K13 is associated with sites of dextran uptake from the RBC. (A) Live-cell imaging of GFP-K13 transfectant-infected RBCs. K13 (GFP, green) and nucleus (DAPI-stained, blue) channels merged with DIC image. Fluorescence channels are displayed as maximum intensity projections while DIC is a single Z-slice. (B) Immunofluorescence assay (IFA) of GFP-K13-imaged using 3D structured illumination microscopy (3D-SIM). Parasites labelled with anti-GFP (K13-WT/, green), anti-GAPDH (cytoplasm, red), and DAPI (nucleus, blue). Maximum intensity projections are displayed; additional images in Figure S5. (C-H) Percoll-purified late-stage parasites cultured in resealed fluorescent dextran-loaded RBCs. (C, D) Fluorescence timelapse imaging of control early-stage parasite-infected RBCs. Timepoints illustrate the decrease of fluorescent dextran signal in the host compartment and its accumulation inside the parasite as the parasite matures. (E) Fluorescence time-lapse imaging of an untreated late-stage parasite-infected RBC. Yellow arrowheads indicate GFP-K13 associated with the peripheral end of a tubular fluorescent dextran structure. The last panel shows a cartoon representation of the 2’ panel. (F, G) Fluorescence timelapse imaging of JAS-treated late-stage parasite-infected RBCs. (F) Timepoints illustrate the repositioning of GFP-K13 together with the repositioning of the peripheral fluorescent dextran structure. (G) Late-stage GFP-K13-infected RBC showing three tubular fluorescent dextran structures, each associated at the peripheral end with a GFP-K13-labelled structure.

K13 is located at the cytostomal neck and its mislocalisation disrupts cytostome formation. (A) Immunoelectron micrograph of a late-stage GFP-K13 transfectant labelled with anti-GFP. Arrows indicate anti-GFP labelling on the neck of a cytostomal invagination (CI). Scale bar: 200 nm. (B, top panel) Molecular system for complete mislocalisation of GFP-K13. Schematic showing N-terminal-tagging of K13 with GFP and FKBP and the mislocaliser plasmid expressing NLS, FRB, and mCherry (Birnbaum et al., 2017). (B, top right) Cartoon representation of GFP-K13 mislocalisation. (B, bottom) Representative live-cell images showing mislocalisation of GFP-K13. Scale bar =2 µm. (C) Representative transmission electron microscopy (TEM) images showing invaginations in DMSO- and rapamycin-treated late-stage parasites. The top panel shows invaginations with shapes typical of a CI with electron-dense rings/collars around the tight necks. Arrows indicate electron dense cytostomal collars. Bottom panel shows invaginations with irregular shapes and wide necks lacking the electron-dense rings/collars. N: nucleus.

Quantitative analysis of atypical invaginations formed upon mislocalisation of K13. Representative single plane Serial Block Face Scanning Electron Microscopy (SBF-SEM) image (section thickness = 50 nm) showing many DMSO-treated K13-100KS-infected RBCs. Additional slices along the Z-axis of the highlighted infected RBC in A (red box). Red arrowhead points to opening of the CI to the RBC cytosol. (C) Comparison of the number of normal cytostomes formed in DMSO- and rapamycin-treated K13-100KS-infected RBCs. Normal cytostome is defined as an invagination with a tight neck in mid to late trophozoites that have not segmented and present as single infections. x-axis: number of cytostomes; y-axis: number of infected RBCs. 100 infected RBCs were counted per treatment.

Tomographic analysis of cytostomal invaginations upon mislocalisation of K13. (A) 3D reconstructions of serial block-face scanning electron microscopy (SBF-SEM) images of DMSO- and rapamycin-treated late-stage K13-100KS-infected RBCs. Yellow arrowheads point at cytostomal invaginations with a tight neck opening to the RBC cytosol. Red arrowheads point at cytostomal invaginations with a wide neck opening to the RBC cytosol. (B) 3D reconstructions of transmission electron tomography images (section thickness = 250 nm; tilt range = ± 60°) of DMSO- and rapamycin-treated late-stage K13-100KS-infected RBCs. Yellow: parasitophorous vacuolar membrane (PVM); Blue: parasite plasma membrane (PPM); Red: PVM surrounding cytostomal invagination; Dark purple: PPM lining cytostomal invagination in DMSO-treated parasites; Light purple: PPM lining cytostomal invagination in rapamycin-treated parasites; Green: electron-dense collar around cytostomal neck; Yellow arrow: invagination with a tight neck surrounded by an electron-dense collar; red arrow: invagination with a wide neck lacking an electron-dense collar. Scale bars = 200 nm.

K13 mislocalisation decreases free haem in ring- and late-stage, and haemozoin in late-stage, parasites. (A) Mislocalisation and haem fractionation workflow. Created with BioRender.com. (B) Graphs showing effect of K13 mislocalisation on haem and haemozoin levels. Top graph shows results from parasites harvested at early stage (next cycle), showing a significant decrease in the free haem fraction (p = 0.0166) in K13-mislocalised parasites compared to untreated controls. Bottom graph shows results from parasites harvested at late stage, showing a significant decrease in the haem (p = 0.0005) and haemozoin (p = 0.0093) fractions in K13-mislocalised parasites (n = 5).

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