Sub-cellular localisation studies of PfOTU in transgenic parasites. (C) Localisation of PfOTU by immune-electron microscopy. Ultra-thin sections of transgenic PfOTU-RFA parasites were labelled with anti-GFP antibody and gold-labelled secondary antibody. The labelling was seen in the cytosolic vesicles or associated with the apicoplast having characteristic four membranes. Scale bar-100 nm. In trophozoite stage parasites, the gold labelling was observed in membrane bound vesicles of 100-200nm diameter. In some sections these labelled vesicles were present near the apicoplast, while in some cases the immuno-staining was observed at the apicoplast surface overlapping with its membrane. (D) Confocal microscopic images showing the mitoTracker staining (red) in PfOTU-RFA transgenic parasites; corresponding three dimensional reconstruction using series of Z-stack images showing GFP labelled vesicles (green) distinct from the mitochondria (red).vStaining of transgenic parasites with MitoTacker Red CMXRos showed elongated/branched mitochondria whereas the GFP-labelled vesicles were present distinct from this organelle,Datta G, Hossain ME, Asad M, Rathore S, Mohmmed A. Plasmodium falciparum OTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with non-canonical role of Atg8. Cell Microbiol. 2017 Apr 18. [Epub ahead of print]

Localisation of PfOTU in transgenic parasites by GFP targeting. Confocal microscopy images of live PfOTU-RFA parasites grown in the presence of TMP at early-, mid-, late-trophozoite and schizonts stages. The nucleus is stained with DAPI (blue). Transgenic parasites showed vesicular localization of the fusion protein; at ring and trophozoite stages two or more GFP labelled vesicle like structures were present in the parasite cytosol. In schizonts, each of the developing merozoites contained one fluorescent vesicle.Datta G, Hossain ME, Asad M, Rathore S, Mohmmed A. Plasmodium falciparum OTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with non-canonical role of Atg8. Cell Microbiol. 2017 Apr 18. [Epub ahead of print]

Down-regulation of PfOTU effects apicoplast development and segregation. (A) Fluorescent microscopy images of PfOTU-RFA parasites immuno-stained with anti- EF-Tu (red) antibodies at different developmental stages (ring, tropozoite and schizonts) during first and second growth cycle for +TMP and −TMP set. The growth and development of the apicoplast in the –TMP culture was indistinguishable from that of +TMP during the first cell cycle; the apicoplast showed elongated morphology in trophozoite stages, branched pattern in the early-schizont-stage parasites and divided normally. However, during the second cell cycle the growth and development of apicoplast was severely affected in −TMP set, the EF-Tu staining was observed to be faint and apicoplast was observed as a single punctate structure even in late stages.Datta G, Hossain ME, Asad M, Rathore S, Mohmmed A. Plasmodium falciparum OTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with non-canonical role of Atg8. Cell Microbiol. 2017 Apr 18. [Epub ahead of print]

Confocal microscopy images of transgenic parasites PfOTU-RFA (green) and immune-stained for apicoplast targeted protein ClpP (A) and apicoplast resident protein EF-Tu (B). For each set, a three-dimensional reconstruction was developed using series of Z-stacks from confocal images with Imaris software. ClpP and EF-Tu staining are shown in red; the ClpP/EF-Tu and GFP co-localising area is seen as yellow staining. In all the cases the PfOTU-GFP labelled vesicles were observed to be in close proximity or partially overlapping with apicoplast staining. The 3D image reconstruction based upon confocal Z-stacks showed these vesicles to merge into the apicoplast in some cases.Datta G, Hossain ME, Asad M, Rathore S, Mohmmed A. Plasmodium falciparum OTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with non-canonical role of Atg8. Cell Microbiol. 2017 Apr 18. [Epub ahead of print]