LD lipid phase transitions characterized by cryo-FIB and cryo-ET (corresponds to Fig. 1 ). (A–J) Electron dose series (“bubblegrams”) for LDs from cryo-FIB–milled WT yeast in log phase (A–E) or after AGR (F–J); series of 2D cryo-EM images were recorded of the same LDs exposed to increasing electron dose (1–400 e−/Å2). Note that LCLs (see box in G magnified in J) and excessive bubbling in LD centers (starting at an electron dose <30 e−/Å2) occurred only under AGR. Even at 400 e−/Å2 electron dose, minimal bubbling (white arrowheads in E) was observed in log WT. (K–M) Representative tomographic slices from cryo-FIB–milled and cryo-ET reconstructed WT in log phase (K), nvj1Δ yeasts after 4 h AGR (L), and are1are2Δ yeast after 4 h AGR (M). The nucleus-vacuole junction (black arrowheads in K and M) was observed in WT and are1are2Δ yeasts, but absent in nvj1Δ yeast (white arrowhead in L). No LDs were found in are1are2Δ yeast. V, vacuole; N, nucleus; L, LD; M, mitochondrion. (N) Yeast stained with LD marker MDH in log and 4 h AGR. Scale bars: 50 nm (A–I), 200 nm (K–M), 25 nm (J).
