Cells were grown overnight at 30°C in complete synthetic medium (CSM; MP Biomedicals) with 2% dextrose to mid–log phase (106–107 cells/ml). For mating mixtures, strains were mixed to obtain a 1:1 ratio unless otherwise indicated. Cell mixes were mounted on CSM slabs with 2% dextrose solidified with 2% agarose and sealed with petroleum jelly. For Ste5-CTM induction, cells were pretreated with 20 nM β-estradiol (Sigma-Aldrich) for 3 h and mounted on slabs containing 20 nM β-estradiol. Imaging was performed at 30°C.
Images were acquired with an Andor Revolution XD spinning-disk confocal microscope (Andor Technology) with a CSU-X1 5000-rpm confocal scanner unit (Yokogawa) and a UPLSAPO 100×/1.4 oil-immersion objective (Olympus) controlled by MetaMorph software (Molecular Devices). Images were captured by an iXon 897 EMCCD camera (Andor Technology) or an iXon Life 888 EMCCD camera (Andor Technology).
Z stacks with 15 z steps of 0.48 μm were acquired at 2 or 4 min intervals. Laser power was set to 10% of maximum for 488 nm and 13% of maximum for 561 nm. Exposure time was 250 ms and electron-multiplying gain was 200.
Images were acquired with an Andor Revolution XD spinning-disk confocal microscope (Andor Technology) with a CSU-X1 5000-rpm confocal scanner unit (Yokogawa) and a UPLSAPO 100×/1.4 oil-immersion objective (Olympus) controlled by MetaMorph software (Molecular Devices). Images were captured by an iXon 897 EMCCD camera (Andor Technology) or an iXon Life 888 EMCCD camera (Andor Technology).
Z stacks with 15 z steps of 0.48 μm were acquired at 2 or 4 min intervals. Laser power was set to 10% of maximum for 488 nm and 13% of maximum for 561 nm. Exposure time was 250 ms and electron-multiplying gain was 200.