Glucose starvation induces the release of vacuolar calcium into the cytoplasm. (A and B) Cells expressing cytoplasmic-anchored jGCaMP7f plasmid in WT, cch1∆, csg2∆, or yvc1∆ yeast strains were subjected to nitrogen starvation or glucose starvation and observed using a fluorescence inverted microscope (Olympus, IX83). Images were captured at 4-s intervals using time-lapse microscopy and shown in 24 fps movies. ImageJ software was used to calculate the relative fluorescence intensity of cells to reflect cytoplasmic calcium signaling. The data are representative of three independent experiments. (C) The image data at 88 s from A and B. Scale bar, 2 µm. (D) Cells co-expressing ER-anchored Stt3-jGCaMP7c (ER Ca²⁺ probe) and Sec63-mCherry were grown to the early log-growth phase and then subjected to either nitrogen or glucose starvation. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (E) Relative fluorescence intensity from D calculated by ImageJ software. n = 60 cells were pooled from three independent experiments. Data are presented as means ± SD. ns, no significance; two-tailed Student’s t tests were used. (F and G) Cells co-expressing Vacuolar-localized Atg15-jGCaMP7f (test vacuolar Ca²⁺ level) and Vph1-mCherry in wild-type (WT) or yvc1∆ yeast strains were grown to the early log-growth phase, and then subjected to nitrogen or glucose starvation. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (H) Relative fluorescence intensity from F and G, calculated by ImageJ software. n = 60 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (I) Cells expressing GFP-Atg8 in WT, atg1∆, or yvc1∆ yeast strains were cultured in either SD-N or SD-G for 4 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (J) Cells from I were quantified for the vacuolar localization of GFP-Atg8. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (K) Cells expressing GFP-Atg8 in WT, atg1∆, or yvc1∆ yeast strains were cultured in SD-N or SD-G for 4 h. Autophagic activity was analyzed by Western blot for GFP-Atg8 cleavage. (L and M) The cleavage of GFP-Atg8 from K was quantified and presented as mean ± SD (n = 3). ***P < 0.001; NS, no significance; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData F6.