Fig. 4.

FAZ27 RNAi disrupted the elongation of the new FAZ and positioning of flagellum-associated cytoskeletal structures. (A) Morphology of 2N2K cells from control and FAZ27 RNAi cells. 2N2K cells were immunostained with anti-CC2D and anti-PFR2 (L8C4) antibodies to label the FAZ and the flagellum, respectively. Cells were counterstained with DAPI to stain nuclear (N) and kinetoplast (K) DNA. pK, posterior kinetoplast; aK, anterior kinetoplast; pN, posterior nucleus; aN, anterior nucleus; NF, new flagellum; OF, old flagellum; P, posterior; A, anterior. Scale bar: 5 μm. (B) Diagram depicting a 2N2K cell used for measuring the length of the new FAZ and the unattached new flagellum, the distance from the posterior kinetoplast (pK) to the posterior cell tip and the distance between the two kinetoplasts. (C) Morphometric measurements of control and FAZ27-deficient 2N2K cells. 2N2K cells from control and FAZ27 RNAi (24 h) were immunostained with anti-CC2D and anti-PFR2 antibodies. Shown are the length of the new FAZ, the length of the unattached new flagellum, the inter-kinetoplast distance and the posterior kinetoplast to cell posterior distance for control (n=100) and FAZ27 RNAi cells (n=100). Mean±s.d. values are indicated. ***P<0.001 (Student's t-test). (D) Position of the flagellar pocket collar (FPC) and the basal body in control and FAZ27 RNAi cells. Shown are 2N2K cells that were co-immunostained with YL 1/2 antibody to label the mature basal body (mBB) and anti-TbBILBO1 antibody to label the FPC. Cells were counterstained with DAPI to stain nuclear (N) and kinetoplast (K) DNA. Abbreviations are as in A. Scale bar: 5 μm. (E) Positions of the hook complex (HC) and the centrin arm (CA) in control and FAZ27 RNAi cells. Shown are 2N2K cells that were co-immunostained with anti-TbMORN1 antibody and 20H5 antibody to label the HC and the CA, respectively, and counterstained with DAPI for nuclear and kinetoplast DNA. Abbreviations are as in A. Scale bar: 5 μm. Images in D,E are representative of three experiments.