transmitted to mosquitoes in the form of Plasmodium gametocytes. Within the mosquito these sexual cells exit the infected red blood cell and differentiate rapidly into gametes, which fuse to form a zygote. Particularly striking is the differentiation of the male gametocyte into eight gametes, which constitute very simple sperm cells containing only an axoneme and a nucleus surrounded by a plasma membrane. Formation of male gametes and their egress is tightly coupled but not well understood. In this project we intend to elucidate molecular mechanisms of this coupled process by genetic modification, biochemical experiments and 3D electron tomography. After intracellular genome multiplication gametes need to pull out part of a nucleus containing a set of chromosomes, a process that appears error prone as only about 50% of male gametes contain a nucleus. Nuclear uptake of forming gametes is likely facilitated by a bipartite microtubule organizing center (MTOC) that links cytoplasmic axoneme to nuclear spindle formation. Through analogy to daughter cell formation in Toxoplasma gondii and Plasmodium sporozoite formation in the oocysts, we hypothesize that the striated rootlet fiber provides a link between the two MTOCs. For exit the membranes surrounding the parasite, the parasitophorous vacuole and the red cell plasma membrane, need to be lysed. Several proteins have been identified as being essential for gamete formation and membrane disruption. Yet, we still have only limited mechanistic understanding of the function of these proteins as few protein-protein interactions have been established. We have previously shown that a membrane spanning adhesion protein called MTRAP is important for gamete egress from red blood cells in the rodent infecting parasite Plasmodium berghei. To further understand MTRAP function we will take two main approaches: For functional studies of MTRAP we will genetically delete key structural domains of MTRAP for functional studies, and investigate interaction partners using MTRAP-fusions with biotinylating enzymes. We generated transgenic parasite lines for both approaches during the first funding period and now aim to thoroughly analyse these lines. Lastly, we will investigate the function of striated fiber assemblins, the likely building blocks of the rootlet fiber, with gene deletions and use 3D electron tomography to shed light on these coupled events of gamete egress.