Subcellular distribution of proteins predicted to be involved in invasion. All proteins were localized in schizonts (s) and free merozoites (m) using GFP-fusion proteins and grouped according to their predominant GFP localization. Apical. Nuclei were stained with DAPI (blue).Hu G, Cabrera A, Kono M, Mok S, Chaal BK, Haase S, Engelberg K, Cheemadan S, Spielmann T, Preiser PR, Gilberger TW, Bozdech Z. Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum. Nat Biotechnol. 2010 28(1):91-8.

Stage-specific expression of CyRPA in late asexual blood-stage parasites. Indirect immunofluorescence stainings of asexual bloodstage parasites confirmed stage-specific expression in schizont stages and free merozoites. Methanol/acetone fixed P. falciparum 3D7 parasites were probed with anti-CyRPA mAb c06 (red) and anti-GAPDH mAb (green). Exposure times were identical for all pictures of the same channel. Nuclei were stained with DAPI (blue). Original magnification x1008.Dreyer AM, Matile H, Papastogiannidis P, Kamber J, Favuzza P, Voss TS, Wittlin S, Pluschke G. Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen. J Immunol. 2012 188(12):6225-37.

Localization of CyRPA to the merozoite apex by immunofluorescence staining. P. falciparum 3D7 merozoites (A) or schizont stages (B) were coimmunostained with anti-CyRPA mAb c06 (red) and anti-GAPDH Abs (marker for cytosol), AMA-1 (marker for micronemes), RAP-1 (marker for rhoptry bulbs), MSP-1 (marker for merozoite surface), or MSP-5 (green). Nuclei were stained with DAPI (blue). Original magnification x1008. CyRPA is localized at the merozoite apex.Dreyer AM, Matile H, Papastogiannidis P, Kamber J, Favuzza P, Voss TS, Wittlin S, Pluschke G. Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen. J Immunol. 2012 188(12):6225-37.

Left: Immunofluorescence microscopy detected CyRPA to colocalize with micronemal proteins (EBA-175, PfRipr). (Scale bar, 2 μm.)Right: PfRH5/PfRipr/CyRPA complex is GPI-anchored to the merozoite surface. (A) 3D reconstruction of IFA z-stacks during merozoite invasion, coimmunostained with MSP-1 detected PfRH5, PfRipr, and CyRPA at the apical end of the merozoite surface. In the 3D images, the γ settings were altered for visual representation only,Reddy KS, Amlabu E, Pandey AK, Mitra P, Chauhan VS, Gaur D. Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion. Proc Natl Acad Sci U S A. 2015 Jan 12.

Previous attempts to disrupt PfRipr and CyRPA in P. falciparum have not been successful, suggesting their function is essential. To understand their function, we constructed P. falciparum lines in which pfripr and cyrpa could be conditionally deleted using dimerizable Cre recombinase (DiCre) by generating RiprloxCre and CyRPAloxCre. The absence of PfRipr and CyRPA expression was analyzed by immunofluorescence, and a significant proportion of the populations showed no detectable expression (E and F). These results show that rapamycin-treated RiprloxCre and CyRPAloxCre have substantially decreased expression of PfRipr and CyRPA, respectively. The decrease in expression had no effect on expression and localization of other proteins involved in merozoite invasion.Volz JC, Yap A, Sisquella X, Thompson JK, Lim NT, Whitehead LW, Chen L, Lampe M, Tham WH, Wilson D, Nebl T, Marapana D, Triglia T, Wong W, Rogers KL, Cowman AF. Essential Role of the PfRh5/PfRipr/CyRPA Complex during Plasmodium falciparum Invasion of Erythrocytes. Cell Host Microbe. 2016 Jun 30 [Epub ahead of print]

Subcellular Localization of PfRipr, CyRPA, and PfRh5 in Schizonts and Invading Merozoites(A) Top panel: Overlay of CyRPA (green) and PfRipr (red) with DAPI and DIC fields. Middle panel, overlay of CyRPA (green) and PfRh5 (red) with DAPI andDIC fields. Lower panel, overlay of PfRipr (green) and PfRh5 (red) with DAPI and DIC images.(B) Merozoites were mixed with erythrocytes and CyRPA, PfRipr, and PfRh5 visualized. Top panel, colocalization of CyRPA (green) and PfRipr (red) with DAPI and DIC images. Two merozoites invading a single erythrocyte. Middle panel, overlay of CyRPA (green) and PfRh5 (red) with DAPI and DIC fields with a single merozoite. Lower panel, overlay of PfRipr (green) and PfRh5 (red) withDAPI and DIC images. White bar indicates 5 mm.Volz JC, Yap A, Sisquella X, Thompson JK, Lim NT, Whitehead LW, Chen L, Lampe M, Tham WH, Wilson D, Nebl T, Marapana D, Triglia T, Wong W, Rogers KL, Cowman AF. Essential Role of the PfRh5/PfRipr/CyRPA Complex during Plasmodium falciparum Invasion of Erythrocytes. Cell Host Microbe. 2016 Jun 30 [Epub ahead of print] PMID: 27374406

Subcellular Localization of PfRipr, CyRPA, and PfRh5 in Schizonts and Invading Merozoites(A) Top panel: Overlay of CyRPA (green) and PfRipr (red) with DAPI and DIC fields. Middle panel, overlay of CyRPA (green) and PfRh5 (red) with DAPI andDIC fields. Lower panel, overlay of PfRipr (green) and PfRh5 (red) with DAPI and DIC images.(B) Merozoites were mixed with erythrocytes and CyRPA, PfRipr, and PfRh5 visualized. Top panel, colocalization of CyRPA (green) and PfRipr (red) with DAPI and DIC images. Two merozoites invading a single erythrocyte. Middle panel, overlay of CyRPA (green) and PfRh5 (red) with DAPI and DIC fields with a single merozoite. Lower panel, overlay of PfRipr (green) and PfRh5 (red) withDAPI and DIC images. White bar indicates 5 mm.Volz JC, Yap A, Sisquella X, Thompson JK, Lim NT, Whitehead LW, Chen L, Lampe M, Tham WH, Wilson D, Nebl T, Marapana D, Triglia T, Wong W, Rogers KL, Cowman AF. Essential Role of the PfRh5/PfRipr/CyRPA Complex during Plasmodium falciparum Invasion of Erythrocytes. Cell Host Microbe. 2016 Jun 30 [Epub ahead of print] PMID: 27374406