Fig. 2: KIAPs have distinct localisation and development patterns during adhesion of in vitro haptomonad-like promastigotes.

mNG-tagged KIAP1 (a), KIAP2 (b) and KIAP3 (c) development. Arrowheads denote bright spots of KIAP1::mNG or mNG::KIAP3, or additional focus of KIAP2::mNG associated with flagellum membrane deformation. Representative images from n = 3 independent sample preparations are shown. Merge shows phase contrast, mNG (green) and DNA (blue) signals. Sequential frames (at ~20 (d) or ~2 (e) min intervals) from a time-lapse movie showing in vitro haptomonad-like promastigote adhesion and mNG::KIAP3 (d) or KIAP2::mNG (e) development. Adhesion starting point (arrowheads) remain fixed. White bars show KIAP2::mNG signal range. Representative images from n = 2 independent sample preparations are shown. f Confocal microscopy of KIAP localisation in an attached flagellum includes three image sets showing KIAP1::mNG with SMP1::mCh, KIAP1::mNG with mCh::KIAP3 and mNG::KIAP3 with KIAP2::mCh (mNG: green, mCh: magenta). Representative images from n = 3 independent sample preparations are shown. The schematic shows viewing directions (bottom view: X–Y, side view: X–Z). g Serial tomogram slice and 3D reconstruction of an in vitro haptomonad-like promastigote. Representative slice from n = 3 different cells is shown. Vesicles near the attachment plaque (green) are shown in red. MtQ: microtubule quartet. FAZ: flagellum attachment zone. h Magnified slice showing vesicles (white arrowheads) near the attachment plaque and interruption of the plaque by vesicle-flagellar membrane fusion (black arrowhead). i Magnified 3D reconstruction showing vesicles (white arrowheads) near the attachment plaque (green) and interruption of the plaque by the vesicle-flagellar membrane fusion (black arrowheads), with side (left) and bottom (right) view. FRAP experiments of KIAP1::mNG (j) and SMP1::mCh (k), graphs show relative fluorescence intensity changes before and after photobleaching (average fluorescence intensity before photobleaching as 1). Mean ± SD (n = 4 independent experiments) data with green (j) or magenta (k) dots indicating values from each experiment. Photobleaching timing is marked by an arrow, and sequential frames (at ~1 s intervals) of KIAP1::mNG and SMP1::mCh are displayed, with dotted white boxes indicating photobleached areas. Source data are provided as a Source Data file.