Dissecting mechanisms of cell division by the malaria parasite

疟原虫是顶复门的一员，顶复门是专性细胞内寄生虫的一个大门。这些独特的真核生物的进化细胞生物学研究的最新发现表明，典型的真核生物成分的适应性非常不同。我们最近的工作表明，恶性疟原虫同源保守的内溶酶体蛋白分拣蛋白作为一个护送运输货物之间的高尔基体和顶端复合体。出乎意料的是，PfSortilin的不存在不仅阻止了顶端复合物的产生，而且导致子寄生虫的产生完全阻断。我们现在建议调查这一令人兴奋的发现，这将是NSERC提案的重点。我们的假设是，顶端复合体的生物发生和细胞分裂的过程是联系在一起的，棒状细胞器作为一个支架，以指导裂殖子出芽的启动。这反过来又解释了如何从一个母细胞同时形成多达32个高度极化的子细胞。因此，我们这项新研究计划的长期目标是了解疟疾寄生虫的细胞分裂过程。我们将深入研究这些问题，使用最新的遗传学工具来修改寄生虫的基因组和用于寄生虫蛋白质的条件表达的尖端系统，我们将这些与高分辨率成像配对。我们建议在下一个五年任期内追求的三个不同但相关的具体目标是：* 目标1。恶性疟原虫裂殖子体内出芽过程的成像 * 实验：产生表达标记的细胞分裂效应子的寄生虫系 *1B。免疫荧光和免疫电子显微镜分析，以定位标记的标志物在出芽过程中 *1C。裂殖子出芽过程的活细胞成像 **AIM 2.研究PfSortilin在裂殖子出芽过程中的作用在其中PfSortilin表达可被条件性调节的寄生虫菌株中产生表达细胞分裂效应物的标记标志物的寄生虫系。2B.在不存在PfSortilin的情况下，细胞分裂过程的免疫荧光测定、免疫电子显微镜和活细胞成像。** AIM 3.恶性疟原虫细胞分裂机制的新组分的鉴定 *产生表达用生物素连接酶BirA标记的细胞分裂效应物的寄生虫系。3B.通过免疫沉淀和质谱鉴定相互作用的伴侣。**通过这项研究计划获得的知识将揭示一种不寻常的细胞分裂形式，并将允许与真核细胞分裂的其他模式进行比较研究，以揭示保守和寄生虫特异性过程，从而有助于进化细胞生物学和细胞微生物学领域的进步。*****

The malaria parasite is a member of the Apicomplexa, a large phylum of obligate intracellular parasites. Recent findings from the study of the evolutionary cell biology of these distinctive eukaryotes demonstrated exquisitely divergent adaptations of canonical eukaryotic components. Our recent work has demonstrated that the Plasmodium falciparum homologue of the conserved endolysosomal protein Sortilin acted as an escorter to transport cargo between the Golgi apparatus and the apical complex. Unexpectedly, the absence of PfSortilin not only prevented the generation of the apical complex but also resulted in a complete block in the generation of daughter parasites. We now propose to investigate this exciting discovery and this will be the focus of this NSERC proposal. Our hypothesis is that the biogenesis of the apical complex and the process of cell division are linked and that the rhoptry organelle acts as a scaffold to guide the initiation of merozoite budding. This would in turn explain how up to 32 highly polarized daughter cells can form simultaneously from a single mother cell. Our long-term goal for this new research program is thus to understand the process of cell division in the malaria parasite. We will delve into these questions using the latest genetics tools to modify the parasite's genome and cutting-edge systems for the conditional expression of parasite proteins and we will pair these with high resolution imaging.******The three distinct but related Specific Aims that we are proposing to pursue over the next 5-year term are:***AIM1. Imaging the P. falciparum merozoite budding process in vivo***Experiments: 1A. Generate parasite lines expressing tagged effectors of cell division ***1B. Immunofluorescence and immunoelectron microscopy assays to localize the tagged markers during the budding process***1C. Live-cell imaging of the merozoite budding process******AIM 2. Investigate the role of PfSortilin in the merozoite budding process***Experiments: 2A. Generate parasite lines expressing tagged markers of cell division effectors in a parasite strain where PfSortilin expression can be conditionally regulated.***2B. Immunofluorescence assays, immunoelectron microscopy and live-cell imaging of the cell division process in absence of PfSortilin.******AIM 3. Identification of novel components of the P. falciparum cell division machinery***Experiments 3A. Generate parasite lines expressing cell division effectors tagged with the biotin ligase BirA.***3B. Identification of interacting partners by immunoprecipitation followed by mass spectrometry.******The knowledge gained through this research program will shed light on an unusual form of cell division and will allow comparative studies with other modes of eukaryotic cell division to uncover conserved and parasite specific processes thus contributing to the advancement of the fields of evolutionary cell biology and cellular microbiology. *****