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doi: 10.1242/10.1242/jcs.00031

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Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plo1 to the spindle pole body and a functional spindle assembly checkpoint

Department of Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (59K): [in a new window]   Fig. 1. Myo2 rings form after the onset of mitosis. (A) A series of images from a movie of Myo2 ring contraction in a myo2-gc cell. In the CAR shown, contraction took place in 24 minutes. (B) myo2-gc cut12-gsp; SPB separation (upper cell) occurs before Myo2 ring formation (lower cell). Left hand panel: Myo2-GFP and Cut12 GFP fluorescence; middle panel: DAPI- phase; right hand panel: DNA (blue) and Myo2-GFP and Cut12-GFP signal overlay (green). (C) Timing of cell cycle events in myo2-gc cut12-gfp cells synchronised by size selection. Cells with two SPBs (filled red triangles) appear slightly ahead of cells also containing Myo2 rings (filled green circles), that is, Myo2 rings are seen only in cells in which SPB duplication and separation has occurred. Binucleate cells (filled blue diamonds) and septa (filled black squares) appear after Myo2 ring formation. (D) Timing of cell cycle events in myo2-gc cut12-gfp cdc25-22 cells synchronised by transient temperature shift. The relationship between Myo2 ring formation and SPB separation is as in (B). Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 2. CAR formation occurs after entry into anaphase. (A) The timing of cell cycle events in myo2-gc cen1-gfp cdc25-22 cells synchronised by transient temperature shift. The peak of cells containing separated centromeres (gold filled triangles) precedes that of cells also containing Myo2 rings (filled green circles), that is, Myo2 rings are seen only in cells in which centromere separation has occurred. Binucleate cells (filled blue diamonds) and septa (filled black squares) appear subsequently. (B) myo2-gc cen1-GFP cdc25-22 cells from the 40 minute time point in A. Left panel: Myo2-GFP and Cen1 GFP fluorescence; the middle cell contains two Cen1 dots but no Myo2 ring; middle panel: DAPI staining of DNA; right hand panel: DNA (blue) and GFP (green) signals overlayed. (C) myo2-gc cells expressing Mad2 for 16 hours from the plasmid pREP3Xmad2+ contain no Myo2 rings. The left panel shows Myo2-GFP signal; middle panel, DAPI staining (arrows point to condensed chromatin); right hand panel shows combined DAPI staining of DNA and phase contrast. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 3. Actin is required for Myo2 ring formation but not for the recruitment of Myo2 to the medial cell cortex. myo2-gc cdc25-22 cells were synchronised by temperature shift and allowed to enter mitosis either in DMSO (A,B) or 10 µM latrunculin B. (C-E). A and C show the timing of cell cycle events in the two cultures. In control cells (A), Myo2 rings (green filled circles) formed before the appearance of binucleate cells (filled blue diamonds) or septa (filled black squares). (C) Myo2 rings and binucleate cells failed to appear in the absence of actin but septal material (shown as a dashed line to distinguish it from true septa in A), appears at the cell equator on cue. (B,D) Micrographs of Myo2-GFP (left panels) and DAPI-calcofluor staining (right panels) from the 40 minute time points in (A,C). Myo2 rings failed to form in latrunculin B but Myo2 nevertheless accumulated at the medial cell cortex (E) Detail from (D): arrows point to colocalisation of Myo2 and cell wall material. Bar, 10 µm.

View larger version (14K): [in a new window]   Fig. 4. CAR formation is delayed in the absence of microtubules. Temperature-arrested myo2-gc cdc25-22 cells were released into DMSO (A, filled symbols), 100 µg/ml thiabendazole (B, open symbols); 25 µg/ml MBC (C, yellow filled symbols); or 10 µM latrunculin B. (D, dashed lines). Graphs show the timing of cell cycle events in each case. The appearance of cells containing Myo2 rings (green circles) and septa (black squares) was delayed in both (B) and (C) and no binucleate cells (blue diamonds) were observed in the absence of microtubules. (D) In the absence of actin, Myo2 accumulated at the cell equator and the deposition of septal material occurred but complete septa failed to form.

View larger version (34K): [in a new window]   Fig. 6. Delay in CAR formation is dependent upon Cdc16, Mad2 and Zfs1. A and B: synchronisation by transient arrest in S phase using 12 mM hydroxyurea and release at 25°C (A) or 35.5°C (B). (C-E): (C-E) synchronisation by cdc25-22 temperature arrest and release. (A) Synchronised myo2-gc cdc16-116 cells were released in the presence (open symbols) or absence (filled symbols) of 100 µg/ml TBZ. The microtubule-depolymerisation-induced delay in the appearance of Myo2 rings (green circles) was also seen using this synchronisation technique (compare open green circles in A with the closed ones), as expected, no binucleate cells (blue diamonds) were observed in TBZ. (B) In the absence of functional Cdc16 there was no delay in the appearance of single septa [compare filled (DMSO) and open (TBZ) black squares] or multiple septa [filled (DMSO) and open (TBZ) red squares]. Synchronised zfs1 myo2-gc cdc25-22 (D) or mad2 myo2-gc cdc25-22 (E) cells were released into DMSO (filled symbols), 100 µg/ml TBZ (open symbols) or 25 µg/ml MBC (yellow filled symbols). In contrast to control myo2-gc cdc25-22 cells (C), no delay was seen.

