(a–d) Schizonts were stained with anti-PfPLP1 followed by anti-mouse Alexa-Fluor 488 (green) and membrane-specific linker dye (red) at different time points (~44–48 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. (e,f) Localization of PfPLP1 in E64-treated schizonts. (e) PfPLP1 colocalizes with linker in E64-treated schizonts suggesting that PfPLP1 localizes to PVM in schizonts. (f) E64-treated schizonts did not show staining with anti-band3 antibody where PfPLP1 showed ring-like staining suggestive of its presence on the membrane. Nuclei were counterstained with DAPI. Scale bar, 2 μm. Garg S, Agarwal S, Kumar S, Yazdani SS, Chitnis CE, Singh S. Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites. Nat Commun. 2013; 4:1736. PMID: 23591903.