mcherry atg13  (Addgene inc)

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Analysis of whole cell lysates (WCL) by SDS-PAGE and western blotting for NIX/BNIP3 double knockout clones #6 and #10, with and without induction of mitophagy by 24 h of DFP treatment. ( B ) Mitophagy flux was measured by flow cytometry of wild-type (WT) or NIX/BNIP3 double knockout (DKO) HeLa cells (clone #6), left untreated or treated with DFP for 24 h. ( C ) Analysis of knockdown efficiency for ATG13. HeLa cells were transfected 72 h prior to the FACS experiment, treated with DFP for 24 h to induce mitophagy, and analyzed by flow cytometry. Cells were collected after the experiment and analyzed by SDS-PAGE and western blotting. The concentration of 10 nM was used for the FACS experiment represented in the manuscript. ( D ) As in (C), but for HeLa cells transfected with siRNAs against FIP200, ULK1 or scrambled as a control (-). Two-way ANOVA with Tukey’s multiple comparisons test. ****P<0.0001. ns, not significant.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: SDS Page, Western Blot, Double Knockout, Clone Assay, Flow Cytometry, Knockdown, Transfection, Concentration Assay, Control

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: ( A ) Representative maximum intensity projection images of wild-type (WT) HeLa cells stably expressing Fis1-FRB and FKBP-GFP-WIPI2. Cells were left untreated (- Rapalog) or treated with Rapalog for 16 h (+ Rapalog) and immunostained for anti-ATG13. Scale bars: overviews, 20 µm; insets: 10 µm. ( B ) Immunoblotting for phosphorylated ATG13 in HeLa cells overexpressing Fis1-FRB and FKBP-EGFP-WIPI2d, treated with Rapalog for the indicated time. ( C ) Mitophagy flux was measured by flow cytometry in wild-type HeLa cells transfected with siRNAs targeting FIP200 or ATG13, and expressing Fis1-FRB, FKBP-GFP-WIPI1/2/3, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. ( D-E ) As in (C) but with or without the addition of (D) the ULK1/2 inhibitor MRT68921, or (E) the Vps34-inhibitor VPS34-IN1. ( F-G ) Wild-type HeLa cells expressing mt-mKeima and transfected with siRNAs targeting ATG13, FIP200, or ULK1, and treated with DFP for 24 h. ( H-I ) As in (F) but with the kinase inhibitors GSK8612 for TBK1, MRT68921 for ULK1/2, VPS34-IN1 for Vps34, or Bafilomycin A1 (BafA1). Two-way ANOVA with Dunnett’s multiple comparisons test in (C-E, I) or One-way ANOVA with Dunnett’s multiple comparisons test (F-H). **P<0.005, ***P<0.001, ****P<0.0001. ns, not significant.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Mitophagy flux was measured by flow cytometry in wild-type HeLa cells expressing Fis1-FRB, FKBP-GFP-WIPI2 wild-type (WT) or ATG16L1-binding mutant R108E/R125E, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. (B) Microscopy-based bead assay of agarose beads coated with GFP-tagged ULK1 complex (composed of FIP200-GFP, ULK1, ATG13, ATG101) and incubated with mCherry-tagged WIPI proteins. (C) As in (B) but with GFP-tagged ATG13/101 subcomplex and incubated with mCherry-tagged WIPI proteins. ( D ) As in (B) but with GFP-tagged FIP200 coated beads and incubated with mCherry-tagged WIPI proteins. ( E) As in (B) but with GFP-tagged kinase dead ULK1 (K46I) coated beads and incubated with mCherry-tagged WIPI proteins. (F) As in (B) but with GFP-tagged ATG13/101 coated agarose beads incubated with mCherry-tagged full-length (FL) or IDR-only (residues 364-425) WIPI2d. ( G ) Mitophagy flux was measured by flow cytometry in wild-type HeLa cells expressing Fis1-FRB, full-length (FL) or IDR-only (364-425aa) FKBP-GFP-WIPI2, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Two-way ANOVA with Šídák’s multiple comparisons test in (A,G). ****P<0.0001. ns, not significant.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: Flow Cytometry, Expressing, Binding Assay, Mutagenesis, Microscopy, Incubation

