Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei
Fig 3
FPC4 is a hook complex and an FPC protein.
(A) (a) Immunolabelling of endogenous FPC4 on cytoskeleton extracted cells from Tb427 29–13 cells using the rat anti-FPC4 antibody. (b) Cytoskeletons from the cell line expressing endogenously myc-tagged FPC4 probed with anti-myc. (c and d) Cytoskeletons from the cell line overexpressing myc-FPC4 using anti-myc. Co-labelling with anti-BILBO1 (a, b, c) or anti-MORN1 (d). (B) Anti-myc immuno-electron microscopy on flagella isolated from myc-FPC4 expressing cells (a), and the respective enlargements (b, c and d). (C) FPC4 immunolabelling using anti-FPC4 on CK extracted Tb427 90–13 BSF cells. (D) Co-labelling on cytoskeleton extracted Tb427 29–13 cells using anti-FPC4 and anti-MORN1 (a-d), and rat anti-FPC4 and anti-BILBO1 (e-h). (E) BILBO1RNAi was induced for 36 h and cells were detergent extracted and probed with rat anti-FPC4 (green) and anti-MORN1 (red). The white arrowheads indicate the area that is enlarged in the zoom image and highlight the labelling of FPC4 and MORN1 within the new detached flagellum (a) and at the base of the axoneme of the new detached flagellum (b). Kinetoplasts and nuclei were DAPI stained in A, C, D and E. Scale bars in A, C, D, and E represent 5 μm. In A c and d where the cells were induced for 24 h. All the experiments were performed more than three independent times.