Atlas of Plasmodium falciparum intraerythrocytic 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 parasite Plasmodium falciparum during 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 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the microtubule organizing center (MTOC) 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 MTOC until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an MTOC association during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Methods
This dataset comprises 647 images of Plasmodium falciparum parasites acquired using Airyscan microscopy on either a Zeiss LSM 800, or Zeiss LSM 900 microscope using a 63x Plan Apochromat 1.4NA objective lens. All images in this dataset have been Airyscan processed in 3D using the "standard" filter setting. Raw, non-airyscanned, images are available upon request, but have not been included in this dataset as they cannot be used in many of the most commonly used microscopy image analysis programs. Channels for fluorescent markers have been changed to reflect the colouring system used in the corresponding manuscript. While image colour combinations vary for some images, they broadly follow the trend: Cyan = SYTOX Deep Red (DNA stain), Magenta = antibody stain of interest (typically Alexa Fluor 488), Inverse greyscale = NHS ester Alexa Fluor 405 (protein stain), White = BODIPYTexasRed Ceramide (lipid stain), Yellow = second antibody stain of interest (typically Alexa Fluor 555), or MitoTracker CMTMRos (mitochondrial stain).