Fig 2: Myosin-II inhibition reduces autophagosome biogenesis in low confluency cells. a Representative images of endogenous ATG16L1 vesicles in MCF10A cells. Scale bar is 10 µm. Right panel: quantification of ATG16L1 vesicles numbers in LC and HC. At least 20–30 cells were quantified per condition. b ATG9A—LC3 colocalization in MCF10A cells. MCF10A cells plated at low and high confluencies were exposed to either DMSO (vehicle control) or blebbistatin (20 µM) for 4 h. The Pearson’s correlation and Manders’ overlap (the amount of LC3 in the ATG9 compartment) coefficients were used to measure the colocalization of LC3 with ATG9A in >30 cells per condition. c Representative confocal images of LC3 - ATG9A and z-stack images of F-actin and YAP/TAZ double immunostainings in MCF10A cells treated as in b. Scale bar is 10 µm. d Number and size of LC3 dots per cell in MCF10A cells plated at various densities and exposed to either DMSO (vehicle control) or blebbistatin (20 µM) for 4 h. Representative images–see b. More than 30 cells were analyzed per condition using ImageJ. e ATG9A—LC3 colocalization in MCF10A cells plated at LC or HC confluencies and exposed to either LATS1/2_p1 alone or combined with YAP/TAZ_p1 siRNAs. The Pearson’s correlation and Manders’ overlap (the amount of LC3 in the ATG9 compartment) coefficients were used to measure the colocalization of LC3 with ATG9A (for LC: n = 25 cells, while for HC: n = 100 cells). See Supplementary Fig. 10 for representative images. a–e Bars—mean ± s.e.m. (***P < 0.001, **P < 0.01, *P < 0.05, NS, not significant; two-tailed t-test). f Representative LC3-II immunoblots for MCF10A cells treated with blebbistatin (20 µM, 5 h). LC3-II/ GAPDH densitometry in MCF10A cells exposed to blebbistatin (20 µM, 5 h)–bottom panel. Bars—mean ± s.d. (*P < 0.05, NS, not significant; two-tailed one sample t-test). g YAP/TAZ localization in MCF10A cells treated as in b, c. There is no dramatic difference in YAP/TAZ localization between control and blebbistatin-treated cells. h Luciferase assay for YAP/TAZ activity in HeLa and MCF10A cells exposed to increasing concentrations of blebbistatin for 4 and 8 h, respectively. Bars–mean ± s.d. (n = 3, ***P < 0.001, *P < 0.05, NS, not significant; two-way ANOVA). n = number of independent biological replicates unless otherwise stated

Fig 3: CEMM disruption promotes autophagosome biogenesis(A) Electron micrographs of HeLa cells treated as the (1) Ctrl; (2) MBCD 5mM, pre-treated 1 h, then incubated for 2 h; (3) MBCD pre-treated as described in (2), then the cells were incubated with CHO (30 μg/mL) for 2 h; (4) cells were treated in AA for 2 h as positive control. Scale bars, 0.5 μm. (B) Cav1 WT and cav1 KO MEFs were treated with or without wortmannin (Wort, 100 nM) for 2 h. Cells were immunostained by ATG16L1 or WIPI2, and observed under confocal microscope (×600). Scale bars, 5 μm. (C) The number of WIPI2 puncta observed in (B) are presented as means ± SD. ∗∗∗∗p < 0.0001; NS > 0.05. (D) The number of ATG16L1 puncta observed in (B) are presented as means ± SD. ∗∗∗∗p < 0.0001; NS > 0.05. (E) HeLa cells were pre-treated with or without MBCD (5 mM) for 1 h, then incubated in the presence or absence of CHO (30 μg/mL) or Wort (100 nM) as indicated for 2 h. Cells were immunostained by ATG16L1 or WIPI2, and observed under a confocal microscope (×600). Scale bars, 5 μm. (F) The number of WIPI2 puncta observed in (E) are presented as means ± SD. ∗∗∗∗p < 0.0001. (G) The number of ATG16L1 puncta observed in (E) are presented as means ± SD. ∗∗∗∗p < 0.0001. (H) HeLa cells with stable expression of GFP-LC3B were pre-treated with or without MBCD (5 mM) for 1 h, then incubated in the presence or absence of CHO (30 μg/mL) for 2 h, meanwhile Baf A1 (100 nM) was added to all the treatments to induce enough accumulation of autophagic vesicles for observing colocalization between ATG16L1 and GFP-LC3. All the cells were immunostained by ATG16L1. Scale bars, 5 μm. (I) The Pearson correlation coefficient of GFP-LC3B puncta with ATG16L1 from the experiment described in (H). ∗∗∗∗p < 0.0001. (J) HeLa cells with stable expression of GFP-LC3B were treated as described in (H), then immunostained by WIPI2. Scale bars, 5 μm. (K) The Pearson correlation coefficient of GFP-LC3B puncta with WIPI2 from the experiment described in (J). ∗∗∗∗p < 0.0001.

