Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Formation of LC3 puncta and LC3II independently of ATG16L1 requires ATG3, ATG5 and ATG7. Panel A. ATG3-/-, ATG5-/- and ATG7 -/- MEFs were incubated for 2 hours in chloroquine (100 µM) fixed cells were immunostained for LC3. Panel B. LC3 puncta in the indicated cells were counted using Imaris software. Panel C. Cells were incubated as described above and generation of LC3II was assessed by western blot for LC3. Panel D. Densitometric calculation of LC3II/LC3 ratios from duplicate blots.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Incubation, Software, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Osmotic stress induces recruitment of galectin-3 to LC3 puncta generated in the absence of ATG16L1. Control (Panel A) and Atg16L1-/- MEFs (Panel B) were incubated for 2 hours in media containing chloroquine (100 µM i-iv), LLOMes (1 mM v-viii) or monensin (100 µM ix-xii). Fixed cells were immunostained for LC3 (green) and galectin 3 (red). Scale bar 5 mm. Panel C. distribution of LC3 (green) and galectin 3 (red) assessed from line profiles. Panel D. Numbers of small (1.0 um dia.) LC3 puncta counted from 20 cells by Imaris. Panel E. LC3II assessed by western blot of cell lysates. Panel F. LC3II/LC3 ratio calculated by densitometry of duplicate blots.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Generated, Control, Incubation, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Conjugation of LC3 to ruptured lysosomes in the absence of ATG16L1. Control and ATG16L1-/- MEFs were incubated chloroquine (100 µM) for 2 hours as indicated, scale bar 5 um. Panel A. Immunostaining for LC3 (green) and V-ATPase (red). Panel B. distribution of LC3 (green and V-ATPase (red) assessed from line profiles. Panel C. Images of ATG16L1-/- MEFS where small puncta of LC3 co-localise with V-ATPase. Panel D. Co-localisation of LC3 and V-ATPase quantified by Imaris TM. Panel E. Atg16L1-/- MEFs immunostained for PI4P (green), galectin-3 (red) and ATG5 (far red purple). Panel F. Co-localisation of PI4P (green), galectin-3 (red) and ATG5 (purple) quantified by Imaris TM . Panel G. immunostaining for p62 (red) and galectin-3 or LAMP1 as indicated. Panel H. Distribution of p62 (red) and galectin-3 or LAMP1 (green) assessed from line profiles.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Conjugation Assay, Control, Incubation, Immunostaining

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Conjugation of LC3 to damaged lysosomes in the absence of ATG16L1 requires TECPR1. Panel A. Domain map of TECPR-1 showing site of insertion of stop codon by custom CRISPR gRNA. ATG16L1-/- MEFs and gene edited ATG16L1-/- MEFS expressing truncated TECPR1* were incubated in nutrient media with chloroquine (100 µM, panel B) or LLOMes (1 mM panel C). Cells were fixed and immunostained for LC3(green) and galectin-3 (red). Rendered images (v and vi, xi and xii) were used to count puncta. Higher magnification images of boxed ROI are shown in iv and x. The graphs in panels D and F compare numbers of LC3 puncta co-localised with galectin-3 counted from rendered images of 30 cells incubated with chloroquine (panel D) or LLOMEs (panel F), P-values were calculated using multiple t-test (****P < 0.0001). Panels E and G show assessment of LC3II by western blot for LC3 for chloroquine (panel E) or LLOMes (panel G). Scale bar 5 μm.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Conjugation Assay, CRISPR, Expressing, Incubation, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: TECPR-1 facilitates recruitment of LC3 and ATG5 to damaged lysosomes in the absence of ATG16L1. Control (WT), Atg16L1-/- and ATG16L1-/- MEFS expressing truncated TECPR1* were incubated for 2 hours in nutrient media containing chloroquine (100 µM). Panel A. Cells were fixed and immunostained for ATG5 (red) and galectin-3 (green). Panel B. Cells were fixed and immunostained for ATG5 (red) and LC3 (green). Panel C. Quantification and composition of puncta positive for ATG5 and galectin 3. Panel D. Quantification and composition of puncta positive for ATG5 and LC3. n ≥ 30 cells were quantified by Imaris, P-values were calculated using multiple t-test (****P < 0.0001). Scale bar 5 μm. Panel E. ATG16L1-/- MEFS expressing GFP-lysenin (green) and TECPR1-RFP (red) were incubated in nutrient media containing chloroquine (200 µM) for 30 minutes. Scale bar 5 μm.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Control, Expressing, Incubation

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Recruitment of galectin-3 and GFP-lysenin to lysosomes correlates with release of dextran. ATG16L1-/- MEFS expressing GFP-lysenin were cultured in Dextran-Red R (300 mg/ml) to label lysosomes and incubated with chloroquine (200 µM) for 10 or 30 minutes as indicated. Images show distribution of GFP-lysenin, Dextran-Red R and galectin-3 (far red). Asterisks indicate puncta positive for lysenin and galectin-3 that are negative for dextran.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Expressing, Cell Culture, Incubation

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Analysis of LC3 and galectin-3 distribution on damaged lysosomes by structured illumination microscopy: MEFs were incubated nutrient media containing chloroquine (100 µM) for 2 hours and then stained with DAPI (cyan) and immunostained for LC3 (yellow) and galectin3 (magenta) and imaged by 3D-SIM. Panel A: control MEFS. A swollen vacuole (I) recruits LC3 (I-i, maximum intensity projection-MIP) but not galactin3. To visualise this better, using Imaris, a 3D rendered surface was applied (I-ii) and then bisected (I-iii) showing the hollow vacuole interior. Analysis of small dense puncta (II) shows that LC3 (II-i, yellow MIP) and galectin3 (II-ii, magenta MIP) are recruited together as seen in the coloured overlay (II-iii). A 3D rendered surface (II-iv) bisected (II-v) shows the densely packed interior of this puncta. Scale bar = 1 µm. Panel B: ATG16L1-/- MEFs. Analysis of small dense puncta shows that LC3 (i, yellow MIP) and galectin3 (ii, magenta MIP) are recruited together as seen in the coloured overlay (iii). A 3D rendered surface (iv) bisected (v) shows the densely packed interior of the puncta. Scale bar = 1 µm.

Article Snippet: The majority (85%) of the PI4P/galectin-3 positive puncta also contained ATG5 ( ) suggesting recruitment of the ATG5-ATG12 complex required to conjugate LC3 to membranes.

Techniques: Microscopy, Incubation, Staining, Control