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Microscopy-based bead assay of agarose beads coated with GST-tagged WIPI2d or WIPI3 and incubated mCherry-tagged ATG13/101 complex which was composed of full-length ATG13 (mCh-ATG13/101), HORMA-domain only (mCh-HORMA; ATG13 1-191aa/101), or IDR only (mCh-IDR; ATG13 191-517aa). (B) As in (A) but with GFP-tagged ATG13 IDR coated beads, either as full IDR (191-517aa) or fragments (191-230aa), (191-205aa), or (206-230), and incubated with mCherry-tagged WIPI2d or WIPI3. (C) As in (A) but with GFP-tagged ATG13 IDR coated beads, either as full IDR (191-517aa) or with variants containing deletion fragments (Δ191-230aa), (Δ191-205aa), or (Δ206-230), and incubated with mCherry-tagged WIPI2d or WIPI3. ( D ) AlphaFold predicted structure of WIPI2d (orange) and ATG13 (green) plus ATG101 (blue) with zoom in on the interaction interface. Note that the indicated residue numbers for WIPI2 correspond to their residue number in the WIPI2d sequence (which match residue numbers Y113 and R143 in WIPI2b). Structures were trimmed for visual clarity. Displayed are ATG13 (residues 1-223), ATG101 (residues 1-218), and WIPI2d (residues 1-383). ( E ) As in (A) but with GFP-tagged ATG13 IDR (191-517aa) coated beads and incubated with mCherry-tagged WIPI2d or WIPI3. The IDR is composed of the indicated either the wild-type (WT), 3x Ala mutant (3A), or 11x Ala mutant (11A). ( F ) Mitophagy flux was measured by flow cytometry of wild-type (WT) or ATG13 knockout (KO) HeLa cells, where indicated rescued with ATG13 wild-type (WT), ATG13 lacking residues 191-230 (Δ191-230), ATG13 lacking residues 191-205 (Δ191-205), or ATG13 lacking residues 206-230 (Δ206-230), left untreated or treated with DFP for 24 h. One of three representative experiments is shown. Two-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001. ns, not significant.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: Microscopy, Incubation, Residue, Sequencing, Mutagenesis, Flow Cytometry, Knock-Out

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: Microscopy-based bead assay of agarose beads coated with GST-tagged ( A ) WIPI2d or ( B ) WIPI3 and incubated with GFP-tagged ATG13/ATG101 subcomplex or fragments of ATG13 alone.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: Microscopy, Incubation

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Immunoblotting for phosphorylated SQSTM1/p62 in wild-type (WT) or ATG13 knockout (KO) cells (clone #1), where indicated rescued with ATG13 WT or ATG13 lacking residues 190-230 (Δ190-230) (B) Immunoblotting for LC3B in the same cell lines as used in (A) but treated with 2 h starvation and Bafilomycin A1 (BafA1) where indicated. (C) Mitophagy flux was measured by flow cytometry of wild-type (WT) or ATG13 knockout (KO) HeLa cells, where indicated rescued with ATG13 wild-type (WT) or ATG13 lacking residues 190-230 (Δ190-230), left untreated or treated with O/A for 5 h. One of three representative experiments is shown. ( D ) As in (C) but with wild-type HeLa cells transfected with siRNAs targeting ATG13, left untreated or treated with O/A for 5 h. ( E ) Immunoblotting of COXII levels in wild-type (WT) or ATG13 knockout (KO) HeLa cells, overexpressing BFP-Parkin, and where indicated rescued with ATG13 wild-type (WT) or ATG13 lacking residues 190-230 (Δ190-230), left untreated or treated with O/A for 24 h. Densitometric analysis was performed for the percentage of COXII remaining relative to WT cells (mean ± s.d.) (n = 3 biologically independent experiments). One-way ANOVA with Dunnett’s multiple comparison test was performed. One-way ANOVA with Dunnett’s multiple comparisons test (A, E) or Tukey’s multiple comparisons test (B), or a Two-way ANOVA with Tukey’s multiple comparisons test (C-D). *P<0.05, ***P<0.001, ****P<0.0001. ns, not significant.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: Western Blot, Knock-Out, Flow Cytometry, Transfection, Comparison

