Rgf1 is part of actin-dependent machinery that signals growth poles in the absence of Tea1–Tea4 complex.

<p>(A) Representative images of LifeAct-GFP (actin) localization in WT cells untreated or treated with KCl 0.6 M, sorbitol 1.2 M, or 37°C (heat) for 1 h. The maximum-intensity projection of 6 Z-slides (0.5 μm step-size) of fluorescence is shown. (B) Quantitation of the T-shaped cells in the <i>tea1</i>Δ mutant treated with DMSO (Unt.), KCl 0.6 M, sorbitol 1.2 M, or 37°C (heat) for 1 h, then washed and allowed to grow without the drug for 3 h. The graph represents the mean ± SD of >200 cells from 2 independent experiments. Statistical significance was calculated using a two-tailed unpaired Student’s <i>t</i> test. (C) Images of WT and <i>rgf1</i>Δ cells expressing LifeAct-GFP to visualize actin. Maximum-intensity projection of cells (<i>n</i> = 22) of the same size from each strain is included to show the distribution of actin patches along the cells. (D) Cells expressing rgf1-GFP and crn1-GFP (actin patches) or mCherry-atb2 (MTs) cultured separately, mixed, and then treated with DMSO, LatA 100 μm, or MBC 50 μm for 15 min. (E) LifeAct-mCherry (actin) and Rgf1-GFP localization in WT cells treated with KCl 0.6 M for 1 h and then washed and allowed to grow without stress for the indicated times. The localization of Rgf1-GFP in <i>tea4</i>Δ cells under the same conditions is shown in the lower panel. The maximum-intensity projection of 4 Z-slides (0.6 μm step-size) of fluorescence is shown. (F) Quantitation of the T-shaped cells in the WT, <i>tea1</i>Δ, <i>gef1</i>Δ, and <i>tea1</i>Δ <i>gef1</i>Δ cells grown to log phase in YES liquid medium at 28°C. The graph represents the mean ± SD of >500 cells from 2 independent experiments. Statistical significance was calculated using a two-tailed unpaired Student’s <i>t</i> test. (G) GFP fluorescence in WT and <i>rgf1</i>Δ log-phase cells expressing <i>sty1-GFP</i>. Cells either were untreated (upper panels) or treated with 1 mM H<sub>2</sub>O<sub>2</sub> for 15 min (lower panels). The maximum-intensity projection of 6 Z-slides (0.5 μm step-size) of Sty1-GFP fluorescence is shown. (H) Extracts from wild type (WT-unt) and <i>rgf1</i>Δ cells (<i>rgf1</i>Δ-unt) were compared with extracts from WT cells treated with 1 mM H<sub>2</sub>O<sub>2</sub> for 15 min. Proteins were visualized by western blot with antibodies against Atf1. (I) GFP fluorescence in WT and <i>sty1</i>Δ log-phase cells expressing <i>Rgf1-GFP</i>. Cells were either untreated (upper panels) or treated with 0.6 M KCl for 1 h (lower panels). The maximum-intensity projection of 6 Z-slides (0.5 μm step-size) of Rgf1-GFP fluorescence is shown. (J) The percentage of T-shaped cells (<i>n</i> >200) in the indicated strains grown to log phase in YES liquid medium at 28°C. Statistical significance was calculated using a two-tailed unpaired Student’s <i>t</i> test. ns = nonsignificant. The experiment was repeated 4 times. (K) Quantitation of the T-shaped cells in WT cells treated with DMSO (WT DMSO), MBC 50 μg/ml (WT MBC) for 4 h, 100 μm of CK666 for 2 h to eliminate actin patches and then washed and allowed to grow with DMSO (WT CK) or 50 μg/ml of MBC for 4 h (WT CK-MBC); <i>for3</i>Δ cells, which lack actin cables, were treated with DMSO (<i>for3</i>Δ DMSO) or MBC 50 μg/ml (<i>for3</i>Δ MBC) for 4 h. WT cells treated with 50 μm of LatA for 2 h were washed and then treated with 50 μg/ml of MBC for 4 h (WT LatA-MBC). The graph represents the mean ± SD of >200 cells from 3 independent experiments. Statistical significance was calculated using a two-way ANOVA test. ****<i>P</i> < 0.0001; **<i>P</i> < 0.01; ns = nonsignificant. Scale bar, 2 μm. The data underlying the graphs shown in the figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002491#pbio.3002491.s014" target="_blank">S1 Data</a>.</p> <p>(PDF)</p>