(C) Immunofluorescence analysis of red blood cells infected with parental 3D7 parasites co-labelled with a-FIKK4.2 and a-PFB0090c (J-dots) antibodies. Merge and phase overlay images indicate that FIKK4.2 and J-dots do not co-localise in infected red blood cells. In all panels, parasite nuclei are counterstained with DAPI. (D) Immunofluorescence analysis of red blood cells infected with either parental 3D7 or FIKK4.2 knockout (1E2) parasites. Infected red blood cells were incubated with antibodies raised against the knob-associated histidine-rich protein (KAHRP), mature-parasite-infected erythrocyte surface antigen (MESA) or P. falciparum antigen 332 (Pf332). Parasite nuclei were counterstained with DAPI. KAHRP, MESA and Pf332 as three examples of other very well described P. falciparum-encoded exported proteins.Kats LM, Fernandez KM, Glenister FK, Herrmann S, Buckingham DW, Siddiqui G, Sharma L, Bamert R, Lucet I, Guillotte M, Mercereau-Puijalon O, Cooke BM. An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells. Int J Parasitol. 2014 Feb 14. [Epub ahead of print]

Immunofluorescence analysis of red blood cells infected with parental (3D7) or FIKK4.2 knockout (1E2) parasites. Experiments were performed on infected red blood cells that had been synchronised at either the young ring stage or the more mature trophozoite/schizont stage. Air-dried and fixed blood smears on glass slides were dual-labelled with both a-FIKK4.2 and a-SBP1 (Maurer’s cleft) antibodies. Merge and phase overlay images demonstrate clearly that FIKK4.2 and SBP1 do not co-localise in infected red blood cells. Specific punctate fluorescence was observed in the cytoplasm of RBCs infected with ring-stage 3D7 parasites. A similar pattern was observed in RBCs infected with trophozoite/schizont-stage parasites, and although the pattern of fluorescence was more diffuse, individual puncta were still clearly discernible. The puncta appeared too small and too numerous to be Maurer’s clefts, and did not co-localise with either the classical Maurer’s cleft marker SBP1.Kats LM, Fernandez KM, Glenister FK, Herrmann S, Buckingham DW, Siddiqui G, Sharma L, Bamert R, Lucet I, Guillotte M, Mercereau-Puijalon O, Cooke BM. An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells. Int J Parasitol. 2014 Feb 14. [Epub ahead of print]

Localization of LyMP in iRBCs. A) Localization of LyMP in trophozoites, with either anti-LyMP (green) or anti-HA (red) showing that LyMP is exported into the RBC cytosol. 4’,6’-Diamidino-2-phenylidole (DAPI) is a nuclear stain (blue). B) Colocalization of LyMP (green) with a variety of iRBC markers (red) that are indicated in the panel. DAPI is a nuclear stain (blue). C) High-resolution structured illumination fluorescence microscopy (OMX) with anti-LyMP (green) and either anti-KAHRP, anti-MESA, or anti-spectrin (red) colocalizations. Corresponding overlay images are shown for all. D) Immunoelectron micrograph of gold-labeled Ha-tagged parasites. Red asterisks indicate knobs and the arrow indicates gold labeling of LyMP at the RBC membrane skeleton. P, parasite; MC, Maurer’s clefts. LyMP showed a pattern of distribution similar to that for MESA, KAHRP. LyMP did not consistently colocalize with MCs or to other punctate structures previously identified in the iRBC cytosol such as those seen for FIKK4.2 (R45) or for J-dots (KAHsp40).Proellocks NI, Herrmann S, Buckingham DW, Hanssen E, Hodges EK, Elsworth B, Morahan BJ, Coppel RL, Cooke BM. A lysine-rich membrane-associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum-infected red blood cells. FASEB J. 2014 Apr 4. [Epub ahead of print]