Planapo n 60 1.42 oil

For lipid droplets staining, cells were treated as previously described [35 (link)]. Briefly, cells were seeded on 11 mm round glass and when they reached 80% confluency, they were washed with PBS and then fixed with 3% paraformaldehyde in PBS for 20 min at room temperature. After fixation, 60% isopropanol was used to wash cells for 5 min and then left to dry. Cells were treated with a 0.5% Oil Red-O/isopropyl alcohol solution for 20 min and then washed abundantly with distilled water. Nuclei were stained with 1 μM diamidino-2-phenylindole (DAPI) for 5 min. Lipid droplets were visualized using EVOS FL Auto Cell Imaging System (ThermoFisher, Waltham, MS, USA) with PlanApo N 60×/1.42 oil (Olympus, Shinjuku, Tokyo, Japan).

Detection of cyan fluorescent protein CFP to yellow fluorescent protein YFP FRET in the AKAR4 sensor was performed as described previously (Depry and Zhang, 2011 (link); Molin et al., 2020 ). CFP was excited at 427/10 nm, YFP was excited at 504/6 nm and emission was monitored using a Semrock dual bandpass filter (part no: FF01-464/547). Images were acquired using an automated epi-fluorescence microscope (Olympus IX81) equipped with a × 60 oil-immersion objective (numerical aperture 1.4, PlanApoN ×60/1.42 Oil, Olympus) and an electron-multiplying charge-coupled device camera (12-bit Hamamatsu camera). The yeast cells were kept in a heated perfusion chamber (FCS2, Bioptechs Inc) at 28°C to avoid heat-induced stress responses. The objective was heated to 26.2°C (according to the manufacturer’s instructions) to maintain a stable temperature in the perfusion chamber. The cover glasses were precoated for 1.5 hr with protein concanavalin A, 0.5 μg μl−1 in 0.01 M PBS, to immobilize yeast cells on the surface.