1. Analysis of the co-localization of PfFC with PfHsp60 using FITC-conjugated (green) secondary antibodies for the former and TRITC-conjugated (red) secondary antibodies for the latter; 1, bright field (dark spots represent the hemozoin pigment); 2, red fluorescence in the parasite due to PfHsp60; 3, green fluorescence in the parasite due to PfFC; 4, merge of 2 and 3 along with Hoescht stain (blue) showing lack of co-localization; 5, projection of two parasites showing lack of co-localization.2. Three different fields showing discrete gold particles in typical apicoplast structures with multiple membranes. 2 part of the whole parasite; 3, elongating apicoplasts in a later-stage trophozoite; 4, a circular apicoplast in an early-stage tropozoite. The signals are localized close to the membrane. Scale bar, 100 μm. AP, apicoplast; M, mitochondrion; R, red cell; MC, Maurer’s cleft; P, parasite; Pv, parasite food vacuole; Pc, parasite cytosol.Reproduced with permission, from Varadharajan S, Sagar BK, Rangarajan PN, Padmanaban G. Localization of ferrochelatase in Plasmodium falciparum. Biochem J. 2004 384:429-36. © the Biochemical Society

Localization of native, functional PfFC in the parasite by immunofluorescence 304 microscopy. Mouse anti-rPfFC IgG, rabbit anti-r(D)PfPBGD (apicoplast marker) IgG and rabbit anti305 rPfHSP60 (mitochondrial marker) IgG were used at 1:200 dilution. The slides were examined using a 306 Fluorescence Microscope (Leica Microsystems) and the images were acquired at 100x 307 magnification using an oil immersion objective and Leica FW 4000 image acquisition 308 software. IE, infected erythrocyte; P, parasite and H, hemozoin. Scale bar = 5 mm.Nagaraj VA, Prasad D, Rangarajan PN, Padmanaban G. Mitochondrial localization of functional ferrochelatase from Plasmodium falciparum. Mol Biochem Parasitol. 2009 168:109-12. Copyright Elsevier 2010

Immunofluorescence images showing multiple localizations of PfTPxGL. PfTPxGL-red signal (anti-mouse Alexa fluor 568). The imaging was carried out with scale bars varying from 5-20 mm. The infected RBCs imaged are 6-8 mm across. Parasites showing localization of PfTPxGl using antibodies purified against rPfTPxGL. In several parasites, PfTPxGL was localized exclusively to only one organelle per parasite. In some parasites, PfTPxGL co-localized with exclusively the mitochondrial markers PfUROD or PfFC. Chaudhari R, Narayan A, Patankar S. A novel trafficking pathway in Plasmodium falciparum for the organellar localization of glutathione peroxidase-like thioredoxin peroxidase. FEBS J. 2012 279(20):3872-88 PMID:

Immunofluorescence images showing PfTPxGl and PfFC (mitochondrial marker protein) trafficking in AlF4−, nocodazole and vineblsatine treated 3D7 parasites(A) PfTPxGl and PfFC co-localization in AlF4−-treated parasites, (B) PfTPxGl and PfFC co-localization in vinblastine-treated parasites, (C) PfTPxGl and PfFC co-localization in nocodazole-treated parasites. In this experiment, PfTPxGl was found to be co-localized with the mitochondrial marker protein PfFC in 40% of the treated parasites suggesting that trafficking of PfTPxGl to the mitochondrion may be partially disrupted by the treatments. Scale Bar: 10 µm. When the parasites from the same treated cultures were analyzed for co-localization of the disrupted PfTPxGl signal with mitochondrial marker protein ferrochelatase (PfFC), we observed that in 35-40% of cells the PfTPxGl signal showed some overlap.Chaudhari R, Dey V, Narayan A, Sharma S, Patankar S. Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum. PeerJ. 2017 Apr 27;5:e3128. PMID: 28462015