Components of the P. falciparum merozoite glideosome localize to the periphery of the parasite. (A) Indirect-immunofluorescence of individual glideosome components in late blood-stage parasites: Thin smears of P. falciparum infected erythrocytes were probed with anti-glideosome sera. The arrow indicates a gap in peripheral staining at the end of the merozoite, suggestive of an inner membrane complex (IMC) localization. (B) Indirect-immunofluorescence of late blood-stage parasites co-stained with antisera to other glideosome components and PfEBA-175: Glideosome antisera were used in co-immunofluorescence assays to assess co-localization. Anti-PfEBA-175 antisera recognize an invasion ligand that is localized to the micronemes, and is used as a marker for the apical end of the merozoite.Jones ML, Kitson EL, Rayner JC. Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex. Mol Biochem Parasitol. 2006 147:74-84. Copyright Elsevier 2009.

Localization of PfGAP proteins to the inner membrane complex. A, PfGAP50 and PfGAP45 co-localize with MTIP (a protein known to localize to the inner membrane complex) and partially with PfMyoA (known to associate with the innermembrane complex and merozoite apical tip (6, 38)). The white arrow in the MTIP inset points to an area of reduced fluorescence at the apical tip in a single z-stack, consistent with the absence of IMC at this position. B, immunoelectron microscopy confirms IMC localization for PfGAP45 (large 25-nm gold particle). The white arrows indicate trilaminar appearance of the IMC and merozoite plasma membrane. i, inner IMC membrane; o, outer IMC membrane; p, plasma membrane R, rhoptry. *, single anti-PfGAP50-conjugated gold particle found in close proximity to PfGAP45 but giving poor reactivity (typically 1 particle/merozoite). Scale bar in electron microscopy, 0.2 mm. C, PfGAP45-GFP shows fluorescence that concentrates to the merozoite periphery late in asexual development, consistent with the Scale bar in IFA, 1 mm.Baum J, Richard D, Healer J, Rug M, Krnajski Z, Gilberger TW, Green JL, Holder AA, Cowman AF. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. J Biol Chem. 2006 281(8):5197-208.

Upper row: PfGAP45 co-localizes with the IMC marker PfMTIP. Immunofluorescence assays were performed using anti-GFP (red) and anti-PfMTIP (green) antibodies on parasites over-expressing PfGAP45-GFP. PfGAP45 shows co-localization with PfMTIP around the periphery of merozoites in mature schizonts. Middle row: PfGAP45 phosphorylation mutants co-localize with endogenous PfGAP45. Immunofluorescence assays were performed using anti-GFP (green) and anti-PfGAP45 (red) antibodies to localize episomally expressing GAP45-GFP and endogenous PfGAP45.Lower row: Live Imaging of PfGAP45 GFP in parasites over-expressing PfGAP45-GFP was performed after labeling the nuclei with DAPI. Thomas DC, Ahmed A, Gilberger TW, Sharma P. Regulation of Plasmodium falciparum Glideosome Associated Protein 45 (PfGAP45) Phosphorylation. PLoS One. 2012;7(4):e35855.

CDPK1 localizes to the plasma membrane of merozoites. A, an anti-CDPK1 antibody detects CDPK1 at the parasite periphery in schizonts (panel i) and free merozoites (panel ii). The second panel shows a merged image of nuclear staining with 4’,6’-diamino-2-phenylindole (blue) and the bright field image, and the third panel shows the bright field image. B, panel i, CDPK1 colocalizes with MTIP around the periphery of merozoites but is also found around the residual body, marked with an arrowhead, from which MTIP is absent. In young (panel ii) and segmented (panel iii) schizonts, CDPK1 colocalizes with the plasma membrane marker MSP1. Both proteins are also detected around the residual body, marked with an arrowhead. CDPK1 does not colocalize with the food vacuole marker PfCRT in either immature (panel iv) or mature (panel v) schizonts. The third panels in all of the images show the merged images of the antibody staining, beside which is a merged image of the bright field image and the nuclei of the parasites stained with 4’,6’-diamino-2-phenylindole (blue), followed by the bright field image alone. C, both CDPK1 (red) and MSP1 (green) can be detected on residual bodies that are released upon schizont rupture. The merged images of the antibody staining are shown in the third panels, followed by the bright field image. In all cases the white scale bar in the first panel of each set of images represents 1 mm.Green JL, Rees-Channer RR, Howell SA, Martin SR, Knuepfer E, Taylor HM, Grainger M, Holder AA. The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. J Biol Chem. 2008 7;283(45):30980-9. PMID:

PfELC expression matches that of known glideosome components in P. falciparum schizonts. Immunofluorescent detection of HA-tagged ELC and either MTIP or GAP45 in mature schizonts and merozoites. Merged colour images are also shown, with ELC-HA (green) and MTIP or GAP45 (red). Nuclei are stained with DAPI (blue). Scale bar: 2 μm. Immunofluorescence analysis of fixed blood-stage P. falciparum with HA-specific antibodies showed co-localisation of the protein with GAP45 and MTIP in late, segmented schizonts.Green JL, Wall RJ, Vahokoski J, Yusuf NA, Ridzuan MAM, Stanway RR, Stock J, Knuepfer E, Brady D, Martin SR, Howell SA, Pires IP, Moon RW, Molloy JE, Kursula I, Tewari R, Holder AA. Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility. J Biol Chem. 2017 Sep 11.

The location of MyoA during P. falciparum merozoite invasion. (A) MyoA-GFP is located at the periphery of developing intracellular (top row of each pair of images) and free extracellular merozoites (bottom row), and is colocalized with antibodies specific for IMC proteins MTIP, GAP45 and GAP50. In the merged colour image the MyoA-GFP signal is green and antibodies specific for the IMC proteins are red; nuclei are stained blue with DAPI. The DIC image is also shown. (B) Individual merozoites are captured at different stages of invasion from initial attachment, through early and late invasion to the intracellular ring stage. MyoA-GFP remains peripheral whereas RON4, initially in the apical rhoptry neck, relocates during invasion. Merged colour images with MyoA-GFP (green), RON4 (red), and nuclei (blue) and DIC images are also shown, together with a schematic of each cell-pair. Scale bar: 2 μm.Green JL, Wall RJ, Vahokoski J, Yusuf NA, Ridzuan MAM, Stanway RR, Stock J, Knuepfer E, Brady D, Martin SR, Howell SA, Pires IP, Moon RW, Molloy JE, Kursula I, Tewari R, Holder AA. Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility. J Biol Chem. 2017 Sep 11.