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First published online 17 October 2006
doi: 10.1242/jcs.03204

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Dynactin targets Pavarotti-KLP to the central spindle during anaphase and facilitates cytokinesis in Drosophila S2 cells

CNRS UMR 6061 `Génétique et Développement', Groupe Cycle Cellulaire, Faculté de Médecine, IFR 140 Génomique Fonctionnelle et Santé, Université de Rennes I, 2 avenue du Pr. Léon Bernard, CS 34317, F-35043 Rennes CEDEX, France

View larger version (34K): [in a new window]   Fig. 1. p150Glued antibodies. (A) S2 cell extracts were probed with pre-immune (PI), immune (I) sera Rb1477 (left), Rb1478 (middle) or affinity-purified antibodies (right, AP). The position of the p150Glued protein is indicated with the arrowhead. (B) Extracts from S2 cells treated (+) or not (–) with Glued dsRNA were analysed with CP190, aurora A, cyclin B and dynein intermediate chain (DIC) antibodies (left) as loading controls, as well as with the affinity-purified anti-p150Glued antibodies (right).

View larger version (69K): [in a new window]   Fig. 2. p150Glued localises to centrosomes, spindle and interphase microtubules. S2 cells (A and B) or early Drosophila syncitial embryos (C) were fixed and stained with the affinity-purified anti-p150Glued antibodies (red and lower panels in monochrome), the anti--tubulin antibody (green and middle panels in monochrome) and DAPI (DNA staining in blue). (A) Mitotic cells. (B) Interphase cells: the right column shows a 10x magnified view of the border of a flat S2 cell. Note the punctuate staining along microtubule fibres. a, anaphase; c, cytokinesis; m, metaphase; p, prophase; t, telophase. (D) A mitotic cell treated with colchicine to depolymerise the mitotic spindle was stained for polo kinase (red and upper right panel in monochrome) and p150Glued (green and lower panel on the right in monochrome). The lower left panel shows a 10x magnification view of the kinetochore region boxed in the panel above. Bars, 10 µm (A-C); 1 µm (D).

