Fig. 4. Tea1 localisation in the presence of pheromone in G2 and G1. A cdc25-22cyr1sxa2 strain was arrested in G2 at 36°C for 90 minutes; pheromone was added and cells were incubated for a further 2 hours; cells were then released into cell cycle progression at 25°C in the continued presence of pheromone. Samples were taken at three different time points: (A) after cells had been arrested in G2 at 36°C in the absence of pheromone; (B) after having been incubated with pheromone for 2 hours at 36°C; and (C) after cells had been allowed to enter G1 at 25°C in the continued presence of pheromone. Cells from each time point were treated with MBC, a microtubule depolymerising drug, to totally depolymerise microtubules. The drug was then washed out and microtubules were allowed to repolymerise for 50 seconds. Cells were fixed in methanol and processed for tubulin and Tea1p immunofluorescence. (D,E) The same experiment was repeated with a cdc25-22cyr1sxa2tea1GFP strain, timepoints a, b and c correspond to points A, B and C. (D) Cells were fixed in methanol and processed for tubulin while Tea1GFP was imaged live on a confocal microscope (E; see Materials and Methods for details). (F) Cells from C (after the release into G1 in the presence of pheromone) were also immunostained for Tip1p and tubulin. Bars, 5 µm.