Journal: The EMBO Journal

Article Title: Ca²⁺ leakage is a conserved signal for non-canonical ATG8/LC3 lipidation and membrane repair

doi: 10.1038/s44318-026-00741-z

Figure Lengend Snippet: ( A ) STED super-resolution imaging of THP-1 macrophages stably expressing Halo-LC3, labelled with Halo dye, under 10 min LLOMe treatment. White arrowheads indicate the LC3-TVS underwent dynamic fusion. ( B – D ) Time-lapse images showing membrane remodelling of LC3-positive vesicles induced by lysosomal damage. THP-1 macrophages stably expressing GFP-LC3B were under normal condition or treated with LLOMe. Cells were imaged using VT-iSIM super-resolution microscopy at 15-second intervals. Images are z-maximum projections covering entire cells (0.5 µm per stack, 5–7 µm total). Time 0 indicates the frame acquired at the onset of LLOMe treatment, following a 10-min stabilization period. LC3-positive structures under untreated conditions ( B ). Representative regions of interest (ROIs) showing dynamic LC3-positive vesicles from three independent experiments (related to Movie ) under LLOMe treatment ( C , D ). Yellow, pink, and white arrows indicate distinct docking and fusion events observed over time. ( E ) Quantification of percentage of single-, double-, and multiple-membrane RFP⁺GFP⁺ LC3-positive vesicles relative to the total number of RFP⁺GFP⁺ LC3-positive vesicles determined by CLEM ( n = 3 cells, 167 vesicles were analysed), related to Fig. and F–H. ( F ) CLEM analysis showing RFP-LC3 and GFP-LC3 double-positive multimembrane structures following LLOMe treatment (related to Fig. and Movie ). Yellow arrowhead indicates the LC3-positive multimembranes. ( G , H ) CLEM analysis reveals complex membrane structures positive for both RFP-LC3 and GFP-LC3 after LLOMe treatment. Yellow arrowheads indicate the LC3-positive multimembranes. Pink arrowheads indicate the LC3-positive single membrane. Scale bars: ( A – D ), 1 μm; ( F – H ), 1 μm (main images), 200 nm (zoomed-in areas).

Article Snippet: Cells were imaged using a VT-iSIM superresolution imaging system (Visitech International) with an Olympus IX83 microscope, or a Leica STELLARIS 5 confocal microscope equipped with an environmental chamber (Okolab) maintained at 37 °C with 5% CO2.

Techniques: Imaging, Stable Transfection, Expressing, Membrane, Super-Resolution Microscopy