Figure S2.
Structure and functional validation of ATG9A-pHluorin construct. (A) Left: Topology of human ATG9A protein, adapted from Guardia et al. (2020), displaying four transmembrane domains (α2, α6, α14, and α15) and two domains (α11 and α19) that are partially embedded in the membrane. The ATG9A-pHluorin fusion protein was produced by inserting the pHluorin sequence (depicted by a cartoon of GFP) into the first luminal domain, between amino acids Leu102 and His103 of the human sequence. Right: The pHluorin fluorescence is expected to be quenched at the acidic endosomal pH. Upon fusion of ATG9A-containing endosomes with the plasma membrane, pHluorin fluorescence will sharply increase at the contact of extracellular physiological pH. (B) Left: Validation of functional activity of ATG9A-pHluorin on autophagosome biogenesis. U87 MG cells were transfected with nontargeting siRNA (siCont) or siRNA targeting ATG9A (siATG9A #2; this siARN specifically targets endogenous ATG9A, without affecting the expression of recombinant ATG9A-pHluorin, due to the codon-optimization procedure of the recombinant sequence introducing several siRNA/target mRNA mismatches), together with an empty vector or the ATG9A-pHluorin construct and a construct encoding the mCherry-LC3B protein (marker of autophagosomes). Transfected cells were placed in serum-free DMEM for 6 h, in the presence or absence of CQ (5 × 10−5 M), as indicated. Cells were fixed, and the number of autophagosomes (mCherry-LC3B fluorescent dots) per cell was quantified. Data represent means and SEM (n = 85–111 cells per group; cells from independent experiments were color-coded). Scale bar, 20 µm. Note that the marked reduction of autophagosome biogenesis induced by the knockdown of endogenous ATG9A was totally rescued by overexpression of recombinant ATG9A-pHluorin, indicating that insertion of the pHluorin sequence within the IL1 loop of ATG9A does not preclude its pro-autophagic function. (C) Validation of functional activity of ATG9A-pHluorin on cell migration. U87 MG cells were transfected with nontargeting siRNA (siCont) or siRNA targeting ATG9A (siATG9A #2), together with an empty vector or the vector encoding ATG9A-pHluorin. Transfected cells were loaded in the upper chamber of Transwells, with or without EGF (50 ng/ml) in the lower chamber, as indicated. After 24 h, cells that migrated onto the lower surface of the membrane were fixed, stained, and counted. Data represent means and SEM (n = 5 Transwells). Note that inhibition of chemotactic migration induced by the knockdown of endogenous ATG9A was rescued by overexpression of recombinant ATG9A-pHluorin. (D) Validation of ATG9A-pHluorin topology. Representative U87 MG cell coexpressing ATG9A-pHluorin and ATG9A-mCherry was imaged by epifluorescence (one frame every 30 s) before and after treatment with BafA1 (100 nM), a selective inhibitor of the vacuolar-type ATPase, an essential proton pump for maintaining vesicular acidic pH. Left: Images extracted from the time-lapse sequence before (−BafA1) and at the end (+BafA1; 1-h incubation) of BafA1 treatment. Scale bar, 20 µm. Middle: mCherry and pHluorin fluorescence signal intensities measured in the perinuclear area and plotted against time. Right: Analysis of the pHluorin/mCherry fluorescence intensity ratio, plotted against time. The increased ratio value following BafA1 treatment indicates dequenching of the pHluorin signal in endosomes, suggesting that the ATG9A-pHluorin fusion protein has the expected topology, with the pHluorin facing the vesicular lumen. (E) Left: Representative U87 MG cell expressing ATG9A-pHluorin recorded using TIRF microscopy (one frame every 390 ms), before (pH 7.4) and after (pH 5.5) acidification of the extracellular medium. Note that most ATG9A-pHluorin puncta rapidly dimmed after extracellular acidification, indicating that ATG9A-pHluorin proteins concentrating in these puncta are located at the plasma membrane, with the pHluorin facing the extracellular medium. Right: pHluorin fluorescence signal intensity measured before and after extracellular acidification and plotted against time. Scale bar, 20 µm; magnified views, 10 µm. (F) ATG9A static puncta colocalize with the endocytic marker clathrin. Left: Representative TIRF images extracted from a time-lapse sequence showing a U87 MG cell coexpressing ATG9A-pHluorin (green) and clathrin-mCherry (red). Note that ATG9A-pHluorin puncta frequently localize with clathrin (red arrowheads), suggesting that they likely represent ATG9A-pHluorin proteins trapped in forming endocytic structures. Right: Line profile plot indicates the fluorescence intensity distribution of green and red channels, through the white line shown in the merged image. Scale bar, 2 µm. Statistical significance was evaluated using one-way ANOVA followed by Tukey post hoc test (B and C). *, P < 0.05; ***, P < 0.001.

