Fig. 4. -tubulin ablation in T. brucei results in growth rate reduction, cell paralysis, aberrant basal bodies and disrupted CP orientation. Growth (A) and motility (B) of -tubulin RNAi mutant cells with (closed squares) and without (open circles) tetracycline induction. (C) Western blot analysis of -tubulin depletion. Total cell lysates prepared at various time-points after -tubulin RNAi induction were separated by SDS-PAGE and analysed with mAb LAZ1 (raised against -tubulin) or mAb KMX (reacts with ß-tubulin). Each lane contains the protein from 2x10⁶ cells. (D) The microtubule number in the basal bodies of the T. brucei -tubulin RNAi cell line. Non-induced basal bodies possess the canonical nine-triplet arrangement at the proximal region, and the normal nine-doublet arrangement at the transition zone. Induced basal bodies were reduced to a mixture of triplets, doublets and singlets. Arrows indicate singlet microtubules. Bar, 100 nm. (E-G) The position of microtubules in transverse sections of induced T. brucei -tubulin RNAi axonemes. Representative individual micrographs illustrate (E) a 9+2 axoneme, (F) an axoneme that lost the B-tubule of doublet number 8 (9+2-B), and (G) an axoneme that lost doublet number 7 (8+2), and the corresponding position of microtubules within the respective nonagons. The number, N, indicates the number of independent micrographs combined in the diagram. Bars, 50 nm.