Ixplore spinsr system

Cells were seeded onto 35 mm glass-bottomed dishes (Corning). Immediately before imaging, cells were incubated with MitoTracker Green for 30 min at 37 °C, washed three times in HBSS and transferred into imaging media (as above). Imaging was performed using an Olympus IXplore SpinSR system at 37 °C, using 488 and 561 nm laser lines. A z-stack was taken with 10 z-slices, taken every 5 min with 10 time points.

Yeast strains were grown as described above for the high-throughput microscopy with changes in the selection required for each strain (See yeast strain information in Table S3). For imaging performed in amino acid depletion or lysine-deprived media, cells were washed twice with corresponding medium prior to imaging.
Imaging was performed at 30 °C using Olympus IXplore SpinSR system, composed of an Olympus IX83 inverted microscope scanning unit (SCU-W1) in addition to a high-resolution spinning disk module (Yokogawa CSU-W1 SoRa confocal scanner with double microlenses and 50-µm pinholes), operated by ScanR. Cells were imaged using a 60× oil lens (NA 1.42) and Hamamatsu ORCA-Flash 4.0 camera. Fluorophores were excited by a laser, and images were recorded in two channels: GFP (excitation wavelength 488 nm, emission filter 525/50 nm) and mCherry (excitation wavelength 561 nm, emission filter 617/73 nm). For all micrographs, a single focal plane is shown.
For high-resolution imaging, cells were imaged using Z-stacks and deconvoluted in the cellSens software (Olympus, Shinjuku, Tokyo, Japan). Representative focal planes are shown.