Figure 6.

Characterization of the Cell Motility Phenotype Induced by TbTTC29 RNAi-Induced Knockdown in T. brucei

(A) Comparative growth curve of parental, non-induced, and RNAi-induced TbTTC29_::TY1/RNAi^TbTTC29 cells. Inset: Western blot analysis of RNAi knockdown by immunolabelling of TbTTC29_::TY1 at 0, 72, 96, and 120 h of induction with tetracycline. Anti-enolase was used for loading normalization.

(B) Sedimentation assays at 48 and 120 h post-RNAi^TbTTC29 induction. Percentages of sedimentation were normalized to basal levels of sedimentation in the parental cells.

(C) Comparative growth curves for parental, non-induced, and RNAi-induced TbTTC29_::TY1/TbTTC29-Nter_::myc/RNAi^TbTTC29 cells.

(D) Western blot analysis of TbTTC29_::TY1 and TbTTC29-Nter_::myc in protein extracts from whole cells (WC), cytoskeleton (CSK), and flagella fractions (FG), showing the presence of TbTTC29_::TY1 in all preparations while TbTTC29-Nter_::myc is detected only in the whole cell fraction. Anti-PFR2 was used as a control for CSK and FG fractions, anti-enolase was used as a control for the cytosolic fraction.

(E) Representative picture of of TbTTC29-Nter_::myc (anti-myc) and of TbTTC29_::TY1 (anti-TY1) immunolabelling on whole cells or detergent-extracted cytoskeleton preparations, showing the absence of axonemal localization for the truncated N terminus protein. The kinetoplasts and the nuclei are stained with DAPI. Scale bars represent 5 μm.

(F) Mobility graphs obtained from movies of non-induced (Video S1) and RNAiTbTTC29 (Video S2). The positions of individual cells are plotted at 0.2 s intervals. Open circle: starting position of each cell. Arrowhead: ending position.

Bars represent 10 μm. In (A), (B), and (C), data are represented as the mean ± SEM.