Figure 4. LC3A interacts with ATG2B... | Rockefeller University Press

Figure 4.

$LC3A interacts with ATG2B during lysosome-induced CASM. (A) Western blot analysis of GFP-IPs and input from WT and ATG13 KO MCF10A cells expressing either GFP or GFP-LC3A-treated ± LLOMe (250 μM, 30 min). Samples were probed for ATG2B, GFP, ATG13, and GAPDH. (B) Western blot analysis of input and GFP-IPs from GFP-LC3A expressing MCF10A cells treated with LLOMe (250 μM, 30 min) or PP242 (1 μM, 1 h) ± Vps34 IN-1 (5 μM). Samples probed for ATG2B, ATG2A, GFP, and GAPDH. (C) Western blot analysis of GFP-IPs and input from MCF10A ATG16L1 cell line panel expressing GFP-LC3A treated ± LLOMe (250 μM, 30 min). Samples were probed for ATG2B, GFP, ATG16L1, and GAPDH. (D) Western blot analysis of input and HA-IPs from U2OS ATG2A/B DKO cells re-expressing WT, mutant LIR (mLIR), or mutant WIPI4 binding (mYFS) forms of HA-ATG2A. Cells were treated with LLOMe (250 μM, 30 min), samples were probed for ATG2A and LC3B. (E) Western blot analysis of input and LysoIP fractions from control or ATG13 KO cells expressing TMEM192-3xHA treated with LLOMe (250 μM, 20 min) ± ATG7 inhibitor ATG7i (10 μM). Samples probed for ATG2B, ATG2A, LAMP1, and LC3B. (F) Representative confocal images of ATG13 KO MCF10A cells expressing GFP-VAPA and LAMP1-RFP treated with LLOMe (250 μM, 20 min) ± ATG7 inhibitor ATG7i (10 μM). Insert shows LLOMe-induced contacts. Scale bar, 5 μm. (G) Western blot analysis of GFP-IPs and input from ATG13 KO MCF10A cells expressing GFP-LC3A treated with monensin (100 μM, 40 min). Samples probed for ATG2B and GFP. (H) Western blot analysis of GFP-IPs and input from RAW264.7 cells expressing GFP-LC3A following incubation with opsonized zymosan (OPZ) for 40 min. Samples were probed for ATG2B and GFP. (I) Model of ATG8 response to lysosome damage illustrating the ATG8 associated lysosome tubulation and vesiculation, activation of the V-ATPase-ATG16L1 axis, ATG8 conjugation to PE and PS, and the engagement of lipid transfer protein ATG2. Image created using Biorender. Source data are available for this figure: SourceData F4.$

LC3A interacts with ATG2B during lysosome-induced CASM. (A) Western blot analysis of GFP-IPs and input from WT and ATG13 KO MCF10A cells expressing either GFP or GFP-LC3A-treated ± LLOMe (250 μM, 30 min). Samples were probed for ATG2B, GFP, ATG13, and GAPDH. (B) Western blot analysis of input and GFP-IPs from GFP-LC3A expressing MCF10A cells treated with LLOMe (250 μM, 30 min) or PP242 (1 μM, 1 h) ± Vps34 IN-1 (5 μM). Samples probed for ATG2B, ATG2A, GFP, and GAPDH. (C) Western blot analysis of GFP-IPs and input from MCF10A ATG16L1 cell line panel expressing GFP-LC3A treated ± LLOMe (250 μM, 30 min). Samples were probed for ATG2B, GFP, ATG16L1, and GAPDH. (D) Western blot analysis of input and HA-IPs from U2OS ATG2A/B DKO cells re-expressing WT, mutant LIR (mLIR), or mutant WIPI4 binding (mYFS) forms of HA-ATG2A. Cells were treated with LLOMe (250 μM, 30 min), samples were probed for ATG2A and LC3B. (E) Western blot analysis of input and LysoIP fractions from control or ATG13 KO cells expressing TMEM192-3xHA treated with LLOMe (250 μM, 20 min) ± ATG7 inhibitor ATG7i (10 μM). Samples probed for ATG2B, ATG2A, LAMP1, and LC3B. (F) Representative confocal images of ATG13 KO MCF10A cells expressing GFP-VAPA and LAMP1-RFP treated with LLOMe (250 μM, 20 min) ± ATG7 inhibitor ATG7i (10 μM). Insert shows LLOMe-induced contacts. Scale bar, 5 μm. (G) Western blot analysis of GFP-IPs and input from ATG13 KO MCF10A cells expressing GFP-LC3A treated with monensin (100 μM, 40 min). Samples probed for ATG2B and GFP. (H) Western blot analysis of GFP-IPs and input from RAW264.7 cells expressing GFP-LC3A following incubation with opsonized zymosan (OPZ) for 40 min. Samples were probed for ATG2B and GFP. (I) Model of ATG8 response to lysosome damage illustrating the ATG8 associated lysosome tubulation and vesiculation, activation of the V-ATPase-ATG16L1 axis, ATG8 conjugation to PE and PS, and the engagement of lipid transfer protein ATG2. Image created using Biorender. Source data are available for this figure: SourceData F4.