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MTOC formation during mitotic exit in fission yeast

School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK

View larger version (105K): [in a new window]   Fig. 1. Immunolocalisation of tubulin. (A) tubulin localisation and cell cycle progression. Cells are arranged in order of mitotic progression from G2 (1) through mitosis (2,3) to septation (4-7), and back into G2 once more (8). tubulin was found on the SPB throughout the cell cycle, appeared at the cell equator during anaphase B (3) and persisted (cells 4-7) until septation was complete (compare 7 and 8). Note that fission yeast do not extrude a structure containing tubulin as mammalian cells do with a mid-body, instead they retain medial tubulin and divide it between the two new daughter cells (7). (B,C) Wild-type cells were stained with a mixture of rabbit anti--tubulin (red) and mouse anti--tubulin (green) antibodies and then processed to stain with DAPI (blue) before imaging. Optical sections were processed by the application of deconvolution algorithms. In each panel, the left hand figure shows a projection of all of the images into a single plane of view, whereas the smaller numbered panels show sections through the series that produced this image from top (1) to bottom (2). (B) Not all regions of the EMTOC that contain tubulin staining are associated with the ends of microtubules (arrow). (C) The upper large arrowhead shows the EMTOC. The two small arrowheads highlight cortical spots of tubulin staining that are associated with microtubule ends. (D) 3D deconvolution of tubulin staining shows that the EMTOC is not a continuous ring. The left hand panel shows a projection of a cell that contains an EMTOC, whereas the right hand, numbered, panels show four snap shots, as a 3D reconstruction of this structure was rotated through 360° in the direction indicated by the red arrow. The EMTOC in this cell is composed of two crescents of tubulin staining that face each other from opposite sides of the cell equator.

View larger version (21K): [in a new window]   Fig. 2. The EMTOC appears towards the end of anaphase B. Wild-type cells (IH365) were cultured to mid-log phase in YES media at 25(C before processing for immunofluorescence microscopy. The spindle and cell length were measured. Because the cell structure shrinks back from the cell wall during preparation, the spindle length figures were calculated by multiplying measured spindle length by the length of the cell wall shell and dividing by the length of the shrunken cell structure.

View larger version (55K): [in a new window]   Fig. 3. The EMTOC and the F-actin ring. Wild-type (A) or dmf1.6 cells (B,C) were fixed and stained with antibodies specific to tubulin, F-actin (A,B) or tubulin (C). (A) tubulin (left), actin (middle) and a DAPI/phase contrast image of a wild-type cell during the late stages of cytokinesis. (B) tubulin (left), actin (middle) and a DAPI image of a dmf1.6 cell during cytokinesis at 36°C. The arrows indicate that the EMTOC forms along the sloping F-actin ring. (C) A dmf1.6 cell during cytokinesis at 36°C. From left to right: tubulin; tubulin; tubulin (red) and chromatin (green); tubulin (green) and tubulin (red); combined DAPI/phase contrast image. Note the microtubule nucleation from the asymmetrically located EMTOC. (D) A wild-type (IH365) culture was synchronised with respect to cell cycle progression by centrifugal elutriation and the indicated features were scored as the population underwent a synchronous cell division. Cells were maintained at 25°C throughout the experiment. The proportion of cells with F-actin rings increased before the appearance of cells with EMTOCs and calcofluor-stained septa, which increased simultaneously. The F-actin ring and EMTOC disappeared together at the end of mitosis when F-actin ring staining was replaced by more general dot-like staining at the junction of the two separating cells. (E,F) An exponentially dividing culture of cdc25.22 cells was shifted to the restrictive temperature for 225 minutes and returned to the permissive temperature (at 0 minutes) to generate a synchronous mitosis. Indicators of mitotic progression in an untreated control culture are shown in the graph. Cells were sampled and treated with LAT-A dissolved in DMSO to inhibit F-actin polymerisation, or the solvent DMSO alone just after the peak of EMTOC formation (arrowhead). Both of these cultures were fixed and stained 15 minutes later to visualise tubulin and F-actin (arrow). F-actin rings were not observed after LAT-A addition to the media (Table 2, n=400 cells). Similarly, EMTOCs were observed in the control cells but not in those exposed to LAT-A (n=400 cells). Anaphase was scored as cells in which the SPBs are on diametrically opposite sides of the two daughter nuclei. (F) The frequency of cells containing Dmf1/Mid1 rings showed a sharp decline as EMTOCs appeared.

View larger version (84K): [in a new window]   Fig. 4. EMTOC formation does not require microtubules. nda3.1828 cells were manipulated by cold shock followed by treatment with 300 µg ml–1 TBZ at 36°C, as described in the text before staining for tubulin (lower panels) and chromatin (DAPI/DIC) (upper panels). EMTOC formation (arrows) accompanied septation, as septation occurred in the absence of microtubules.

