Characterizing the GTPase activity of a novel contractile ring protein in Plasmodium falciparum - Plasmodium falciparum is a parasite with great public health importance as the deadliest causative agent of malaria. The parasite has a complex life cycle, that requires a human and mosquito host. The clinical symptoms associated with malaria are caused by the asexual blood stage which undergoes a divergent form of cell division called schizogony. During schizogony, the parasite develops in a red blood cell (RBC) for 48 hours to generate enough copies of every organelle to create 20-36 daughter cells in a single shared cytoplasm. The final step of schizogony is segmentation, the synchronous cytokinesis event that creates dozens of daughter cells with high fidelity. Segmentation is mainly driven by the basal complex, the putative contractile ring. The basal complex is a massive multiprotein complex essential for proper daughter cell formation. However, the exact mechanism of the basal complex is still unknown, and few components of the complex have been characterized. CINCH is a highly essential member of the basal complex that has been shown to possess GTPase activity. CINCH is predicted to act as a dynamin-superfamily protein (DSP) which are large mechanochemical GTPases that are often involved in membrane contraction and scission. This is the first member of the complex identified that could clarify the energetic force generated by the putative contractile ring. I will further assess the GTPase activity of CINCH in vitro with a sensitive fluorescence-based assay to evaluate optimal conditions for activity and test our ability to inhibit CINCH with broad-spectrum inhibitors. All DSPs form homodimers to stabilize GTP hydrolysis. DSPs involved in membrane contraction will additionally form large polymers and wrap around membranes. As the DSPs hydrolyze GTP, conformational changes in the protein constrict the underlying membrane. Isolating CINCH for in vitro experiments allows us to determine if it will form ring-like structures in the absence of other basal complex components. I will assess the oligomeric state of CINCH with size-exclusion chromatography, blue native gels, and negative stain electron microscopy. The structures CINCH forms in isolation will have implications on the formation of the basal complex in the parasite and the mechanism of contraction. While CINCH is highly essential for parasite survival, the GTPase activity has not yet been shown to be the essential function of the protein. I will determine the essentiality and role of the GTPase activity of Cl NCH using an in trans complementation system. I will generate and test 10 mutants I predict will interfere with the enzymatic activity of CINCH with conditional knockdown systems commonly used in our lab. All mutants will be assessed for protein expression levels, localization, and parasite viability. Any defects in the mutants will be further evaluated by super-resolution microscopy to determine when the defect occurs during segmentation and to what severity. Together, these experiments will expand our understanding of the contractile ring of Plasmodium by evaluating the role of an essential member of the basal complex.
