PROJECT SUMMARY Malaria is an important cause of illness and death worldwide, with most of these deaths resulting from Plasmodium falciparum infection. Successful completion of the P. falciparum life cycle and infection of a new human host requires transmission. During the asexual blood stage in human red blood cells, a small population of parasites differentiates into transmission forms known as gametocytes. These gametocytes can complete the sexual stage of the parasite life cycle following ingestion by a mosquito. Gametocyte maturation in human red blood cells occurs over 10-12 days and is associated with major changes in cellular morphology and rigidity. A newly discovered protein PfBLEB (for Baso-Lateral Expansion Boundary) is essential for mature gametocyte formation. In asexual parasites, PfBLEB is part of the basal complex, a ring-like multi-protein complex at the leading edge of the inner membrane complex. While PfBLEB is dispensable for both asexual replication and gametocyte commitment, it is essential for gametocyte development. PfBLEB-knockdown or knockout gametocytes arrest during maturation and are non- transmissible. Furthermore, the PfBLEB-deficient gametocytes have gross morphologic changes with defects in major cytoskeletal features of the maturing transmission-stage parasite, including the inner membrane complex and subpellicular microtubules. In gametocytes with normal PfBLEB expression, PfBLEB defines a new subcellular compartment within the parasite, demarcating the regions of the parasite plasma membrane that are devoid of the underlying inner membrane complex. The PfBLEB-compartment is essential for gametocyte development, but the function and protein constituents of this newly discovered subcellular compartment remain unknown. The goal of the current application is to define and genetically evaluate the protein components of the PfBLEB compartment and to understand the functional defects in PfBLEB-deficient gametocytes. The first aim will utilize proximity labeling and reverse genetics to explore the PfBLEB-containing compartment. The second aim will utilize multiple imaging and microfabrication techniques to gain a functional understanding of what processes are abnormal in PfBLEB-deficient gametocytes. Together, the proposed studies will add a new layer to our molecular understanding of gametocyte development in P. falciparum.