Cryo-ET structure of the Plasmodium basal complex - PROJECT SUMMARY Malaria remains an important global cause of morbidity and mortality. The majority of the burden of this disease is due to infection with the Plasmodium falciparum parasites and affects children and pregnant women. Asexual replication of the parasite, during the human blood stage, causes the signs and symptoms of the disease when repeated rounds of asexual replication generate a dramatic increase in the number of parasites. P. falciparum asexual replication occurs via schizogony, wherein the nuclei and organelles for 20-36 daughter cells are produced within a common cytoplasm without cytokinesis. Then, in a complex and highly organized process known as segmentation, the parasite partitions the nuclei and organelles into each daughter parasite with remarkable fidelity. There is a major knowledge gap surrounding this divergent method of cell division. We aim to use cryo-electron tomography to learn new molecular insights into this process. Segmentation is largely driven by two parasite-specific structures, the inner membrane complex (IMC) and the basal complex. The IMC consists of two lipid bilayers and multiple associated proteins that form adjacent to an underlying cytoskeletal network of intermediate-like filaments. Together, the IMC and associated cytoskeleton are critical for the parasite morphology and rigidity. The basal complex is a multi-protein molecular machine that forms at the posterior/basal end of the IMC. It is highly essential for parasite cell division and is hypothesized to be the contractile ring that mediates abscission during cytokinesis. Importantly, the basal complex is unlike the contractile rings of model organisms and lacks dependence on actin-myosin interactions. Thus, the mechanism of action for this presumed contractile ring is fully unknown. Several basic questions are ready for answers – does the basal complex form a ring or a coil, are concentric structures present or is it a single “ring”, is the complex symmetrical or are there distinctly different regions with it. In the current proposal, we will utilize cryo-electron tomography (cryo-ET) to gain a molecular understanding of basal complex function. We have generated a parasite strain where an essential component of the basal complex, PfCINCH, has been tagged with 3xFLAG and mNeonGreen. This allows visualization of the basal complex in intact parasites and purification of the basal complex from lysed parasites. In the first aim, we will determine the native structure of the basal complex using in-situ cryo-electron tomography. In the second aim, we will use affinity-purification to obtain basal complexes from lysed parasites. Cryo-ET will be applied to these isolated basal complexes to obtain a high-resolution structure via sub-tomographic averaging. Together, the proposed experiments will provide the first structural information about this essential multi-protein machine. The resulting structural insights will begin to reveal the molecular mechanism of the basal complex – and potentially lead to possible interventions to block its essential activity.
