PfAIP localizes to the rhoptry neck. (A) Schematic representation of pSLI-PfAIP-FKBP-GFP vector integration in the genomic PfAIP locus via homolog recombination resulting in endogenous tagging of PfAIP with FKBP-GFP (PfAIP-FKBP-GFP). Gray: PfAIP coding region, turquoise: 2xFKBP, green: GFP, blue: T2A skip peptide [64,65] orange: neomycin resistance gene. Arrows (P1–P4) are indicating position of oligonucleotides used for B. (B) PCR analysis of the rendered genomic locus using primers as shown in the schema using gDNA of 3D7 wild type and PfAIP-FKBP-GFP parasites. (C) Western blot analysis using anti-GFP antibody detects an approx. 110 kDa protein in PfAIP-FKBP-GFP (expected size MW 106kD) and no protein in the parental cell line. Anti-Aldolase antibody was used as loading control. (D) PfAIP-FKBP-GFP (green) can be localized in unfixed cells at the apical pole of schizont and merozoite stage parasites. DAPI (blue) was used to stain the nuclei. Scale bar, 2 μm; DIC, differential interference contrast; 5x zoom is indicated by a white square. (E-G) Colocalization of PfAIP-FKBP-GFP with PfARO-mCherry using confocal microscopy reveals a more restricted distribution of PfAIP (green) on the rhoptries with only minimal overlap with PfARO-mCherry (red). DAPI (blue) was used to stain the nuclei. In (E) a single confocal image section acquired with a Leica SP8 microscope is displayed, whereas in (F) a full three-dimensional reconstruction of a cell is shown. For (G) a Zeiss Airyscan microscope was used. (H) SIM image from parasites expressing PfARO-GFP [12] (green) and stained with anti-GAPM2 antibodies that visualize the inner membrane complex (red). Geiger M, Brown C, Wichers JS, Strauss J, Lill A, Thuenauer R, Liffner B, Wilcke L, Lemcke S, Heincke D, Pazicky S, Bachmann A, Löw C, Wilson DW, Filarsky M, Burda PC, Zhang K, Junop M, Gilberger TW. Structural Insights Into PfARO and Characterization of its Interaction With PfAIP. J Mol Biol. 2020. PMID: 31877322.