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. 2024 Jun 4;12:RP92189. doi: 10.7554/eLife.92189

Figure 3. Phosphatidylinositol 4-phosphate (PI4P) is enriched in the autophagosomal membrane during maturation.

(A) Mouse embryonic fibroblasts (MEFs) stably expressing GFP–CERT(PHD) and mRuby3–STX17TM or mRuby3–ATG5 were cultured in starvation medium for 1 hr. GFP intensities of mRuby3-positive structures (n>60) were quantified. In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t-test. (B–D) Time-lapse analysis of MEFs stably expressing GFP–CERT(PHD) and mRuby3–ATG5 (B), WIPI2B–mRuby3 (C), or mRuby3–STX17TM and HaloTag–LC3B (visualized with SaraFluor 650T HaloTag ligand) (D) cultured in starvation medium. Autophagosomes are indicated by arrows. Experiments were performed three times independently. Scale bars, 10 μm (A [main]), 1 μm (A [inset], B–D).

Figure 3—source data 1. Data used for graphs presented in Figure 3A and Figure 3—figure supplement 2C.

Figure 3.

Figure 3—figure supplement 1. Localization of phospholipids in mature autophagosomes.

Figure 3—figure supplement 1.

(A) Mouse embryonic fibroblasts (MEFs) stably expressing the indicated GFP-tagged phospholipid probe and mRuby3–STX17TM were cultured in starvation medium for 1 hr. The following phospholipid probes were used: phosphatidic acid (PA), Spo20(PABD); PS, Evectin-2; diacylglycerol (DAG), PKD C1ab; PI3P, 2×FYVE; PI4P, CERT(PHD); PI5P, ING2(PlantHD); PI(3,4)P2, TAPP1(PHD); PI(4,5)P2, PLCd1(PHD); PI(3,5)P2, TRPML1(PHD); and PIP3, Btk(PHD). (B) MEFs stably expressing GFP–CERT(PHD) or TRPML1(PHD) were cultured in starvation medium containing LysoTracker Deep Red for 1 hr. (C) MEFs stably expressing GFP–PI4KB or GFP–PI4K2A and mRuby3–LC3B were cultured in starvation medium for 1 hr. Experiments were performed three times independently. Scale bars, 10 μm (main), 1 μm (inset).
Figure 3—figure supplement 2. Phosphatidylinositol 4-phosphate (PI4P) is enriched in mature autophagosomes before fusion with lysosomes.

Figure 3—figure supplement 2.

(A and B) Mouse embryonic fibroblasts (MEFs) stably expressing the indicated GFP-tagged PI4P probe, CERT(PHD)(W33A), FAPP(PHD), OSBP(PHD) or P4M-SidMx2, and mRuby3–STX17TM or mRuby3–ATG5 were cultured in starvation medium for 1 hr. (C) GFP intensities of mRuby3-positive structures (n>50) in (B) were quantified. In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t-test. (D) Time-lapse analysis of MEFs stably expressing GFP–CERT(PHD) cultured in starvation medium containing LysoTracker Deep Red. (E) U2OS cells stably expressing GFP–CERT(PHD) and mRuby3–LC3B were transfected with siSTX17 and siYKT6. After 3 days, cells were cultured in starvation medium for 1 hr, and immunostained with anti-LAMP1 antibodies. (F) WT and ATG8 hexa KO HeLa cells stably expressing GFP–STX17TM and transiently expressing mRuby3–CERT(PHD) were cultured in starvation medium for 1 hr. Experiments were performed three times independently. Scale bars, 10 μm (A, B, and E [main]), 1 μm (A, B, E [inset], and D).