Rotor bench top colony arrayer

Cells were moved from agar plates into liquid 384‐well plates using the RoToR bench‐top colony arrayer (Singer Instruments). Liquid cultures were grown overnight in synthetic medium with 2% glucose (SD) in a shaking incubator (LiCONiC Instruments) at 30°C. A Tecan freedom EVO liquid handler (Tecan), which is connected to the incubator, was used to back‐dilute the strains to ∼ 0.25 OD₆₀₀ in plates containing the same medium. Plates were then transferred back to the incubator and were allowed to grow for 4 h at 30°C to reach logarithmic growth phase. The liquid handler was then used to transfer strains into glass‐bottom 384‐well microscope plates (Brooks Bioscience) coated with Concanavalin A (Sigma‐Aldrich) to allow cell adhesion. Wells were washed twice in a low fluorescence synthetic medium (Formedium) to remove floating cells and reach a cell monolayer. Plates were then transferred into the automated microscopy system using a KiNEDx robotic arm (Peak Robotics).
Imaging was performed using an automated Olympus SpinSR system using a Hamamatsu flash Orca 4.0 camera and a CSUW1‐T2SSR SD Yokogawa spinning disk unit with a 50 μm pinhole disk. Images were acquired using a 60× air lens NA 0.9 (Olympus), 100 mW 488 nm OBIS LX laser system (Coherent), GFP Filter set [EX470/40, EM525/50] (Chroma).
Images were manually inspected using Fiji‐ImageJ software (Schindelin et al, 2012 (link)).

Query strain JEY06 expressing ER-GFP was constructed on the synthetic genetic array compatible strain YMS721 [83 (link)] and was integrated into yeast deletion [34 (link)] and DAmP libraries [35 (link)] following synthetic genetic array methodology [88 (link),89 (link)]. Mating was performed on 1536-colony format YPD plates using a RoToR bench top colony arrayer (Singer Instruments; Somerset, UK). Resulting diploids were selected for deletion/DAmP libraries and ER-GFP markers KanR and URA3, respectively. Sporulation was induced by transferring cells to nitrogen starvation media for seven days and haploid cells were selected in histidine deficient SD plates to select for spores with an A mating type using canavanine and thialysine (Sigma-Aldrich Chemie; Munich, Germany) against remaining diploids alongside with previously mentioned selection markers.

The yeast strains used in this study are listed in Table S1. The library for the peroxisomal morphology screen was prepared by mating a roGFP-PTS1 plasmid containing query strain (constructed on the basis of a synthetic genetic array–compatible strain, YMS721; Papić et al., 2013 (link)) with a collection of ∼15 strains in which peroxisomal genes were deleted using a KanMx knockout cassette (Goldstein and McCusker, 1999 (link)). Automated sporulation and selection of haploids was performed using the synthetic genetic array method (Tong and Boone, 2006 (link); Cohen and Schuldiner, 2011 (link)) in high-density format using a RoToR benchtop colony arrayer (Singer Instruments).
A similar synthetic genetic array approach was used to create the library for the mitochondrial morphology screen by mating a TOM20-GFP, Δdnm1 expressing query strain to a collection of ∼100 strains in which genes for mitochondrial outer membrane proteins, and a number of other mitochondria-related proteins, have been modified to be driven by a TET-OFF promoter.

Lro1-GFP, expressed from the NOP1 promoter in a CEN/ARS URA3 vector, was introduced in the KanMX deletion and DAmP collections (Giaever et al., 2002 (link), Breslow et al., 2008 (link)) by Synthetic Genetic Array, by standard procedures previously described (Cohen and Schuldiner, 2011 (link)), using the RoToR bench-top colony arrayer (Singer Instruments, Roadwater, Watchet, UK). Cells were imaged, at room temperature at PDS phase in SC medium lacking uracil, using the automated inverted fluorescent microscopic ScanR system (Olympus, Waltham, Massachusetts, USA), with a 60× air lens, for GFP (excitation, 490/20 nm; emission, 535/50 nm) and brightfield channels. After acquisition, images were manually reviewed using the ImageJ software (NIH, Bethesda, MD). Cells showing increased Lro1-GFP signal in the ER, or overall decreased targeting to the nucleolus without enrichment at other subcellular localizations were selected for further analysis. The data obtained were analysed with GO (gene ontology) term finder of Saccharomyces Genome Database to determine GO term enrichment (https://www.yeastgenome.org/goTermFinder), and selecting the “Process” ontology aspect. Based on this analysis, Lro1-GFP localization was then manually inspected in mutants of interest (see Table S1).