PI(4,5)P₂ is asymmetrically localized in budding and filamentous cells with a steep gradient emanating from the cell tip. (A) PI(4,5)P₂ is asymmetrically localized in budding cells. False-colored sum projections (8–12 z-sections) of representative wt budding cells expressing the PI(4,5)P₂ reporter from different fields of view. Signal concentration over the long axis of each cell (in relative units, set to 100, determined by the BP program) starting from the bud (n_i.ex. = 4). (B) A steep gradient of PI(4,5)P₂ is observed in filamentous cells. Images of representative cells as described in A, incubated with FCS for indicated times from different fields of view (n_i.ex. = 5). (C) Quantification of PI(4,5)P₂ gradients in cells responding to FCS. Signal concentration (in arbitrary units) was quantified over the cell long axis starting from the cell body using the HP program. Average (n = 25 cells) with SD in gray, individual profiles shown in Fig. S2 A. (D) A PI(4,5)P₂ gradient occurs concomitant with germ tube emergence. Time-lapse confocal microscopy of wt cells expressing PI(4,5)P₂ reporter in the presence of FCS. Sum projections of 18 deconvolved z-sections. (E) Probe signal increases at germ tube tip concomitant with a decrease at the back of cell. Signal intensity determined in a 1-µm-radius area at the germ tube tip (or where it will form) and at the opposite end of the cells from sum projections as described in D. Average fold change in intensity relative to t = 0 (n = 25 cells). The average slope of the intensity at the back to the cell, −0.0160 ± 0.0037 relative intensity/min was two times greater than that due to photobleaching (determined with GFP-Rac1), −0.0076 ± 0.0017 relative intensity/min (n = 25 cells).