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First published online 12 October 2004
doi: 10.1242/jcs.01401

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Elongation of centriolar microtubule triplets contributes to the formation of the mitotic spindle in -tubulin-depleted cells

ISTMT, Centre de Recherche en Pharmacologie-Santé, UMR 2587 CNRS-P. Fabre, 3 rue des Satellites, 31 400 Toulouse, France

View larger version (45K): [in a new window]   Fig. 1. Specific depletion of 23C -tubulin by RNA-mediated interference (RNAi). (A) Depletion kinetics. Control cells (C) and cells subjected to 23C -tubulin RNAi treatment (23C) were harvested after 5 days, 7 days and 9 days of culture. Total-protein extracts (50 µg) were analysed by immunoblotting. From day 5 to day 9, a strong and specific depletion of 23C -tubulin was observed. (First and second rows) Monoclonal GTU -tubulin antibody staining. The 37CD -tubulin band was not visible when the signal from 23C -tubulin was not overexposed (first row) and could be visualized only by reacting blots with more antibody and then overexposing the membranes (second row). (Third row) Purified polyclonal R70 antibody staining. Here, 37CD -tubulin also appeared to be less abundant than the 23C -tubulin. (Fourth row) The monoclonal antibody mab1501 was used to detect actin as an internal loading control. (B) Quantification of the depletion of -tubulin. The -tubulin content of 40 µg of an extract from cells submitted to a 7-day RNAi (23C) was compared by immunoblotting with that of serial loadings (20-1.25 µg) of protein extracts from control cells (C) and quantified by densitometry analysis. 23C -tubulin RNAi reduced the -tubulin level by at least 95%. (C) Reversibility of -tubulin depletion. Extracts (50 µg), prepared from control cells (C), 7-day-treated cells (23C) or from cells that had been treated with the 23C RNA for 7 days, then washed and maintained in culture for a further 7 days (R7) and 14 days (R14) without RNAi were analysed by immunoblotting with the GTU (against 23C -tubulin) and mab1501 (against actin) antibodies. The 23C -tubulin signal reappeared 14 days after the removal of the 23C RNA by washing. (D) Depletion specificity. Extracts (50 µg) from cells submitted to a 7-day RNAi treatment against either 23C -tubulin (23C) or the transcriptional factor Cdk8 (cdk8) were immunoblotted with the antibodies GTU (against 23C -tubulin) and mab1501 (against actin). Depletion of Cdk8 did not affect the levels of 23C -tubulin and actin.

View larger version (39K): [in a new window]   Fig. 2. Prometaphase stages observed after 23C -tubulin depletion. Three characteristic mitotic figures were observed after 23C--tubulin depletion: monopolar, bipolar and elongated spindles. Chromosomes were stained with DAPI (blue), microtubules with antibody against -tubulin (red), whereas 23C--tubulin, Bub1 or 37CD--tubulin immunostaining appeared in green. Scale bar, 5 µm. (A,A') Control metaphase, showing -tubulin and 23C -tubulin merged staining (A) and -tubulin staining only, showing the presence of 23C -tubulin at the two poles and in the two half-spindles (A'). All chromosomes were localized to the chromosomal plaque. The two poles exhibited astral microtubules (arrowheads). (B-M) -Tubulin-depleted cells. (B-E) Monopolar figures. No -tubulin signal could be detected (B,C). Generally the microtubules radiated preferentially from a single region, giving an overall conical shape. The fully condensed chromosomes constituted a plaque (B) or were dispersed in the spindle (C-E). The shape of the single pole was variable (compare B and C) but always devoid of astral microtubules. In all cases, the kinetochores were labelled by Bub1 antibodies (D,E). (F-I) Bipolar figures. The two opposite poles defined by the microtubules were devoid of astral microtubules and of a 23C--tubulin signal (F,G). Although the condensed chromosomes could constitute a typical chromosomal plaque (F,I), more often, some chromosomes were not aligned on the plaque (H) or were even scattered over the spindle. Their kinetochores were always labelled by Bub1 antibodies (H,I). (J,K) Elongated figures. The chromosomes were either scattered (J) or unequally distributed at each pole (K). In all cases, the kinetochores were labelled by Bub1 antibodies. (L,M) 37CD--Tubulin staining, which was present at the pole(s) of formed spindles.

View larger version (56K): [in a new window]   Fig. 3. Post-metaphase figures observed after 23C--tubulin depletion. Chromosomes appear in blue, microtubules in red, and the staining in green corresponds to -tubulin. (A,A') Control cytokinesis showing -tubulin and -tubulin merged staining (A) and -tubulin staining only, showing the presence of 23C -tubulin at the two poles and in the mid-body (A'). (B-E) Anaphase (B), telophase (C) and cytokinesis (D,E) after -tubulin depletion. None of these figures exhibited -tubulin signals (absence of green staining). Cytokinesis was generally symmetrical (D) and exceptionally asymmetrical (E). Scale bar, 5 µm.

View larger version (43K): [in a new window]   Fig. 4. Characterization of the poles in monopolar and bipolar figures after 23C--tubulin depletion. (A) Chromosomes appear in blue and microtubules in red, and the staining in green corresponds to the polar mitotic markers Asp (a,b,g,h), CP190 (c,d,i,j) or centrosomin (e,f,k,l). (Aa-f) Monopolar figures. Asp labelling was always exclusively localized to the single pole in several dots corresponding in most cases to sub-spindles (a,b). CP190 labelling was concentrated at the pole as between one and four dots (d). Centrosomin (CNN) was also concentrated at the pole, usually as a large and irregular structure (e,f). (Ag-l) Bipolar figures. Asp was always localized to the two poles, where, in most cases, it constituted a large area composed of two to five dots associated with each sub-spindle (g,h). CP190 and centrosomin labelling were present as one (i) or several (k) dots located at both poles or at one pole (j,l). (B) Renucleation in -tubulin-depleted cells. Microtubule arrays (red) renucleated from one or two centrosomin-labelled spots (green) after cold depolymerization. Scale bar, 5 µm.

View larger version (155K): [in a new window]   Fig. 5. Electron microscopic characterization of the poles in monopolar figures after -tubulin depletion. (A) Overall image of a monopolar prometaphase. The microtubules from the polar region defined by short centrioles (arrowheads) interacted with the kinetochores (k) of the chromosomes (ch) or passed through the chromosomal mass. (B1-4) Serial thin sections of a pole. The presence of four short centrioles (labelled 1 to 4) shows that, despite depletion of the major cellular -tubulin, centriole duplication but not separation took place. (C) Elongation of the centriole microtubules. The spindle microtubules resulted at least partly from the elongation of the distal ends of centriolar microtubules (arrowheads). Scale bars, 0.2 µm.

View larger version (18K): [in a new window]   Fig. 6. Centriole length in -tubulin-depleted cells. The length of interphase and mitotic centrioles was determined on longitudinal sections obtained by electron microscopy from control (A) and treated (T23C) (B) cells. The two length distribution profiles were significantly different as checked by a homogeneity test based on variance analysis: an experimental score of 41 against a theoretical score of 7 for a security of 99% [according to Snedecor's tables (De Muth, 1999)] indicating that -tubulin depletion reduces centriole length.

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