View larger version (61K): [in a new window]   Fig. 3. Glued RNAi leads to anaphase defects. Control (A) or Glued dsRNA-treated (B) mitotic S2 cells were fixed and stained for -tubulin (red), -tubulin (green and middle panels in monochrome) and DNA (blue and lower panels in monochrome). a, anaphase; c, cytokinesis; m, metaphase; p, prophase; t, telophase. In p150Glued-depleted cells, cells are delayed in metaphase (see also Table 1). 50% of these cells show weak centrosome connection to spindle poles (panel 2) whereas others show a normal shape (panel 1). Note the segregation defect and the absence of microtubule bundling during anaphase (panels 3 and 4). During telophase, a central spindle forms but with strongly disorganised microtubules and a lack of well-defined microtubule bundles (panel 5). (C) FACS analysis of Glued dsRNA-treated cells does not show any accumulation of polyploid cells but evidences an increase of apoptotic or aneuploid cells (arrow) compared with control cells. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 4. Cyclin B, BubR1 and MEI-S322 behaviour in Glued-depleted anaphases. Control or Glued dsRNA-treated S2 cells were cold fixed in methanol (A and B) and stained for Cyclin B (A, green and lower panels in monochrome) and BubR1 (B, green and lower panels in monochrome), tubulin (red) and DNA (blue). Metaphase and anaphase cells are shown. (A) Cyclin B levels are elevated in metaphase and decrease during early anaphase after chromosome segregation. (B) A pool of BubR1 protein remains at the kinetochores of Glued or control dsRNA-treated cells during metaphase and is released from the kinetochore at anaphase onset in control cells. Note that BubR1 signal remains elevated at the kinetochore of chromatids that have not segregated in Glued-depleted anaphases. (C) MEI-S322 protein releases from the centromeres of p150Glued knockdown anaphase cells. Control (left) or Glued dsRNA-treated (right) cells were stained for MEI-S322 protein (red in bottom panels), -tubulin (red in top panels) and DNA (blue). Note that the MEI-S322 antigen is released in early anaphase cells but persists at the centromeres of unsegregated chromatids during asynchronous chromosome segregation. Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 5. Aurora B behaviour in p150Glued-depleted anaphases. Control (A) or Glued dsRNA-treated (B) S2 cells (B) were fixed and stained for aurora B (green and lower panels in monochrome), tubulin (red and monochrome in top right panel 4) and DNA (blue). In control or p150Glued-depleted metaphase cells (panel 1), aurora B is recruited to the centromeres. During anaphase A (A, panel 2), note the disappearance of aurora B from the centromeres and its progressive relocalisation to the central spindle during anaphase B (panel 3) and telophase (panel 4). In p150Glued-depleted anaphase cells (panels 2 and 3), aurora B is detected at the centromeres (white arrowhead) that have not segregated (see also supplementary material Fig. S2) and there is no recruitment to the microtubules. Panel 4 shows a p150Glued-depleted anaphase cell in which aurora B signal was overexposed (lower left panel) to show that aurora B signal is not detected on the few microtubules seen between the two chromatin masses. Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 6. Polo protein kinase behaviour in p150Glued-depleted anaphases. Wild-type (A,B) and Glued RNAi (C,D) cells during anaphase were fixed and stained for cyclin B (green), polo (red, in panels B and D, and lower panels in monochrome), and -tubulin (red in panels A and C, and lower panels in monochrome. In control anaphase (B), polo localises to the kinetochores and on central spindle microtubules. In Glued dsRNA-treated cells (D), polo is detected on the kinetochores but not on the microtubules present between the two chromatid masses. Note also the abnormal cortex contraction in this cell (white line). Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 7. Pavarotti-KLP targeting to the plus ends of microtubules and central spindle formation is delayed in Glued dsRNA-treated cells. Control or Glued dsRNA-treated mitotic cells were stained for Pavarotti-KLP (red and lower panels in monochrome), DNA (blue) and microtubules (green). (A) Pavarotti-KLP faintly localises to the mitotic spindle during metaphase (panel 1) and strongly accumulates at the plus ends of microtubules during anaphase (panel 2), telophase (panel 3) and the midbody during cytokinesis (panel 4). (B) Pavarotti-KLP localises to the mitotic spindle in a p150Glued knockdown metaphase cell (panel 1) but is not relocalised to microtubule plus ends during anaphase and transiently accumulates at the cell periphery where cortex contractions are detected (panel 2). As the cell progresses through anaphase-B-telophase, Pavarotti-KLP starts to accumulate on the microtubules present between the two chromatid masses (panel 3) allowing a reduced central spindle to form (panel 4) and cytokinesis to occur (panel 5). Bar, 10 µm.

View larger version (108K): [in a new window]   Fig. 8. Following Glued RNAi, centrosomes are not properly attached to spindle poles during spindle formation and microtubule bundling is defective after anaphase. Selected frames from time-lapse series of a control cell (A, see also supplementary material Movie 1) or a Glued dsRNA treated cell (B,C and supplementary material Movies 2 and 3) expressing GFP-tagged tubulin during mitosis. (A) Mitosis of a control cell. (B) Mitotic spindle formation following nuclear envelope breakdown until metaphase in a Glued dsRNA-treated cell. Note the poor centrosome connection to the spindle poles. (C) Metaphase-to-anaphase transition in a Glued dsRNA-treated cell. To avoid long light exposure and radiation damage, the acquisition started at metaphase. Note the lack of microtubule bundles during anaphase (60:48). The cell is also subjected to strong cortex contraction but is able to organise a central spindle region and a midbody (115:4). The panels are maximum projections of Z series and note that at time 66:48 the two poles are not in the same focal plane. The time scale (minutes: seconds) is shown at the bottom of each panel. Bars, 10 µm.

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