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. 2010 Jun 14;107(26):11805–11810. doi: 10.1073/pnas.1005817107

Fig. 1.

Fig. 1.

Fluorescent reporters reveal highly polarized PtdIns(4,5)P2 distribution during pheromone response and do not perturb signaling. (A) Exponentially-growing wild-type cells (strain BY4741) carrying a 2-μm DNA vector expressing from the GAL1 promoter the PtdIns(4,5)P2-binding GST-GFP-PHPLCδ1 probe were induced by addition of galactose for 30 min and then harvested by brief centrifugation, washed, and resuspended in glucose-containing medium to shut off any further probe expression. After 15 min, the cells were treated with 5 μM α-factor, and samples were withdrawn every 15 min, as indicated, and examined by standard epifluorescence microscopy. Each panel depicts a collage of representative cells at each time point; arrows denote the tips of mating projections that exhibited marked asymmetric distribution of the probe. (B) Strain YCS234 carrying a 2-μm DNA vector (pRS426) expressing from the constitutive PRC1 promoter an alternative PtdIns(4,5)P2-binding probe (GFP-2XPHPLCδ1) was treated with 0.1 μM α-factor for 1 h, spread on an agar pad, and examined by deconvolution microscopy. False color image represents the indicated scale of measured pixel intensity. (C) Upper, strain BY4741 was transformed with an empty 2-μm DNA vector or the same plasmid expressing GST-GFP-PHPLCδ1 from the GAL1 promoter (pPP1872), as indicated and tested for sensitivity to pheromone-induced growth arrest by halo bioassay on galactose-containing medium. Lower, strain BYB88 (ste4Δ ste5Δ) was cotransformed with a URA3-marked 2-μm DNA vector expressing from the GAL1 promoter the hyperactive Ste5(P44L) allele and a LEU2-marked CEN vector expressing GST-GFP-PHPLCδ1 from the GAL1 promoter (pLG99) and tested for mating proficiency by patch mating assay on galactose-containing medium. (D) Strain YLG36 carrying a FUS1prom-lacZ reporter gene was transformed with an empty 2-μm DNA vector or the same plasmid expressing GST-GFP-PHPLCδ1, as indicated. Representative transformants (n = 3) of each type were grown to early exponential phase, induced with galactose for 2 h, and treated with 3 μM α-factor for 90 min, and expression of β-galactosidase was measured (mean ± SD).