Colocalization studies of RALP1 with rhoptry and microneme marker proteins. (A and B) RALP1-C-specific antibodies (green) colocalize with the rhoptry protein RAP1 (red) (A) and predominantly colocalize with CLAG9 a rhoptry-specific marker, (red) (B) in fixed schizonts (s) and free merozoites (m) using RAP1- and CLAG9-specific antibodies, respectively. (C and D) RALP1-C-specific antibodies (green) visualize a different compartment within the parasite than the microneme marker proteins EBA-175 (red) (C) and EBA-181 (red) (D), as is evident in the merge of the two fluorescence photomicrographs. Nuclei were stained blue (DAPI).Haase S, Cabrera A, Langer C, Treeck M, Struck N, Herrmann S, Jansen PW, Bruchhaus I, Bachmann A, Dias S, Cowman AF, Stunnenberg HG, Spielmann T, Gilberger TW. Characterization of a conserved rhoptry-associated leucine zipper-like protein in the malaria parasite Plasmodium falciparum. Infect Immun. 2008 76:879-87.

Apical expression of RhopH1/Clag members -2, -3.1, and -9 in P. falciparum segmented schizonts. Schizont-infected erythrocytes were duallabeled with a-CL3.1B and a-RhopH2 mAb 61.3 in the 3D7 parasite line (Panel A); a-CL3.1A and a-CL9M (Panel B) and a-CL3.1A and a-CL2N (Clag2) (Panel C) in Dd2 parasite line. Overlaid images are shown in the right-hand panels. All segmented schizont-stage parasites are positive for the antisera against Clag2, -3.1, and -9. Nuclei are counterstained with either DAPI or Hoechst-33342 (Hx). Scale bar represents 5 mm. Overlaid images of the double-staining for anti-Clag3.1 and anti-PfRhopH2 or anti-Clag3.1 and anti-Clag9 sera reacting with segmented schizonts showed indistinguishable patterns of localization for these proteins. Clag3.1 and Clag2 are localized within the rhoptries of segmented schizonts. Kaneko O, Yim Lim BY, Iriko H, Ling IT, Otsuki H, Grainger M, Tsuboi T, Adams JH, Mattei D, Holder AA, Torii M. Apical expression of three RhopH1/Clag proteins as components of the Plasmodium falciparum RhopH complex. Mol Biochem Parasitol. 2005 143(1):20-8.

A. Labeling of CLAG9 in 3D7 and E5KO. Note there is labeling of E5KO (clag9 knockout) rhoptries with mAb7H8/50 that reveals RAP3, but not with anti-CLAG9 specific sera due to gene disruption. Scale bars = 5 mm. B. Trafficking of PfEMP1 and KAHRP by E5KO; labeling was identical to 3D7. Scale bars = 5 mm. CLAG9 was not detected in the knockout clone E5KO. These results show that the clag9 gene product is not directly implicated in cytoadhesion to CD36.Nacer A, Roux E, Pomel S, Scheidig-Benatar C, Sakamoto H, Lafont F, Scherf A, Mattei D. clag9 is not essential for PfEMP1 surface expression in non-cytoadherent Plasmodium falciparum parasites with a chromosome 9 deletion. PLoS One. 2011;6(12):e29039.

VAR2CSA and CLAG9 are present in the IRBC membranes of C4S-adherent parasites. The C4S-adherent FCR3 parasites, at the late ring to early trophozoite stages, were analyzed by immunoelectron microscopy by using rat anti-VAR2CSA and rabbit anti-CLAG9 primary antibodies followed by biotin-conjugated goat anti-rat IgG and 18-nm gold particle-conjugated goat anti-rabbit IgG secondary antibodies. The bound biotin-labeled antibodies were probed with 12-nm gold particles conjugated streptavidin. Arrows and arrowheads indicate, respectively, 12-and 18-nm gold particles. (Scale bars: 200 nm.) a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA.Goel S, Valiyaveettil M, Achur RN, Goyal A, Mattei D, Salanti A, Trenholme KR, Gardiner DL, Gowda DC. Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins. Proc Natl Acad Sci U S A. 2010 107:16643-8.