Fig 4: SPATA33 interaction with ATG16L1 and VDAC2.a, b Coimmunoprecipitation between VDAC2 and deletion mutants of SPATA33. pCherry-FLAG-ATG16L1 was transiently co-transfected with pGFP-SPATA33, pGFP-N-SPATA33, or pGFP-C-SPATA33 in HEK293T cells, respectively. Cell lysates were examined by western blotting using the anti-FLAG or anti-GFP antibody. For coimmunoprecipitation, the lysates were immunoprecipitated with anti-FLAG or anti-GFP, followed by immunoblotting with the anti-GFP or anti-FLAG antibody. Arrowheads indicate the target bands. c, d Coimmunoprecipitation between ATG16L1 and deletion mutants of SPATA33. p3xFLAG-VDAC2 was transiently co-transfected with pGFP-SPATA33, pGFP-N-SPATA33, or pGFP-C-SPATA33 in HEK293T cells, respectively. Cell lysates were examined by western blotting using the anti-FLAG or anti-GFP antibody. For coimmunoprecipitation, the lysates were immunoprecipitated with anti-FLAG or anti-GFP, followed by immunoblotting with the anti-GFP or anti-FLAG antibody. Arrowheads indicate the target bands. e Coimmunoprecipitation analysis of interaction among endogenous SPATA33, ATG16L1, and VDAC2 in GC-1 cells. The GC-1 cell lysates were immunoprecipitated with anti-SPATA33, anti-ATG16L1, or anti-VDAC2 antibody, followed by immunoblotting with the anti-SPATA33, anti-VDAC2, or anti-ATG16L1 antibody, respectively. The whole cell lysates were examined by western blotting using the anti-ATG16L1, anti-VDAC2, or anti-SPATA33 antibody. Arrowheads indicate the target bands. f Colocalization analysis of SPATA33 with VDAC2 and ATG16L1. GC-1 cells were transiently co-transfected with pCherry-SPATA33 and pGFP-VDAC2. After 24 h in normal medium, the cells were cultured in normal (control), CCCP (10 μM, 1 h), EBSS medium (1 h), or EBSS with CCCP (10 μM, 1 h) addition, respectively. Immunofluorescence analysis was performed with anti-ATG16L1 and Dylight 405 Donkey anti-Rabbit IgG (H + L) antibodies. Single channel (red, green, or blue) and merged images were taken by confocal microscopy. Colocalizing structures are indicated in white (merge). Scale bar: 25 μm. g Statistical analysis of colocalized puncta between SPATA33, ATG16L1, and VDAC2. Data are presented as means ± S.D. **p < 0.01 (n = 3 independent experiments, >15 cells per experiment). h SPATA33 mediates mitophagy via interaction with VDAC2 and ATG16L1. SPATA33 interacts with mitochondrial outer membrane protein VDAC2 through its carboxyl terminal, while its amino terminal interacts with WD40 region of ATG16L1 protein. Upon starvation stress, damaged mitochondria are encapsulated by autophagosome through mediator SPATA33 linking between VDAC2 and ATG16L1 to initiate mitophagy.

Fig 5: mATG9 Is Internalized by Clathrin-Mediated Endocytosis(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson’s coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson’s coefficient: 0.413 ± 0.020 (SEM).(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson’s coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson’s coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1–3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.See also Figure S1.