Structure and functional validation of ATG9A-pHluorin construct. (A) Left: Topology of human ATG9A protein, adapted from Guardia et al. (2020), displaying four transmembrane domains (α2, α6, α14, and α15) and two domains (α11 and α19) that are partially embedded in the membrane. The ATG9A-pHluorin fusion protein was produced by inserting the pHluorin sequence (depicted by a cartoon of GFP) into the first luminal domain, between amino acids Leu102 and His103 of the human sequence. Right: The pHluorin fluorescence is expected to be quenched at the acidic endosomal pH. Upon fusion of ATG9A-containing endosomes with the plasma membrane, pHluorin fluorescence will sharply increase at the contact of extracellular physiological pH. (B) Left: Validation of functional activity of ATG9A-pHluorin on autophagosome biogenesis. U87 MG cells were transfected with nontargeting siRNA (siCont) or siRNA targeting ATG9A (siATG9A #2; this siARN specifically targets endogenous ATG9A, without affecting the expression of recombinant ATG9A-pHluorin, due to the codon-optimization procedure of the recombinant sequence introducing several siRNA/target mRNA mismatches), together with an empty vector or the ATG9A-pHluorin construct and a construct encoding the mCherry-LC3B protein (marker of autophagosomes). Transfected cells were placed in serum-free DMEM for 6 h, in the presence or absence of CQ (5 × 10−5 M), as indicated. Cells were fixed, and the number of autophagosomes (mCherry-LC3B fluorescent dots) per cell was quantified. Data represent means and SEM (n = 85–111 cells per group; cells from independent experiments were color-coded). Scale bar, 20 µm. Note that the marked reduction of autophagosome biogenesis induced by the knockdown of endogenous ATG9A was totally rescued by overexpression of recombinant ATG9A-pHluorin, indicating that insertion of the pHluorin sequence within the IL1 loop of ATG9A does not preclude its pro-autophagic function. (C) Validation of functional activity of ATG9A-pHluorin on cell migration. U87 MG cells were transfected with nontargeting siRNA (siCont) or siRNA targeting ATG9A (siATG9A #2), together with an empty vector or the vector encoding ATG9A-pHluorin. Transfected cells were loaded in the upper chamber of Transwells, with or without EGF (50 ng/ml) in the lower chamber, as indicated. After 24 h, cells that migrated onto the lower surface of the membrane were fixed, stained, and counted. Data represent means and SEM (n = 5 Transwells). Note that inhibition of chemotactic migration induced by the knockdown of endogenous ATG9A was rescued by overexpression of recombinant ATG9A-pHluorin. (D) Validation of ATG9A-pHluorin topology. Representative U87 MG cell coexpressing ATG9A-pHluorin and ATG9A-mCherry was imaged by epifluorescence (one frame every 30 s) before and after treatment with BafA1 (100 nM), a selective inhibitor of the vacuolar-type ATPase, an essential proton pump for maintaining vesicular acidic pH. Left: Images extracted from the time-lapse sequence before (−BafA1) and at the end (+BafA1; 1-h incubation) of BafA1 treatment. Scale bar, 20 µm. Middle: mCherry and pHluorin fluorescence signal intensities measured in the perinuclear area and plotted against time. Right: Analysis of the pHluorin/mCherry fluorescence intensity ratio, plotted against time. The increased ratio value following BafA1 treatment indicates dequenching of the pHluorin signal in endosomes, suggesting that the ATG9A-pHluorin fusion protein has the expected topology, with the pHluorin facing the vesicular lumen. (E) Left: Representative U87 MG cell expressing ATG9A-pHluorin recorded using TIRF microscopy (one frame every 390 ms), before (pH 7.4) and after (pH 5.5) acidification of the extracellular medium. Note that most ATG9A-pHluorin puncta rapidly dimmed after extracellular acidification, indicating that ATG9A-pHluorin proteins concentrating in these puncta are located at the plasma membrane, with the pHluorin facing the extracellular medium. Right: pHluorin fluorescence signal intensity measured before and after extracellular acidification and plotted against time. Scale bar, 20 µm; magnified views, 10 µm. (F) ATG9A static puncta colocalize with the endocytic marker clathrin. Left: Representative TIRF images extracted from a time-lapse sequence showing a U87 MG cell coexpressing ATG9A-pHluorin (green) and clathrin-mCherry (red). Note that ATG9A-pHluorin puncta frequently localize with clathrin (red arrowheads), suggesting that they likely represent ATG9A-pHluorin proteins trapped in forming endocytic structures. Right: Line profile plot indicates the fluorescence intensity distribution of green and red channels, through the white line shown in the merged image. Scale bar, 2 µm. Statistical significance was evaluated using one-way ANOVA followed by Tukey post hoc test (B and C). *, P < 0.05; ***, P < 0.001.

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