Tea4 accumulation at the cell ends depends on Rgf1 anchoring to the PM and Rho1 activation.

<p>(A) The graphic represents the mean ± SD of the relative fluorescence intensity of Tea4-GFP measured at the growing and non-growing ends of the strains indicated (<i>n</i> > 100 for each end). Calcofluor staining was used to differentiate the growing poles from the non-growing poles. (B) Protein extracts from cells producing <i>rgf1</i><sup>+</sup><i>-</i>GFP, <i>rgf1</i>ΔPH<i>-</i>GFP, and pact-<i>rgf1</i>ΔPH<i>-</i>GFP were analyzed by western blot with an anti-GFP antibody to visualize Rgf1 levels. An anti-tubulin antibody was used as a loading control. (C) Extracts from cells producing Rho1-HA (pREP4X-Rho1-HA) in the WT, <i>rgf1</i>Δ, <i>rgf1</i>ΔPH, and pact-<i>rgf1</i>ΔPH<i>-</i>GFP cells were pulled down with GST-C21RBD and blotted against anti-HA antibody (Rho1-GTP). Total Rho1-HA was visualized by western blot (WCE). The relative units indicate the fold-differences in Rho1 levels in the mutants compared with the WT strain, with an assigned value of 1 (bottom) from 2 independent experiments. (D) Maximum-intensity projection of 6 Z-slides (0.5 μm step-size) of representative fluorescence images of WT, <i>rgf1</i>ΔPH<i>-</i>GFP, and pact-<i>rgf1</i>ΔPH<i>-</i>GFP cells. (E) The graphic represents the mean ± SD of the relative fluorescence intensity of Tea4-GFP (<i>n</i> > 120) measured at the cellular tips of the WT, <i>rgf1</i>ΔPH, and pact-<i>rgf1</i>ΔPH. Statistical significance was calculated using a two-tailed unpaired Student’s <i>t</i> test. (F) Representative images of Rgf1-GFP and Rgf1ΔPTTR-GFP localization. Cells were stained with calcofluor white (20 μg/ml) to spot areas of growth. Scale bar, 2 μm. (G) The percentage of cells WT, <i>tea1</i>Δ, <i>rgf1</i>Δ, <i>rgf1</i>ΔPH, pact-<i>rgf1</i>ΔPH, and <i>rgf1</i>ΔPH-ΔPTTR cells forming branches 3 h after release to growth after 3 days in stationary phase, in the absence and in the presence of MBC (50 μg/ml). The mean ± SD of >200 cells from 3 independent experiments is shown. Statistical significance of each strain compared to the WT was calculated using a two-way ANOVA test. ****<i>P</i> < 0.0001, *<i>P</i> < 0.05, ns = nonsignificant. 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>