View larger version (106K): [in a new window]   Fig. 5. EMTOC formation requires SIN function. spg1.B8 cdc7.A20 double mutant cells were grown to early log-phase at 25°C before the culture temperature was shifted to 36°C. Panels show tubulin (upper panels in A-E and left in F) and DAPI/DIC images (lower panels in A-E and right in F) of the same fields of cells. EMTOCs clearly formed at 25°C (arrowheads A, B) but were not seen in the first (C) or second division (D,E) at 36°C. The profile of the nuclei in C and D show that the cells are in late anaphase B when the EMTOC would be expected to have already formed. Similarly, the nuclear profiles in E are those of a cell in which the spindle has just depolymerised. Cells at this stage should also have an EMTOC. (F) A mixed culture of cdc7.A20 and wild-type cells was processed for tubulin staining five hours after the temperature of the culture had been changed from 25°C to 36°C. EMTOCs were not observed in the long multi-nucleate mutant cells but were seen in the wild-type control cells (arrowhead).m

View larger version (28K): [in a new window]   Fig. 6. Inappropriate activation of the SIN induces cytokinesis but not EMTOC formation. (A) cdc16.116 cells were cultured in YES at 25°C then size selection was used to generate a synchronous population of late S phase/early G2 cells and the frequency of septa (A) or EMTOCs (B) was scored. The culture was split into two. One portion was constantly cultured at 25°C to monitor the cell cycle synchrony by following the septation index (A, open squares). Hydroxyurea (HU) was added to the second culture 60 minutes after elution, and it was cultured at 25°C for 260 minutes. At this time this second culture was split into two and one aliquot incubated at 36°C (A, filled circles) and the other maintained at 25°C (A, filled triangles). The grey area of the graph represents the timing of the temperature shift. Panel A shows the septation profile of each portion of the culture as indicated on the graph. (B) Failure of EMTOC formation in HU-arrested cells lacking Cdc16 function at 36°C. EMTOC formation in HU-arrested cdc16.116 cells at 36°C (filled circles) was not significantly higher than in those that retained Cdc16 function at 25°C (filled triangles), despite septum formation in over 75% of the cells following Cdc16 inactivation (filled circles in A). The frequency of binucleate cells was scored to gauge the efficacy of the S phase arrest. A minority of cells had leaked through the checkpoint arrest and re-entered the cell cycle and executed mitosis (open triangles and circles), and many of these cells contained EMTOCs (filled triangles and circles). The EMTOCs that were seen are therefore likely to be due to leak through the cell cycle arrest rather than arising from the activation of the SIN by shift to 36°C.

View larger version (21K): [in a new window]   Fig. 7. Anaphase-promoting complex function is required for EMTOC formation. (A-C) Asynchronous cut4.533, cut8.563 and cut9.665 cultures were grown at 25°C, and the temperature of the culture was then shifted to 36°C at 0 minutes. Cells were fixed and stained with antibodies to and tubulin or the SPB component Sad1 every 30 minutes. 200 cells were scored at each time point to determine the frequency of metaphase spindles (open triangles), anaphase (open squares), septation occurring in the absence of anaphase (cuts, filled triangles), and EMTOC formation (filled circles). After approximately 160 minutes at the restrictive temperature, cells began to accumulate in metaphase, and the frequency of cells in anaphase and cells with an EMTOC declined. No EMTOCs were observed after 160 minutes, despite an accumulation of cut cells indicating a successful cytokinesis in any of these strains. (D) Overexpression of plo1+ induced EMTOC formation in interphase cells. A culture of cdc25.22 cells containing pREP1plo1+ was grown in EMM2 containing thiamine at 25°C before being split into two. One of the two cultures was extensively washed and re-inoculated to EMM2 medium lacking thiamine for 15 hours to induce plo1+ overexpression. Centrifugal elutriation was then used to isolate small G2 cells from both cultures, and the cells were immediately shifted to 36°C to inactivate Cdc25 and maintain cells in G2 phase. Different cytological features were monitored in both cultures as shown in the legend. Cells with septa rapidly accumulated in these G2-arrested cells when Plo1 was overproduced (filled squares). The frequency of cells containing EMTOCs peaked around 150 minutes and declined to 0 by 250 minutes (filled circles). In the control culture, septation (open squares) and EMTOC formation (open circles) were negligible. The few cells in this control culture in which plo1+ expression was repressed had binucleate EMTOCs, indicating that they had leaked through the G2/M arrest and executed a normal mitosis.

View larger version (15K): [in a new window]   Fig. 8. Regulation of EMTOC formation. The formation of the EMTOC requires the activity of both the SIN and APC/C. It can also be induced by overexpression of plo1+ but not by activation of the SIN. These observations and data from other studies, such as the ability of Plo1 overexpression to activate the SIN, would be consistent with Plo1 acting upstream of both the APC/C and the SIN (red – for details see text) (A). We favour this model over the alternative possibilities (B) that Plo1 acts independently of both of these pathways (yellow) or functions downstream of either SIN or APC (blue).

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