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) AF3 screen for interaction between all known cargo receptors, soluble and transmembrane, and WIPI2. Predicted interactions are plotted for their ipTM score. (B) Microscopy-based bead assay of agarose beads coated with GST-tagged NIX, CCPG1, FAM134C, TEX264, and FKBP8 or GST alone as negative control, and incubated with mCherry-tagged WIPI2d. (C-D) As in (B), but with GFP-tagged C-terminal region of FIP200 (CTR). The laser power was either very low to visualize CCPG1-FIP200 interaction (C) or with higher laser power to visualize FAM134C, TEX264, FKBP8 and FIP200 interaction (D). In panel (C) we used the Fire LUT to better visualize the difference in binding strength between the different receptors. (E) As in (B), but with mCherry-tagged WIPI2d and/or GFP-tagged C-terminal region of FIP200 (CTR). (F) Schematic overview of the different selective autophagy pathways. Soluble cargo receptors are recruited to ubiquitinylated organelles and recruit the ULK1 complex through FIP200 to initiate autophagosome biogenesis. Transmembrane cargo receptors can initiate autophagosome biogenesis either through recruiting FIP200 or through recruiting WIPI proteins. The latter then recruit the ULK1 complex through interactions with ATG13, and in case of WIPI2 also through interaction with FIP200. Depending on the cargo receptor, autophagosome biogenesis can be initiated through FIP200- and/or WIPI-driven mechanisms.

Article Snippet: To purify mCherry-ATG13/101 HORMA dimer, we expressed mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (RRID:Addgene_223759) together with GST-TEV-ATG101 (RRID:Addgene_171414).

Techniques: Microscopy, Negative Control, Incubation, Binding Assay

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Analysis of whole cell lysates (WCL) by SDS-PAGE and western blotting for NIX/BNIP3 double knockout clones #6 and #10, with and without induction of mitophagy by 24 h of DFP treatment. ( B ) Mitophagy flux was measured by flow cytometry of wild-type (WT) or NIX/BNIP3 double knockout (DKO) HeLa cells (clone #6), left untreated or treated with DFP for 24 h. ( C ) Analysis of knockdown efficiency for ATG13. HeLa cells were transfected 72 h prior to the FACS experiment, treated with DFP for 24 h to induce mitophagy, and analyzed by flow cytometry. Cells were collected after the experiment and analyzed by SDS-PAGE and western blotting. The concentration of 10 nM was used for the FACS experiment represented in the manuscript. ( D ) As in (C), but for HeLa cells transfected with siRNAs against FIP200, ULK1 or scrambled as a control (-). Two-way ANOVA with Tukey’s multiple comparisons test. ****P<0.0001. ns, not significant.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: SDS Page, Western Blot, Double Knockout, Clone Assay, Flow Cytometry, Knockdown, Transfection, Concentration Assay, Control