View larger version (46K): [in a new window]   Fig. 5. TBZ-induced delay in CAR formation is Mad2-dependent. Timing of cell cycle events in myo2-gc cdc25-22 (A,B) or myo2-gc cdc25-22 mad2 (C,D) synchronised by transient temperature shift and release into DMSO (A,C: filled symbols) or 100 µg/ml TBZ (B,D: open symbols). The TBZ-induced delay in the appearance of cells containing Myo2 rings (green circles) and septa (black squares) was substantially reduced in the absence of Mad2, and cell viability showed a corresponding decline (red crosses). As expected, no binucleate cells (blue diamonds) were observed in either strain in the presence of TBZ.

View larger version (108K): [in a new window]   Fig. 7. Other CAR components show a similar delay in recruitment to the medial cortex in TBZ. (A) Temperature-arrested myo2-gc cdc25-22 cells were released into either DMSO (left panels) or 100 µg/ml TBZ (right panels) and stained for actin. Whereas control cells at the 40 minute time point formed rings containing both actin (I,ii) and Myo2 (iii), TBZ-treated cells showed actin remaining at the cell poles and no Myo2 rings. Panel Aiv shows the merged images. (B) mid1-gfp cdc25-22 cells synchronised by transient temperature shift and released in the presence (open symbols) or absence (filled symbols) of 100 µg/ml TBZ. Whereas control cells formed normal Mid1-GFP rings (filled pink circles) followed by septa (filled black squares), both were substantially abolished in TBZ. (C) Control cells from the 40 minute time point in (B) showing Mid1 rings. (D) An equivalent sample from the TBZ-treated culture. In about 20% of TBZ-treated cells, Mid1-GFP localised to the medial cortex but failed to form a ring. Left panels in C and D: Mid1-GFP; right hand panels: DAPI-phase. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 8. Full Plo1 SPB association is microtubule and Mad2 dependent. plo1-gfp cdc25-22 mad2+ (A-C) or plo1-gfp cdc25-22 mad2 (D-F) cells were synchronised by transient temperature shift and released in the presence or absence of 100 µg/ml TBZ. (A,D) Time course of appearance of cells with Plo1 on the SPB (filled green diamonds, DMSO control; open green diamonds, TBZ) and septa (filled black squares, DMSO control; open black squares, TBZ). Initial recruitment of Plo1 to the SPB is microtubule independent. Disassociation of Plo1 from the SPB is, however, microtubule dependent, and this relationship is Mad2 dependent. (B,E) Fluorescence intensity of Plo1-GFP at the SPBs. Fluorescence was measured in 100 cells at each time point in the absence (green bars) or presence (open bars) of TBZ. Plo1 is only partially loaded onto the SPBs in TBZ, and this inhibition is Mad2 dependent. (C,F) Micrographs of cells from the 40 minute time points in A and D, respectively, showing the marked difference in Plo1 ntensity at the SPBs. (Upper panels, in presence of DMSO; lower panels, in presence of TBZ). (G) 10% of normal SBP associated Plo1 fluorescence was observed when plo1-gfp cells bearing the pREP3Xmad2+ plasmid overexpressed the Mad2 protein for 17 hours. Arrows point to the faint Plo1 SPB fluorescence. Bars, 10 µm.

View larger version (82K): [in a new window]   Fig. 9. Cdc7 localisation to the SPBs is co-commitant with Myo2 ring recruitment. myo2-gc cdc7-gfp cdc25-22 cells were synchronised by transient temperature shift and released in the absence (A, filled symbols) or presence (B, open symbols) of 100 µg/ml TBZ. In (A), Cdc7 fluorescence at the SPBs (red squares) is coincident with the appearance of Myo2 rings (green circles). Both precede the appearance of binucleate cells (blue diamonds). Both Cdc7 at the SPBs and Myo2 ring formation are delayed in the presence of TBZ. (C,D) Cells from the 40 minute time point in A and the 100 minute time point in B, respectively (left hand panels: Cdc7 and Myo2-GFP; right hand panels: DNA). Bar, 10 µm.

View larger version (13K): [in a new window]   Fig. 10. Diagrammatic representation of the relationship between the spindle assembly checkpoint, the septation initiation network and the formation of the Myo2 ring. Loading of Plo1 onto the spindle pole body is the key step in CAR formation and SIN activation. For details, see the text.