Observer z 1

The growth analysis was performed in S. cerevisiae acl4Δ strains that were transformed with pRS415 constructs carrying various mCherry-tagged Acl4 variants. Transformed cells were selected twice on SDC-LEU plates, before analysis. Ten-fold dilution series were prepared and 17.5 μl were spotted on SDC-LEU plates and grown at 23, 30 and 37 °C for 2–3 days. Localization assays were performed using pRS415 vectors carrying mCherry-tagged Acl4 variants and a pRS413 vector harbouring eGFP-tagged RpL4. Transformed cells were selected twice on SDC-LEU-HIS plates before analysis. The variants were grown in SDC-LEU-HIS medium at 30 °C to mid-log phase. For heat-shock analysis, cells were grown at 30 °C to mid-log phase before shifting cells to 37 °C for 6 h. For fluorescence microscopy 1 ml of cells was centrifuged at 500g and washed once with 1 ml of water. The cell pellet was resuspended in 100 μl water and 10 μl were analysed using a Carl Zeiss Observer Z.1 equipped with a Hamamatsu C10600 Orca-R2 camera.

The following systems were used: for confocal imaging, Zeiss LSM880-META NLO confocal microscope equipped with Airyscan; for color imaging, Zeiss Observer Z1 with a Hamamatsu ORCA-ER camera; for fluorescence imaging, Zeiss Observer Z1 with an AxioCam MRm camera. All histology analysis and immune-fluorescent staining analysis were quantitated in a blinded way by at least two investigators.

Cells were imaged by differential interference contrast (DIC) or epifluorescence microscopy using a 40× (water immersion, C-Apochromat, 1.1 NA), 63× (oil immersion, Plan-Apochromat, 1.4 NA), or 100× (oil immersion, Plan-Apochromat, 1.4 NA) Zeiss objective mounted on a Zeiss Observer Z.1 with a Hamamatsu Orca Flash 4.0 V2 CMOS camera (C11440-22CU).