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: ( A ) Representative maximum intensity projection images of wild-type (WT) HeLa cells stably expressing Fis1-FRB and FKBP-GFP-WIPI2. Cells were left untreated (- Rapalog) or treated with Rapalog for 16 h (+ Rapalog) and immunostained for anti-ATG13. Scale bars: overviews, 20 µm; insets: 10 µm. ( B ) Immunoblotting for phosphorylated ATG13 in HeLa cells overexpressing Fis1-FRB and FKBP-EGFP-WIPI2d, treated with Rapalog for the indicated time. ( C ) Mitophagy flux was measured by flow cytometry in wild-type HeLa cells transfected with siRNAs targeting FIP200 or ATG13, and expressing Fis1-FRB, FKBP-GFP-WIPI1/2/3, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. ( D-E ) As in (C) but with or without the addition of (D) the ULK1/2 inhibitor MRT68921, or (E) the Vps34-inhibitor VPS34-IN1. ( F-G ) Wild-type HeLa cells expressing mt-mKeima and transfected with siRNAs targeting ATG13, FIP200, or ULK1, and treated with DFP for 24 h. ( H-I ) As in (F) but with the kinase inhibitors GSK8612 for TBK1, MRT68921 for ULK1/2, VPS34-IN1 for Vps34, or Bafilomycin A1 (BafA1). Two-way ANOVA with Dunnett’s multiple comparisons test in (C-E, I) or One-way ANOVA with Dunnett’s multiple comparisons test (F-H). **P<0.005, ***P<0.001, ****P<0.0001. ns, not significant.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Mitophagy flux was measured by flow cytometry in wild-type HeLa cells expressing Fis1-FRB, FKBP-GFP-WIPI2 wild-type (WT) or ATG16L1-binding mutant R108E/R125E, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. (B) Microscopy-based bead assay of agarose beads coated with GFP-tagged ULK1 complex (composed of FIP200-GFP, ULK1, ATG13, ATG101) and incubated with mCherry-tagged WIPI proteins. (C) As in (B) but with GFP-tagged ATG13/101 subcomplex and incubated with mCherry-tagged WIPI proteins. ( D ) As in (B) but with GFP-tagged FIP200 coated beads and incubated with mCherry-tagged WIPI proteins. ( E) As in (B) but with GFP-tagged kinase dead ULK1 (K46I) coated beads and incubated with mCherry-tagged WIPI proteins. (F) As in (B) but with GFP-tagged ATG13/101 coated agarose beads incubated with mCherry-tagged full-length (FL) or IDR-only (residues 364-425) WIPI2d. ( G ) Mitophagy flux was measured by flow cytometry in wild-type HeLa cells expressing Fis1-FRB, full-length (FL) or IDR-only (364-425aa) FKBP-GFP-WIPI2, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Two-way ANOVA with Šídák’s multiple comparisons test in (A,G). ****P<0.0001. ns, not significant.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: Flow Cytometry, Expressing, Binding Assay, Mutagenesis, Microscopy, Incubation

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Microscopy-based bead assay of agarose beads coated with GST-tagged WIPI2d or WIPI3 and incubated mCherry-tagged ATG13/101 complex which was composed of full-length ATG13 (mCh-ATG13/101), HORMA-domain only (mCh-HORMA; ATG13 1-191aa/101), or IDR only (mCh-IDR; ATG13 191-517aa). (B) As in (A) but with GFP-tagged ATG13 IDR coated beads, either as full IDR (191-517aa) or fragments (191-230aa), (191-205aa), or (206-230), and incubated with mCherry-tagged WIPI2d or WIPI3. (C) As in (A) but with GFP-tagged ATG13 IDR coated beads, either as full IDR (191-517aa) or with variants containing deletion fragments (Δ191-230aa), (Δ191-205aa), or (Δ206-230), and incubated with mCherry-tagged WIPI2d or WIPI3. ( D ) AlphaFold predicted structure of WIPI2d (orange) and ATG13 (green) plus ATG101 (blue) with zoom in on the interaction interface. Note that the indicated residue numbers for WIPI2 correspond to their residue number in the WIPI2d sequence (which match residue numbers Y113 and R143 in WIPI2b). Structures were trimmed for visual clarity. Displayed are ATG13 (residues 1-223), ATG101 (residues 1-218), and WIPI2d (residues 1-383). ( E ) As in (A) but with GFP-tagged ATG13 IDR (191-517aa) coated beads and incubated with mCherry-tagged WIPI2d or WIPI3. The IDR is composed of the indicated either the wild-type (WT), 3x Ala mutant (3A), or 11x Ala mutant (11A). ( F ) Mitophagy flux was measured by flow cytometry of wild-type (WT) or ATG13 knockout (KO) HeLa cells, where indicated rescued with ATG13 wild-type (WT), ATG13 lacking residues 191-230 (Δ191-230), ATG13 lacking residues 191-205 (Δ191-205), or ATG13 lacking residues 206-230 (Δ206-230), left untreated or treated with DFP for 24 h. One of three representative experiments is shown. Two-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001. ns, not significant.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: Microscopy, Incubation, Residue, Sequencing, Mutagenesis, Flow Cytometry, Knock-Out

