The malaria parasite Plasmodium falciparum is a single-celled microorganism with a complex life cycle that invades both host hepatocytes and red blood cells (RBCs). It spends a great proportion of its life cycle within the red blood cell (RBC), a cell comprised mostly of hemoglobin and lacking in nuclei and organelles. The invasion and subsequent modification of the RBC is a crucial step for the survival of this pathogenic and lethal parasite. To support the dynamic multiplication of malaria parasites, the parasite takes up large amounts of RBC cytosol into a specialized acidic organelle called the digestive vacuole (DV). This organelle breaks down the RBC cytosol's primary constituent hemoglobin to provide itself with nutrients (i.e. amino acids) that are crucial for its development and growth. The DV has been thought to have similarities to both tonoplasts, an acidic intracellular vacuole of plant cells, and lysosomes of mammalian cells. However, the absence of the typical lysosomal acid phosphatase and glycosidase indicates that the DV of Plasmodium is a specialized organelle that most likely evolved to efficiently degrade hemoglobin. Importantly, the DV does not persist throughout the complete blood stage life cycle, as is seen for other organelles such as the mitochondrion and the apicoplast. The DV is discarded once the merozoite parasites are released from the RBC and is reformed once a new RBC is invaded and the parasite develops within. Consequently, the parasite's DV carries out a variety of specialized and critical functions to ensure the survival of the parasite, including hemoglobin degradation, detoxification of oxygen radicals, ion homeostasis, and nutrient and/or solute transport across its membrane. The underlying biology of this complex organelle is incomplete and poorly understood, possibly due to past experiments that mostly used fixed samples of parasites. Therefore, the long-term objective of this research program is the development of an in-depth understanding of the molecular and cellular processes of the parasite's digestive vacuole using modern imaging techniques available in our lab. With our previous NSERC proposal, we set out to better characterize the PfMDR1 transporter and were able to quantify the kinetics of this transporter. This work was published in several journals. We also discovered a new phenotype, the formation of Hz-containing compartments that form when the parasite infected RBCs are treated with the antimalarial chloroquine. We accomplished an important objective and published this in work in Scientific Reports. The present proposal will focus on a better understanding of the Hz-containing compartments that arise when parasites are treated with certain antimalarials. We will continue to use live cell imaging techniques to get a better understanding of parasite dynamics in situ. This will allow us to better understand these processes in real time within the live parasite.