Visualization of the LCL-LDs promoted by TG lipolysis using in situ cryo-ET. (A) Representative tomographic slice from a cryo-FIB–milled and cryo-ET reconstructed WT yeast cell grown for 4 h under AGR. Note the “bubbled” (lighter) centers of the LDs (L). V, vacuole. N, nucleus. A different tomographic slice of the boxed LD is also shown in C. (B–J) Representative tomographic slices of LDs in yeast from glucose-fed WT in log phase (B), WT after 4 h AGR (C, boxed area magnified in D; E shows line-scan plot of area between yellow arrowheads), WT after 4 h AGR + 0.1% OA (F), tgl3,4,5Δ yeast after 4 h AGR (G), WT cultured with 2% glucose and 0.1% OA (H), nvj1Δ after 4 h AGR (I, boxed area magnified in J). LCLs were only observed in LDs from WT and nvj1Δ yeasts in AGR (C, D, I, and J). White arrows highlight the “bubbles” due to electron radiation in centers of LCL-LDs. (K) Quantification of relative whole-cell TGs and SEs in log and 4-h AGR conditions. (L) Relative TGs in log and 4-h AGR conditions. (MandN) Perecent abundance of LCL-LDs (M) and diameters of LDs (N) under various conditions measured in cryo-tomograms. Note that the observed diameter depends on the plane at which the LDs were sectioned; therefore, for size measurements, only LDs with clearly visible monolayer (indicating a slice through the LD center) were included. *, P < 0.05; ***, P < 0.001. Scale bars: 200 nm (A), 50 nm (B, C, and F–I), and 20 nm (D and J).