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: Microscopy-based bead assay of agarose beads coated with GST-tagged ( A ) WIPI2d or ( B ) WIPI3 and incubated with GFP-tagged ATG13/ATG101 subcomplex or fragments of ATG13 alone.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: Microscopy, Incubation

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Immunoblotting for phosphorylated SQSTM1/p62 in wild-type (WT) or ATG13 knockout (KO) cells (clone #1), where indicated rescued with ATG13 WT or ATG13 lacking residues 190-230 (Δ190-230) (B) Immunoblotting for LC3B in the same cell lines as used in (A) but treated with 2 h starvation and Bafilomycin A1 (BafA1) where indicated. (C) Mitophagy flux was measured by flow cytometry of wild-type (WT) or ATG13 knockout (KO) HeLa cells, where indicated rescued with ATG13 wild-type (WT) or ATG13 lacking residues 190-230 (Δ190-230), left untreated or treated with O/A for 5 h. One of three representative experiments is shown. ( D ) As in (C) but with wild-type HeLa cells transfected with siRNAs targeting ATG13, left untreated or treated with O/A for 5 h. ( E ) Immunoblotting of COXII levels in wild-type (WT) or ATG13 knockout (KO) HeLa cells, overexpressing BFP-Parkin, and where indicated rescued with ATG13 wild-type (WT) or ATG13 lacking residues 190-230 (Δ190-230), left untreated or treated with O/A for 24 h. Densitometric analysis was performed for the percentage of COXII remaining relative to WT cells (mean ± s.d.) (n = 3 biologically independent experiments). One-way ANOVA with Dunnett’s multiple comparison test was performed. One-way ANOVA with Dunnett’s multiple comparisons test (A, E) or Tukey’s multiple comparisons test (B), or a Two-way ANOVA with Tukey’s multiple comparisons test (C-D). *P<0.05, ***P<0.001, ****P<0.0001. ns, not significant.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: Western Blot, Knock-Out, Flow Cytometry, Transfection, Comparison

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) AF3 screen for interaction between all known cargo receptors, soluble and transmembrane, and WIPI2. Predicted interactions are plotted for their ipTM score. (B) Microscopy-based bead assay of agarose beads coated with GST-tagged NIX, CCPG1, FAM134C, TEX264, and FKBP8 or GST alone as negative control, and incubated with mCherry-tagged WIPI2d. (C-D) As in (B), but with GFP-tagged C-terminal region of FIP200 (CTR). The laser power was either very low to visualize CCPG1-FIP200 interaction (C) or with higher laser power to visualize FAM134C, TEX264, FKBP8 and FIP200 interaction (D). In panel (C) we used the Fire LUT to better visualize the difference in binding strength between the different receptors. (E) As in (B), but with mCherry-tagged WIPI2d and/or GFP-tagged C-terminal region of FIP200 (CTR). (F) Schematic overview of the different selective autophagy pathways. Soluble cargo receptors are recruited to ubiquitinylated organelles and recruit the ULK1 complex through FIP200 to initiate autophagosome biogenesis. Transmembrane cargo receptors can initiate autophagosome biogenesis either through recruiting FIP200 or through recruiting WIPI proteins. The latter then recruit the ULK1 complex through interactions with ATG13, and in case of WIPI2 also through interaction with FIP200. Depending on the cargo receptor, autophagosome biogenesis can be initiated through FIP200- and/or WIPI-driven mechanisms.

Article Snippet: To purify the mCherry-tagged or GFP-tagged ATG13/101 subcomplex, we expressed mCherry-tagged ATG13 from a pCAG backbone (RRID:Addgene_223735) together with GST-TEV-ATG101 (RRID:Addgene_171414) or GST-TEV-GFP-tagged ATG13 (RRID:Addgene_223797) together with ATG101 (RRID:Addgene_223796).

Techniques: Microscopy, Negative Control, Incubation, Binding Assay