Spatial relationship of TbRab23 to components of the flagellum. (A) Gallery of IF images showing TbRab23YFP and PFR in BSF cells. Cells were stained with anti-GFP and anti-PFR and counterstained with Alexa-Oregon Green and Alexa-568 (red). DAPI (blue) was used to visualize DNA. Flagellum-associated TbRab23YFP is juxtaposed to PFR. Scale bar 2 μm. Arrows demark where the PFR emerges from the cell; TbRab23 staining extends beyond this point towards the flagellar pocket. For clarity images have been processed to remove cytosolic/plasma membrane staining of TbRab23. Representative images of over 20 cells are shown. (B) TbRab23YFP is coincident with flagella attachment zone protein (FAZ). The region where the flagellum attaches to the cell was visualized using antibodies raised against FAZ counterstained with Alexa-568 (red). TbRab23YFP was indirectly imaged with anti-GFP and Alexa-Oregon Green. DAPI (blue) was used to visualize DNA. Arrows demark where the FAZ originates. The insets have been gated to emphasise a pool of TbRab23YFP that is not associated with the flagellum in relation to FAZ. The proximal end of FAZ extends to the point of TbRab23 foci and was often found juxtaposed, but did not extend beyond it, indicating that TbRab23 foci at the vicinity of the basal bodies. Representative images of over 20 cells are shown. Scale bar 2 μm. (C) BSF parasites expressing TbRab23YFP labelled with anti-GFP antibodies and counterstained with gold-conjugated secondary antibody. Gold particles (15nm) were seen on the flagellum in the majority of sections analyzed (95%; n = 30), in addition to the basal body. Particles were occasionally seen between the kinetoplast and nucleus possibility defining an endosomal pool. 'B' basal body, 'F' flagellum, 'K' kinetoplast, 'P' pro-basal body, 'FP' flagellar pocket. Scale bar 200nm. Representative images are shown.