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.
