Disruption of RAMA leads to selective mislocalisation of some but not all rhoptry proteins.
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(A) IFA showing that the staining profile of RAP2, RhopH1/Clag3.1, Rh5, RhopH2, ARO and RhopH3 was unaltered between DMSO- and RAP-treated RAMAloxP clone 9C10 schizonts. Scale bar, 2 μm. (B) IFA showing that, whilst RhopH3 and RhopH1/Clag3.1 staining was unaffected by RAMA disruption, RON2, RON3 and RON4 staining was modified in RAP-treated RAMAloxP clone 9C10 schizonts. In all cases, the staining was much weaker in the RAMAΔE2 parasites and lacked the characteristically punctate, apically-disposed pattern typical of rhoptry staining. Scale bar, 2 μm. (C) The extent of RON protein mis-trafficking depends on the maturity of the RAP-treated RAMAloxP schizonts. Whilst some signal was still detectable in less mature schizonts (those with fewer nuclei), the RON2, RON3 and RON4-specific signal was absent or much reduced in the mature segmented schizonts (examples indicated with arrows). Note that these images are of lower magnification that those shown in the rest of the figure in order to display multiple schizonts in each field. Scale bar, 10 μm. (D) Schizonts of DMSO- and RAP-treated RAMAloxP clone 9C10 were probed with a polyclonal antibody against AMA1 as well as anti-MSP1 antibodies. AMA1 localisation (upper panel) and discharge onto the merozoite surface in mature schizonts (lower panel) was unaffected by RAMA disruption. Scale bar, 2 μm. Parasite nuclei were visualised throughout by staining with DAPI.