Atlas ofPlasmodium falciparumintraerythrocytic development using expansion microscopy

Abstract

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 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 ofP. falciparumduring its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Detailing organelle division and segregation inPlasmodium falciparum

“…Based on apicoplast visualizations in P. berghei and our observations of the formation of MOCs during oocyst stages, mitochondrial and apicoplast dynamics in these sub-compartments in both oocyst and liver stages resemble the dynamics of these organelles in blood-stage schizogony 11,37,38 . Although the use of new imaging techniques, such as expansion microscopy and 3D volume EM, have revealed new insights in mitochondrial dynamics, many questions about the timing and organization of mitochondrial division remained unanswered 10,31 . In an earlier literature review, we proposed three possible mitochondrial division models: synchronous fission, outside-in fission, or branching point fission 8 .…”

Global Release of Translational Repression AcrossPlasmodium’sHost-to-Vector Transmission Event

“…PyDOZI::GFP expressing female gametocytes were subjected to ultrastructure expansion microscopy (U-ExM) as previously described (68, 99). Cells were stained with chicken anti-GFP IgY fraction (Aves Labs, #GFP-1010) at a 1:1000 dilution, and counterstained with custom rabbit polyclonal antisera against PyCITH, PyPABP1, or PyALBA4 (20, 70, 71) at a 1:5000 dilution.…”

“…Secondary antibodies against chicken IgY (Thermo, #A11039, AF488) or rabbit IgG (Thermo #A11011, AF568) were applied at a 1:500 dilution. Sytox Deep Red (Thermo, #S11380, 1:1000) and NHS-Ester (Thermo, #30000, AF405, 1:250) were used to stain nucleic acids and proteins as before (67, 98). Images were captured on a Zeiss LSM980 series microscope using a Plan-Apochromat 63X/1.40 Oil DIC M27 objective with Airyscan 2 detector using ZEN Blue software.…”

“…The eluted proteins were removed from the beads and used for affinity blot analyses, probed with anti-GFPmut2 primary antibody Cells were prepared for IFA as previously described (22,40). Briefly, the transgenic gametocytes were pelleted at 1,400 Ultrastructure Expansion Microscopy (U-ExM): PyDOZI::GFP expressing female gametocytes were subjected to ultrastructure expansion microscopy (U-ExM) as previously described (67,98). Cells were stained with chicken anti-GFP IgY fraction (Aves Labs, #GFP-1010) at a 1:1000 dilution, and counterstained with custom rabbit polyclonal antisera against PyCITH, PyPABP1, or PyALBA4 (20,69,70) at a 1:5000 dilution.…”

“…Figure 5: Ultrastructure expansion microscopy cross-validates protein-protein interactions in female gametocytes.PyDOZI::GFP-expressing female gametocytes were visualized via ultrastructure expansion microscopy (U-ExM) as previously described(67,98). Parasites were stained with anti-GFP to mark DOZI (yellow) and were counterstained with custom rabbit polyclonal antibodies raised against PyCITH, PyPABP1, and PyALBA4 (magenta).…”