Fig. 8
From: Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human
T. brucei CFAP44 and CFAP43 are necessary for cell survival and proper axonemal organization. a–d Immunofluorescence on detergent-extracted cells of parental T. brucei stained with anti-PFR (red) and anti-myc (green) antibodies. e–h Immunofluorescence on detergent-extracted cells expressing TbCFAP44myc and RNAi induced (24 h) for TbCFAP44 (TbCFAP44 myc RNAi). Cells showed no or weak myc labeling (green) on the new flagellum (NF), while the old flagellum (OF) remained labeled. PFR is labeled in red. Note: cells with a maximum of two flagella were chosen for clear imaging. i–l Similar RNAi experiments as performed in e–h for TbCFAP43 RNAi in cells expressing TbCFAP43myc (TbCFAP43 myc RNAi). Cells showed no or weak myc labeling (green) on the new flagellum (NF), while the old flagellum (OF) remained labeled. PFR is labeled in red. Nuclei and kinetoplasts (mitochondrial genome) are labeled with DAPI (blue). Scale bars=5 μm. Insets are enlargement of images of flagella taken from the main panels and display areas indicated by white arrows (scale bar=1 µm). m Growth curves for parental cells, and TbCFAP44RNAi and TbCFAP43RNAi cells, non-induced or induced with tetracycline. The graph represents the cumulative number of cells per ml. Error bars represent the standard error from three independent experiments. n Flagellum length measurement of the new flagellum in cells bearing two flagella from parental cells, and from cells expressing TbCFAP44myc and TbCFAP44myc and induced (24 h) for TbCFAP44 RNAi and TbCFAP43 RNAi, respectively. Flagellum length was measured in cells showing a clear decrease in myc labeling in the new flagellum. o–t Electron micrographs of stained thin sections of parental cells (o, r) bearing one flagellar pocket (FP) and one flagellum (*), and of 24 h induced TbCFAP44RNAi (p, s) and TbCFAP43RNAi (q, t) cells. s and t are enlargements of p and q, respectively. Scale bars=100 nm in o–q; 200 nm in r–t. Note: in TbCFAP44RNAi and TbCFAP43RNAi cells, the flagellar pocket is enlarged and bears more than two flagella (p, q). Some of these flagella present axonemal defects including displaced and rotated CPC and shifted DMTs (p, white arrows, s, t)
