Fig. 4: Echinocytosis is likely induced by membrane fusion and lipid redistribution from parasite to host cell during rhoptry release.

a Time differences between the start of Ca²⁺ flux to host cell transformation into echinocyte I across different treatments. Untreated (n = 14), MβCD (n = 13, p = 0.005), R1 peptide (n = 19, p = 0.618), cytochalasin D (n = 14, p = 0.04). b Time differences for the transformation from echinocyte I to echinocyte III across different treatments. Untreated (n = 11), MβCD (n = 10, p = 0.032), R1 peptide (n = 20, p = 0.762), cytochalasin D (n = 23, p = 0.01). *p < 0.05; **p < 0.01; two-sided randomization test. a, b The central lines and empty circles represent mean values. c, d Extended XY and YZ views, showing merozoite–erythrocyte interaction in the presence of inhibitors. R1 peptide treatment results in a membrane tether between the parasite and erythrocyte following echinocytosis, observed in three independent experiments. Cytochalasin D treatment results in observable membrane thickening forming intracellular protrusions from the attempted invasion site, observed in two independent experiments. Data were displayed in blend mode and processed using IMARIS. Scale bars: 2 μm. e Representative images from two independent experiments showing RON3 localization on late-stage schizont and free merozoite, captured with structured illumination microscopy. Scale bars: 0.5 μm. f, g Snapshots showing the release of RON3, a rhoptry bulb protein, by untreated and R1 treated parasites. Images captured with LLSM, denoised using content-aware image restoration (CARE)⁶², and displayed as slice view with FIJI. In the control event, RON3 was released onto the invagination pit and incorporated into the parasitophorous vacuole membrane as the parasite invades. With R1 inhibition, RON3 was released onto the erythrocyte membrane instead. This observation was followed up with an immunofluorescence assay in fixed cells (see Supplementary Figure 5). Scale bars: 2 μm.