Dissecting mechanisms of cell division by the malaria parasite

疟疾寄生虫是顶复门的成员，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具有截然不同的适应能力。我们最近的工作表明，保守的内溶酶体蛋白分拣蛋白的恶性疟原虫同源物充当高尔基体和顶端复合体之间运输货物的护航者。出乎意料的是，PfSortilin的缺失不仅阻止了顶端复合体的产生，而且还导致子代寄生虫的产生完全受阻。我们现在提议研究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相关的，并且棒状细胞器充当支架来引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条件表达的尖端系统来深入研究这些问题，并将它们与高分辨率成像配对。我们建议在下一个 5 年期限内追求的三个不同但相关的具体目标是：AIM1。体内恶性疟原虫裂殖子出芽过程的成像实验：1A。生成表达细胞分裂 1B 标记效应子的寄生虫​​系。免疫荧光和免疫电子显微镜检测在出芽过程中定位标记标记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实验：2A。在寄生虫菌株中生成表达细胞分裂效应子标记标记的寄生虫系，其中 PfSortilin 表达可以有条件地调节。2B。在不存在PfSortilin的情况下对细胞分裂过程进行免疫荧光测定、免疫电子显微镜和活细胞成像。目的3.鉴定恶性疟原虫细胞分裂机制的新成分实验3A。生成表达用生物素连接酶 BirA.3B 标记的细胞分裂效应器的寄生虫系。通过免疫沉淀和质谱鉴定相互作用的伙伴。通过该研究计划获得的知识将揭示细胞分裂的不寻常形式，并将允许与其他真核细胞分裂模式进行比较研究，以揭示保守的和寄生虫特异性的过程，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 vivoExperiments: 1A. Generate parasite lines expressing tagged effectors of cell division 1B. Immunofluorescence and immunoelectron microscopy assays to localize the tagged markers during the budding process1C. Live-cell imaging of the merozoite budding processAIM 2. Investigate the role of PfSortilin in the merozoite budding processExperiments: 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 machineryExperiments 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.