Fig. 3.

Overexpression of the E3 ubiquitin ligase Dma2 prevents septin ring splitting, CAR contraction and cytokinesis. a Wild-type and GAL1-DMA2 bud4-G820fs cells were grown in YEPR at 25 °C, arrested in G1 by alpha factor and induced with 1% galactose 30 min before the release. Cells were finally released in YEPRG at 30 °C (time 0). Cells were collected at the indicated time points for FACS analysis of DNA contents. FACS data were plotted after gating out the debris as illustrated in Supplementary Fig. 12. b GAL1-DMA2 BUD4 cells expressing Shs1-mCherry and Myo1-GFP grown in SD-raffinose were induced for 90 min with galactose and then imaged in SD-raffinose/galactose at 30 °C. Arrowheads indicate the appearance of new septin rings (yellow) or CARs (white) before the old structures have been disassembled. DIC: differential interference contrast. Scale bar: 5 µm. c Wild-type and GAL1-DMA2 bud4-G820fs cells were treated as in a. At 240 min after release cells were fixed and processed for transmission electron microscopy. Scale bar: 2 µm. d Wild-type and GAL1-DMA2 BUD4 cells were treated as in a. At the indicated times after release cells were fixed for phalloidin staining of actin structures. Data are means from three independent experiments. Error bars: s.d. Micrographs show representative cells