Analyses of the distribution of GAMA in asexual blood stage parasites by immunofluorescence. . Formaldehyde-fixed P. falciparum 3D7 schizonts were probed with mouse anti-CTD (C-terminal domains; top panel) or anti-NTD (N-terminal domains; lower panel) antisera, and an anti-mouse-FITC conjugate (green). Parasite nuclei were stained with DAPI (blue). Staining with both antisera resulted in a strong punctuate pattern, with a single point of fluorescence located away from the nucleus, and consistent with a location in the apical organelles. Scale bars represent 2 mm. (B) Formaldehyde-fixed free P. falciparum merozoites were probed with rabbit anti-CTD antiserum, and either mouse polyclonal anti-AMA1 (PF11_0344)-antibodies (i, iii and iv) or mouse mAb 61.3 (ii). Mouse antibodies were detected with an anti-mouse Alexa Fluor 594 conjugate (red), and rabbit antibodies with an anti-rabbit Alexa Fluor 488 conjugate (green). Parasite nuclei were stained with DAPI (blue). Scale bar in (i) 5 μm, in remainder 2 μm. In (i) and (ii) there is a clear apical localization of AMA1, RhopH2 (PFI1445w) and GAMA, with AMA1 and GAMA appearing to be co-localized; (iii) shows a circumferential localisation of both GAMA and AMA1 on the surface of free merozoites; (iv) shows a ‘cap’-like distribution of GAMA on the surface of free merozoites (arrowed), in contrast to the circumferential distribution of AMA1. Hinds L, Green JL, Knuepfer E, Grainger M, Holder AA. A novel putative GPI-anchored micronemal antigen of Plasmodium falciparum that binds to erythrocytes. Eukaryot Cell. 2009 8:1869-1879. Copyright 2010.

Newly invaded ring-stage parasites were probed with the mouse monoclonal anti-MSP119 (PFI1475w) antibody 1E1, and either rabbit anti-CTD (C-terminal domains; top panel) or anti-NTD (N-terminal domains; lower panel) antisera. Mouse antibodies were detected with an anti-mouse Alexa Fluor 594 conjugate (red) and rabbit antibodies with an anti-rabbit Alexa Fluor 488 conjugate (green). Parasite nuclei were stained with DAPI (blue). Scale bars are 2 μm. There is a clear reactivity of anti-CTD antibodies, but not anti-NTD antibodies, with ring-stage parasites. Anti-CTD and anti-MSP119 signals appear to co-localize (i), suggesting that ‘stub’ part of GAMA that is produced upon shedding of the p42-p37 complex is carried into the host cell and remains associated with the ring-stage parasite. Scale bar 2 μm. Hinds L, Green JL, Knuepfer E, Grainger M, Holder AA. A novel putative GPI-anchored micronemal antigen of Plasmodium falciparum that binds to erythrocytes. Eukaryot Cell. 2009 8:1869-1879. Copyright 2010.

Localization of GAMA in asexual blood stage parasites. (A) GAMA localization using immuno-fluorescence assay. Acetone-fixed P. falciparum 3D7 mature schizonts (top panel) and free merozoites (bottom panel) were probed with rabbit anti-FL (green) and mouse anti-PfAMA1 (microneme marker)(red). Parasite nuclei were stained with DAPI (blue). Scale bars represent 2 μm. (B) GAMA localization using immunoelectron microscopy. The two sections of merozoites in schizont-infected erythrocytes were probed with purified rabbit anti-Tr3 antibody and subsequently by secondary antibody conjugated with gold particles. The arrows indicate the micronemal localization of signals from gold particles. Bars represent 200 nm. Arrows mark micronemes.Arumugam TU, Takeo S, Yamasaki T, Thonkukiatkul A, Miura K, Otsuki H, Zhou H, Long CA, Sattabongkot J, Thompson J, Wilson DW, Beeson JG, Healer J, Crabb BS, Cowman AF, Torii M, Tsuboi T. Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen. Infect Immun. 2011 79(11):4523-32