PROJECT SUMMARY Malaria is an important cause of illness and death worldwide, with most of these deaths resulting from Plasmodium falciparum infection. Clinical malaria results from the asexual replication of parasites in human red blood cells. During the blood stage, P. falciparum replicates via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. The inner membrane complex (IMC), a unique structure within the parasite composed of parasite proteins and a double lipid bilayer that is closely associated with the plasma membrane, and associated basal complex are hypothesized to orchestrate daughter parasite assembly and division. The focus of the current application is on the molecular mechanisms of schizogony and segmentation. Directed experiments will determine the biogenesis, composition, and function of the IMC and basal complex. We have discovered two novel parasites that are essential for schizogony and segmentation. The first, PfMOP, localizes initially near the centrosome and later to the apical end of the parasite and is critical for IMC biogenesis. The second, PfCINCH, localizes to the basal complex and is critical for parasite cytokinesis. These two proteins allow interrogation of the IMC and basal complex from the apical and basal ends of the parasite, respectively. The recently discovered P. falciparum Merozoite Organizing Protein is essential for both asexual and gametocyte development. In PfMOP-deficient parasites, the IMC does not form properly, resulting in a failure of segmentation, and the incompletely segmented merozoites remain in an agglomerate with a common cytoplasm. The molecular function of PfMOP and its link to the progression of schizogony and IMC biogenesis remain unknown. The proposed studies address these critical knowledge gaps. The first aim is divided into three independent subaims. In Aim 1.1, the link between PfMOP and IMC biogenesis in late schizogony will be investigated using a cell biologic approach with live video microscopy. In Aim 1.2, the function of PfMOP in early schizonts will be investigated, testing the hypothesis that PfMOP recruits a critical protein complex for chromosome condensation. In Aim 1.3, the PfMOP protein interactions in late schizonts will be determined, validated, and functionally evaluated by reverse-genetics. PfCINCH (Coordinator of nascent cell detachment) is a novel and essential component of the basal complex. In PfCINCH-deficient parasites, the final stages of segmentation are disrupted. In the second aim of this proposal, we focus on the cellular function of PfCINCH and its protein-protein interactions. The long-term objectives and public health implications of these studies are to identify critical biologic process pathways in the malaria parasite that could be targeted by future therapeutics.