Dissecting mechanisms of cell division by the malaria parasite

疟疾寄生虫是顶体复合体的一员，顶体复合体是专性细胞内寄生虫的一个大门。最近对这些独特的真核生物进化细胞生物学的研究结果表明，规范的真核细胞成分具有微妙的不同适应能力。我们最近的工作表明，恶性疟原虫保守的内溶体蛋白sortilin的同源物作为护卫者在高尔基体和心尖复合体之间运输货物。出乎意料的是，PfSortilin的缺失不仅阻止了顶端复合体的产生，而且还完全阻止了子寄生虫的产生。我们现在提议对这一令人兴奋的发现进行调查，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过程是联系在一起的，棒状细胞器作为支架来指导裂殖子萌发的启动。这将反过来解释为什么多达32个高度极化的子细胞可以同时从单个母细胞形成。因此，我们对这一新研究项目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深入研究这些问题，以修改寄生虫的基因组和用于寄生虫蛋白质条件表达的尖端系统，并将这些与高分辨率成像配对。*我们提议在下一个5年任期内追求的三个截然不同但相关的具体目标是：*AIM1。体内恶性疟原虫裂殖子萌发过程成像实验：1。产生表达标记的细胞分裂效应器的寄生系*1B。免疫荧光和免疫电子显微镜分析在萌发过程中定位标记的标记*1C。目的研究PfSortilin在裂殖子萌发过程中的作用*实验：2A。在PfSortilin表达可被有条件调控的寄生虫株中，产生表达细胞分裂效应器的标记标记的寄生虫系。*2B。在没有PfSortilin的情况下，细胞分裂过程的免疫荧光分析、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。产生用生物素连接酶BILA.*3B标记的表达细胞分裂效应器的寄生虫系。通过免疫沉淀和质谱学鉴定相互作用的伙伴。*通过这项研究计划获得的知识将有助于揭示一种不寻常的细胞分裂形式，并将允许与其他真核细胞分裂模式的比较研究，以揭示保守的和寄生虫特有的过程，从而促进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. *****