Atlas ofPlasmodium falciparumintraerythrocytic development using expansion microscopy

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample ∼4.5x. Here, we apply U-ExM to the human malaria parasitePlasmodium falciparumduring the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 differentP. falciparumstructures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis ofP. falciparumduring its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.