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]