Figure 6. TbFUT1 is essential for procyclic and bloodstream form cell growth in vitro.

The cell numbers (± standard deviation) for TbFUT1 cKO under permissive (plus tetracycline, dotted line) and non-permissive (minus tetracycline, solid line) conditions are shown for three procyclic (A) and bloodstream form (C) clones, as well as for three bloodstream clones carrying a tetracycline-inducible ectopic TbFUT1 gene with a C-terminal MYC₃ tag (B). For each clone procyclic form clone, two biological repeats were analysed and three for bloodstream form clones. (D, E) Corresponding TbFUT1 mRNA levels were determined by northern blots. Alpha-tubulin (TUB) was used as a loading control. (F) TbFUT1-MYC₃ and untagged TbFUT1 are detected by western blot analysis in the respective bloodstream form cKO cell lines under permissive conditions (+Tet). The left panel shows an anti-MYC (αMYC) blot and the right panel an anti-recombinant TbFUT1 antibody (αFUT1) blot. Membranes were stained with Ponceau S (PS) to ensure equal loading.

Figure 6—figure supplement 1. Comparison of TbFUT1 and TbGMD conditional null mutant growth.

TbFUT1 and TbGMD cKO were grown in parallel under permissive (solid) or non-permissive (dotted) conditions. Cells were split and counted every 2 days in biological duplicates (procyclic form) or triplicates (bloodstream form) using a haemocytometer. Growth is depicted in cumulative curves over several passages of (A) procyclic form TbFUT1 conditional knockout. (B) Three different clones of bloodstream form BSF TbFUT1 cKO cells expressing the MYC₃-tagged ectopic copy. (C) Procyclic form TbGMD cKO cells. (D) Two different clones of bloodstream form TbGMD cKO cells.

Figure 6—figure supplement 2. TbFUT1 and TbGMD cKO have increased cell size.

(A) The average cell volume was measured using a Cazy cell counter (day 10–13) for both cell lines grown in permissive (+Tet) and non-permissive (–Tet) conditions. The average cell volume (circles) for each day is shown. The boxes mark the interquartile range, the black lines the median, while the whiskers mark the maximum and minimum values. Values outside the whiskers are considered outliers. (B) Number of cells with detached flagella in TbFUT1 and TbGMD cKO cells grown in permissive and non-permissive conditions compared to wild type (dm). The average of two biological replicates (± standard deviation) is shown. In each replicate, 200 cells/cell line were counted using a haemocytometer at an inverted light microscope. (C) Scanning electron microscopy (SEM) images of PCF TbFUT1 cKO grown in the presence or absence of tetracycline for 6 and 12 days. No flagellar detachment was observed, but cells grown in non-permissive conditions appear longer. Scale bars: 2 μm.

Figure 6—figure supplement 3. The paraflagellar rod (PFR) and flagellar attachment zone (FAZ) appear normal in PCF TbFUT1 cKO.

Cells were grown in permissive (+Tet) or non-permissive (-Tet) conditions and harvested at days 10 and 13. They were fixed, permeabilized, and stained as described in Materials and methods. The antibodies were used at the following dilutions: anti-FAZ 1:2, anti-PFR 1:10. Scale bars: 4 μm.

Figure 6—figure supplement 4. TbFUT1 and TbGMD cKO procyclic form cells show no defect in motility.

(A) Growth curve from days 6–13 of the cell lines used in the sedimentation assay. (B) Sedimentation assays on days 8, 10, 12, and 13. A reduction in motility could be observed in the PCF TbFUT1 and TbGMD cKO cells, grown under non-permissive conditions, only after the start of the growth phenotype (day 12 and onwards), not before. This suggests that the reduction in motility may be an effect of the growth defect and not its cause, as would be expected in a true flagellar attachment mutant (Bastin et al., 1999; Demonchy et al., 2009). TbGMD cKO is marked in grey while TbFUT1 cKO is marked in black. Cultures grown in non-permissive conditions are marked with dashed lines.