Change in localization of PfPLP1 to host cell membrane during egress of merozoites. (a–d) Schizonts were stained with anti-PfPLP1 followed by anti-mouse Alexa-Fluor 488 (green) and membrane-specific linker dye (red; PKH26 Red Fluorescent Cell Linker Dye) at different time points (B44–48 hpi) during egress. Three-dimensional reconstruction of confocal z-stack images of schizonts was done using Imaris software. PfPLP1 is represented as green dots and membrane dye is shown as two concentric rings corresponding to RBCM and PVM in red. The insets show a bright field and single-slice images with deconvolution. (a) Early schizonts do not show PfPLP1 on the two limiting membranes but shows punctate staining within parasite. (b) Mature schizonts show ring-like staining of PfPLP1 that colocalizes with the membrane dye, suggesting its presence on the membrane. (c) Upon rupture, linker stains the merozoite cell membrane and PfPLP1 shows punctate staining in merozoites with linker staining its cell membrane. (d) After egress, free merozoites show apical localization of PfPLP1. PfPLP1 localizes to RBCM and PVM during egress.Garg S, Agarwal S, Kumar S, Shams Yazdani S, Chitnis CE, Singh S. Calcium-dependent permeabilization of erythrocytes by a perforin-like proteinduring egress of malaria parasites. Nat Commun. 2013 Apr 16;4:1736.

Subcellular localization of PfPLP1 in schizonts and merozoites of P. falciparum. (a) Co-immunostaining of PfPLP1 (green) with merozoite surface protein MSP1 (red). (b) Co-immunostaining of PfPLP1 (green) with micronemal protein EBA-175 (red). PfPLP1 colocalizes with EBA-175 (colocalization coefficient being more than 0.75). (c) Co-immunostaining of PfPLP1 (green) with rhoptry protein CLAG3.1 (red). PfPLP1 is not localized in rhoptry. Nuclei were counterstained with DAPI and Scale bar, 2 mm. (d) Immunoelectron microscopy of PfPLP1. Ultrathin sections of merozoites and late-stage schizonts were labelled with anti-PfPLP1 mouse sera followed by gold-labelled (15 nm) anti-mouse IgG secondary antibodies. Gold particles were detected in elongated, membrane-enclosed micronemes towards the apical end in schizonts and merozoites. Scale bar, 500 nm. PfPLP1 is localized to the micronemes of merozoites.Garg S, Agarwal S, Kumar S, Shams Yazdani S, Chitnis CE, Singh S. Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites. Nat Commun. 2013 Apr 16;4:1736.

Stage-specific expression of PfPLP1 and PfPLP2 in the blood stage of P. falciparum. Expression of PfPLP1 and PfPLP2 starts in trophozoite stage, with maximal expression in schizonts. In mature schizonts, PfPLP1 and PfPLP2 show punctate staining at the apical end of merozoites. PfPLP1 also shows a ring-like staining, suggesting its localization to RBC membrane. Scale bar, 2 mm.Garg S, Agarwal S, Kumar S, Shams Yazdani S, Chitnis CE, Singh S. Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites. Nat Commun. 2013 Apr 16;4:1736.