Figure 1.
IQGAP1 plays a role in autophagosome closure. (A) Scatter plot of ATG9A proximity proteome comparing starvation-induced autophagy (EBSS, 90 min) and treatment with CCCP (20 µM, 6 h) identified by LC-MS/MS in FLAG-APEX2-ATG9A Flp-In T-REx HEK293(TetON) cells. For highlighted proteins (colors defined in inset legend), functions are separated by the stage of autophagosome biogenesis. (B) Schematic ATG9A was modified from the AlphaFold entry AF-Q7Z3C6 by rotating the unstructured C-terminal loop to avoid clashes with the membrane. Successive stages of autophagosome biogenesis: initiation (X), expansion (Y), and closure (Z). (C) Schematic representation of quantitative high content microscopy HaloTag (HT)-LC3B based closure assay (MIL/MPL HCM) encompassing incubation with membrane-impermeant HT ligand (MIL) to stain and saturate HT-LC3B-II accessible to the cytosol followed by membrane-permeant HT ligand (MPL) to stain LC3B-II (protected from and free of MIL because of sequestration within sealed membranes). (D) IQGAP1 knockdown (siRNA pool) in Huh7 HT-LC3B cells, immunoblot analysis. (E) MIL/MPL HCM quantification in Huh7 HT-LC3B control cells or cells knocked down for IQGAP1. Starvation in EBSS, 90 min incubation ± 100 nM BafA1. (i) MPL+ puncta (red symbols), closed autophagosomes. (ii) MIL+ puncta (green symbols), unclosed phagophores, and other accessible HT-LC3B; (iii) Ratio of MIL+ and MPL+ profiles (puncta/cell; gray symbols) in i and ii. Circles, siRNA control cells; squares, cells knocked down for IQGAP1. (F) Immunoblot of IQGAP1 KD with individual siRNAs, MIL/MPL HCM quantification in Huh7 HT-LC3B control cells or cells knocked down for IQGAP1 (siRNA1, squares; siRNA2, triangles) and complementation with siRNA resistant constructs pDest-3xFLAG-iQGAP1Res1 (diamonds) or pDest-3xFLAG-IQGAP1Res2 (inverted triangles) against siRNA1 and siRNA2, respectively. Starvation in EBSS, 90 min incubation ± 100 nM BafA1. (i–iii) MPL+ puncta (red symbols), (ii) MIL+ puncta (green symbols), (iii) Ratio of MIL+ and MPL+ profiles (puncta/cell; gray symbols). HCM parameters: 60 fields/well, >500 primary objects (cells)/well; 6 (E) or 4 (F) wells per sample/plate. Statistical significance was determined by one-way ANOVA and post-hoc Tukey’s multiple comparison test. Data, means ± SD, n = 5 (E) or 3 (F) biologically independent experiments per condition. Source data are available for this figure: SourceData F1.

IQGAP1 plays a role in autophagosome closure. (A) Scatter plot of ATG9A proximity proteome comparing starvation-induced autophagy (EBSS, 90 min) and treatment with CCCP (20 µM, 6 h) identified by LC-MS/MS in FLAG-APEX2-ATG9A Flp-In T-REx HEK293(TetON) cells. For highlighted proteins (colors defined in inset legend), functions are separated by the stage of autophagosome biogenesis. (B) Schematic ATG9A was modified from the AlphaFold entry AF-Q7Z3C6 by rotating the unstructured C-terminal loop to avoid clashes with the membrane. Successive stages of autophagosome biogenesis: initiation (X), expansion (Y), and closure (Z). (C) Schematic representation of quantitative high content microscopy HaloTag (HT)-LC3B based closure assay (MIL/MPL HCM) encompassing incubation with membrane-impermeant HT ligand (MIL) to stain and saturate HT-LC3B-II accessible to the cytosol followed by membrane-permeant HT ligand (MPL) to stain LC3B-II (protected from and free of MIL because of sequestration within sealed membranes). (D) IQGAP1 knockdown (siRNA pool) in Huh7 HT-LC3B cells, immunoblot analysis. (E) MIL/MPL HCM quantification in Huh7 HT-LC3B control cells or cells knocked down for IQGAP1. Starvation in EBSS, 90 min incubation ± 100 nM BafA1. (i) MPL+ puncta (red symbols), closed autophagosomes. (ii) MIL+ puncta (green symbols), unclosed phagophores, and other accessible HT-LC3B; (iii) Ratio of MIL+ and MPL+ profiles (puncta/cell; gray symbols) in i and ii. Circles, siRNA control cells; squares, cells knocked down for IQGAP1. (F) Immunoblot of IQGAP1 KD with individual siRNAs, MIL/MPL HCM quantification in Huh7 HT-LC3B control cells or cells knocked down for IQGAP1 (siRNA1, squares; siRNA2, triangles) and complementation with siRNA resistant constructs pDest-3xFLAG-iQGAP1Res1 (diamonds) or pDest-3xFLAG-IQGAP1Res2 (inverted triangles) against siRNA1 and siRNA2, respectively. Starvation in EBSS, 90 min incubation ± 100 nM BafA1. (i–iii) MPL+ puncta (red symbols), (ii) MIL+ puncta (green symbols), (iii) Ratio of MIL+ and MPL+ profiles (puncta/cell; gray symbols). HCM parameters: 60 fields/well, >500 primary objects (cells)/well; 6 (E) or 4 (F) wells per sample/plate. Statistical significance was determined by one-way ANOVA and post-hoc Tukey’s multiple comparison test. Data, means ± SD, n = 5 (E) or 3 (F) biologically independent experiments per condition. Source data are available for this figure: SourceData F1.

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