Fig. 3: The punctate Ca²⁺ flux marks the formation of a fusion pore during host membrane recoil and acts as a temporal reference for analysis of downstream inhibitors.

Images represent parasites labeled with MitoTracker Red, cyan, interacting with erythrocytes labeled with Di-4-ANEPPDHQ, magenta, and loaded with Fluo-4AM Ca²⁺ reporter, yellow, displayed using IMARIS in extended XY view and XZ view. a Sequence of invasion steps including the punctate Ca²⁺ flux localized at the parasite apical tip (n = 25). Parasite-induced deformations lead to a final erythrocyte membrane recoil that coincides with the Ca²⁺ flux prior to internalization. Time-lapse of parasite-host interaction b in the presence of R1 peptide (n = 22), which blocks tight junction formation, and c cytochalasin D (n = 17), which disrupts actin polymerization on the parasite. Parasites were still able to trigger Ca²⁺ flux and induce echinocytosis despite failing to internalize. a–c Scale bars: 2 μm. d Parasite-associated host membrane (PAM) time plot demonstrating a conserved timing of the Ca²⁺ flux coinciding with host membrane recoil, marking the transition from deformation (i) to internalization (ii) (n = 12). For the R1 peptide (n = 9) and cytochalasin D-treated (n = 6) events, plots were shifted in time accordingly with the Ca²⁺ flux being the reference point. Line with shaded error band represent mean ±95% CI. PAM values were normalized to a fully internalized parasite from the untreated data sets. e Representative XY slices and 3D images of Gaussian curvature mapping on erythrocyte with red-blue look-up table from the untreated and R1 treated parasite–erythrocyte interaction reported in a, b. The untreated parasite induced a highly negative curvature around the aperture of the invagination pit, which was absent from the deformation induced by the R1 peptide-treated parasite. Scale bars: 1 μm.