VAR2CSA and CLAG9 localize predominantly to IRBC membranes of C4S-adherent parasites. Immunofluorescence and light micrographs (LM) of membrane-permeabilized (A and C) and intact (B and D) IRBCs of C4S-adherent FCR3 parasites, harvested at the late ring/early trophozoite stage, were stained by using rabbit anti-CLAG9 (A and B), rabbit anti-VAR2CSA (C and D), and mouse anti-KAHRP (control) antibodies at dilutions given in SI Materials and Methods. (E and F) Immunofluorescence and LM of permeabilized (E) and intact (F) C4S-adherent 3D7 IRBCs stained as above with anti-VAR2CSA antibodies. (G and H), Immunofluorescence and LM of permeabilized clag9−/− parasites stained with anti-VAR2CSA antibodies (G) and anti-CLAG9 antibodies (H). Anti-CLAG9 antibodies showed a strong staining of the IRBC membrane (A). A substantial level of CLAG9 is located at or in close proximity to the erythrocyte membrane. Nonpermeabilized IRBCs also showed onsiderable levels of staining of the erythrocyte surface with anti-CLAG9 antibodies (B). Anti-VAR2CSA antibodies also localized VAR2CSA on the erythrocyte membrane of both permeabilized and nonpermeabilized IRBCs of the C4S-adherent parasites (C and D)Goel S, Valiyaveettil M, Achur RN, Goyal A, Mattei D, Salanti A, Trenholme KR, Gardiner DL, Gowda DC. Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins. Proc Natl Acad Sci U S A. 2010 107:16643-8.

Localization of Clag9 to the rhoptry organelles. A–D. An identical field from a 1% formaldehyde fixed thin smear of 3D7 schizonts: (A) rabbit anti-Clag9, TRITC; and (B) mAb 4E10, Oregon Green. Each antibody bound to P. falciparum schizonts gave a distinctive punctate pattern typical of apical proteins. The two antibodies were co-localized within schizonts (C) visualized as yellow; (D), DAPI, a nuclear stain. Control normal mouse and rabbit sera produced no fluorescence. E–J. Electron micrographs illustrating immunogold labelling for Clag9 (E–G) and RhopH2- (H–J). Labelling in (E), (F), (H) and (I) shows antigen localization to the basal region of developing rhoptries within schizonts (C10), and (G) and (J) depict similar basal labelling in released merozoites (3D7). No staining was detected in the neck of the rhoptries. Negative controls gave no distinctive pattern. A scale bar is indicated in (E–J).Ling IT, Florens L, Dluzewski AR, Kaneko O, Grainger M, Yim Lim BY, Tsuboi T, Hopkins JM, Johnson JR, Torii M, Bannister LH, Yates JR 3rd, Holder AA, Mattei D. The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion. Mol Microbiol. 2004 52:107-18. PMID:

Immunofluorescence co-localization assay of Plasmodium falciparum clones 3D7 and the isogenic CLAG 9 mutant clone 11E. Antibody raised in mice to CLAG 9 peptide C were used at 1/1,000 dilution in conjunction with antibodies raised in rabbits to the rhoptry protein, RhopH3, at a dilution of 1/600. This was followed by anti-mouse antibodies conjugated to cy2 and anti-rabbit antibodies conjugated to Texas red at a dilution of 1/150. The parasite nuclei were visualized with Hoechst dye (DAPI). The slides were examined with a microscope with filters appropriate for the three fluorescent dyes,and the images merged. A Specific green staining of a schizont of clone 3D7 with the CLAG 9 antiserum, and the specific red staining of the schizont with RhopH3 antiserum. The merging of these two giving a yellow staining indicates that CLAG 9 colocalizes with RhopH3. B Rupturing schizonts of 3D7 showing multiple nuclei of individual merozoites and the co-localization of CLAG 9 and RhopH3 to the rhoptries. C CLAG 9 isogenic mutant clone 11E showing that there was no staining of the rhoptries in this parasite with the CLAG 9 antisera, although there still was specific staining with the RhopH3 antisera.Gardiner DL, Spielmann T, Dixon MW, Hawthorne PL, Ortega MR, Anderson KL, Skinner-Adams TS, Kemp DJ, Trenholme KR. CLAG 9 is located in the rhoptries of Plasmodium falciparum. Parasitol Res. 2004 93:64-7. Copyright Springer 2010