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First published online 23 May 2006
doi: 10.1242/jcs.02969

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Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes

C. Gadelha^1,*, B. Wickstead^1,*, P. G. McKean² and K. Gull^1,

¹ Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
² Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK

View larger version (47K): [in a new window]   Fig. 1. (A) An elliptical section is formed if a perfect cylinder with longitudinal axis z', is cut at an angle to the x'y' plane. (B) The ellipse can be transposed onto the x'y' plane by a transformation, T, to generate a true transverse section. (C) A thin-section transmission electron micrograph showing a cross-section through a T. brucei flagellum. The section angle was corrected by the transformation T to generate a transverse section. (D) Ninefold rotation of the corrected axoneme shown in C. Bars, 50 nm.

View larger version (12K): [in a new window]   Fig. 2. (A) The positions of microtubules in transverse sections of axonemes from T. brucei. Points show the positions of the centres of either the CP microtubules or the A-tubules of the outer doublets. Dotted lines show the x' and y' axes of the axoneme (see Materials and Methods) and also a regular normal nonagon. The number, N, indicates the number of independent micrographs combined in the diagram. (B) Radial distribution of the doublets and the CP microtubules in transverse sections of axonemes from the wild-type trypanosomes. Since the CP are indistinguishable on the electron micrographs, their distribution can be anywhere in the range 0-180°, whereas the distribution of the doublets is 0-360°.

View larger version (133K): [in a new window]   Fig. 3. The orientation of the CP microtubules is fixed, and at the same position, in Kinetoplastida species. Representative cross-section micrographs through the flagellum of (A) procyclic-form T. brucei, (B) bloodstream-form T. brucei, (C) epimastigote-form T. cruzi, (D) promastigote-form L. major, (E) promastigote-form H. megaseliae, and (F) choanomastigote-form C. fasciculata. These micrographs had the section angle corrected by the transformation T to become true transverse section, and they show the CP microtubules always aligned with respect to the doublets 3 and 8. This orientation does not vary in either attached or partially attached flagella (trypanosomes), or free flagella (Leishmania, Herpetomonas, Crithidia). Bars, 50 nm.

View larger version (77K): [in a new window]   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.

View larger version (26K): [in a new window]   Fig. 5. CP orientation in -tubulin, PACRG and PFR2 RNAi mutant cell lines. (A) The remaining CP microtubule in 9+1 axonemes resulting from -tubulin ablation has a random orientation. (B) Knockdown of PACRG severely disrupts outer doublet microtubules, but only partially removes CP constraint in X+2 axonemes. (C) Immotility as a result of PFR2 ablation does not affect CP orientation. (D) Radial distribution of the doublets and the CP microtubules in transverse sections of axonemes from the induced -tubulin and PACRG RNAi trypanosomes.