Fluorescence microscopy

S. cerevisiae cells were imaged in indicated media at room temperature in 96-well glass-bottom microplates (Greiner Bio-One). Images and semi-three-dimensional time-lapse images were acquired using a Dragonfly 500 spinning disk microscope (Andor) attached to an inverted Ti2 microscope stand (Nikon) with a CFI Plan Apo Lambda 60×/1.4 oil immersion objective (Nikon) and a Zyla 4.2 sCMOS camera (Andor). Fluorophores were excited with excitation lasers 405, 488, and 561 nm; emission was collected using 450/50, 525/50, and 600/50 nm bandpass filters. Image processing was performed with Fiji version 2.1.0 (Schindelin et al., 2012).