Dissecting mechanisms of cell division by the malaria parasite

疟疾寄生虫是顶复合体的一员，顶复合体是专性细胞内寄生虫的一大门。这些独特的真核生物的进化细胞生物学研究的最新发现证明了典型真核生物成分的精致的不同适应。我们最近的工作表明，保守的内溶酶体蛋白Sortilin的恶性疟原虫同源物在高尔基体和根尖复合体之间运输货物时起着护送作用。出乎意料的是，PfSortilin的缺失不仅阻止了顶端复合体的产生，而且导致子代寄生虫的产生完全受阻。我们现在提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，顶复合体的生物发生与细胞分裂过程是联系在一起的，茎生细胞器作为一个支架来指导裂殖子出芽的开始。这反过来解释了一个母细胞如何同时形成多达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寄生虫蛋白条件表达的尖端系统，我们将这些与高分辨率成像配对。

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.