Fig. 2. Localization of TbCentrin4. (A-D) Cells stably expressing TbCentrin4-YFP (green) were fixed and labeled for Golgi (anti-GRASP; red), kinetoplasts (DAPI; small blue dots) and nuclei (DAPI; large blue structures). The outline of each cell is indicated by a white line. Note the presence of TbCentrin4 on both basal bodies (open arrowheads) and the bi-lobe structure (filled arrowheads; large arrowheads indicate the lobe next to the old Golgi, small arrowheads the lobe that becomes associated with the new Golgi), which was closely associated with the Golgi throughout the cell cycle, as previously observed for TbCentrin2. The same cells are shown at higher magnification in supplementary material Fig. S2. (E-G) TbCentrin4-YFP cells were also fixed for cryo-iEM and labeled with an anti-GFP antibody followed by protein-A–gold (10 nm) labeling. TbCentrin4 was found at both the basal bodies (indicated by open arrowheads in E; shown also in F) and the Golgi/ERES region (bracketed region in G). K, kinetoplast; G, Golgi; ERES, ER export site. (H-M) Representative confocal images of a 1K1N (cell containing one kinetoplast and one nucleus) (H-J) and a 2K2N cell (containing duplicated kinetoplasts and nuclei) (K-M) representing early and late cell cycle stages showing that 20H5 (H,K) and anti-TbCentrin4 (I,L) both label basal bodies (open arrowheads) and the bi-lobe structure (filled arrowheads). Scale bars, 5 µm (A-D); 0.5 µm (E,G); 0.2 µm (F); 1 µm (H-M).