Rgf1 is required for proper localization of the Tea1-Tea4 complex at the cell tip.

<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 WT cells (<i>n</i> > 150 for each end) and <i>cdc10-129</i> cells (<i>n</i> > 150 for each end) grown for 4 h at 37°C. Calcofluor staining was used to differentiate the growing poles from the non-growing poles. (B) Wild-type cells expressing rgf1-GFP were stained with Calcofluor white (20 μg/ml) to spot areas of growth. The arrows indicate the localization of Rgf1 at the growing tip in monopolar cells. (C) Maximum projection images of the cells from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002491#pbio.3002491.g001" target="_blank">Fig 1C</a> show the merged localization of Tea4-GFP (green) and Tea1-tomato (red) in wild-type (WT) and <i>rgf1</i>Δ cells (left panels). The fluorescence intensity profiles for green and red channels along a line across the poles, covering the cell width, are shown for cells 1 and 2 of WT and <i>rgf1</i>Δ cells, respectively, in the right panels. Scale bar, 2 μm. (D) GFP fluorescence in cells expressing <i>tea1-GFP rgf1</i><sup><i>+</i></sup> and <i>tea1-GFP rgf1</i>Δ grown to log-phase at 28°C in YES medium. The maximum-intensity projection of 6 Z-slides (0.5 μm step-size) of Tea1-GFP fluorescence is shown. The graphic represents the mean ± SD of the relative fluorescence intensity of Tea1-GFP measured at the growing and non-growing ends of WT (<i>n</i> > 80 for each end) and <i>rgf1</i>Δ (<i>n</i> > 80 for each end) cells. (E) Quantitation of the number of Tea4 dots associated to the MTs in WT and <i>rgf1</i>Δ strains per cell. The mean ± SD of >80 cells is shown. (F) The WT and <i>rgf1</i>Δ cells expressing <i>tea4</i>-GFP were treated with the translation inhibitor cycloheximide (CHX, 100 μg/ml) for the indicated times. Proteins were visualized by western blot with antibodies against GFP (Tea4) or tubuline (Tub), as a loading control (upper). The graphic represents the quantification of Tea4 levels at different times (hours) after the treatment relative to time 0, which was assigned a value of 1 (bottom). (G) The graphics show the MT polymerization (left) and depolymerization (right) rates in WT and <i>rgf1</i>Δ cells. The mean ± SD of >75 cells is shown. Statistical significance was calculated using two-tailed unpaired Student’s <i>t</i> test. *<i>P</i> < 0.05; ****<i>P</i> < 0.0001; 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>