Additional file 23 of A heat-sensitive Osh protein controls PI4P polarity

Figure S8. Osh3 regulates the polarized localization of the exocyst subunit Exo70 and polarized secretion of the chitin synthase Chs3. (a) Wild type and temperature conditional stt4-4 mutant cells expressing Exo70-GFP at 26 °C were grown to log phase at 26 °C, shifted 10 min at 32 °C, and then imaged by spinning disk confocal microscopy. Representative confocal sections showing Exo70-GFP localization in wild type and stt4-4 mutant cells and corresponding Nomarski images are provided. Arrows point to non-polarized Exo70-GFP foci in stt4-4 mother cells. Mother (m) and daughter (d) cells are indicated. Scale bar, 2 μm. (b) Exponentially growing wild type or osh3∆ cells expressing Exo70-GFP at 26 °C were imaged by spinning disk confocal microscopy. Representative confocal sections showing Exo70-GFP localization in wild type and osh3∆ mutant cells are provided. Arrows point to non-polarized Exo70-GFP foci in mother cells lacking Osh3 (osh3∆). Mother (m) and daughter (d) cells are indicated. Scale bar, 2 μm. (c) Quantitative analysis of Exo70-GFP polarization in small-budded cells at 26 °C. Total number of cells analyzed: wild type 26 °C n=212, osh3∆ 26 °C n=208. The graph shows the means and standard deviations from three independent experiments (t test, *p< 0.015). (d) Exponentially growing wild type and osh3∆ cells were stained with calcofluor white (a dye that stains cell wall chitin enriched at sites of cell division known as bud scars). Representative Z projections showing bud scar distribution are provided. Arrow shows a non-polarized bud scar in an osh3∆ mutant cell. Scale bar, 2 μm.