Fig. 6: WIPI1-, WIPI2- and LC3-decorated autophagic membranes migrate through tunnelling nanotubes (TNTs) to neighbouring cells with limited autophagic activity.

a Scheme displaying a TNT connecting two cells. Created with BioRender.com. b U2OS cells stably expressing GFP-WIPI1 form TNTs. U2OS GFP-WIPI1 cells were costained with phalloidin-AF546 to visualize F-actin and with WGA-AF647 to visualize the plasma membrane. Merged channels (left panels) are shown as well as split channels displaying F-actin (middle panels) and WGA only (right panels). Boxes (upper panels) indicate magnified sections (lower panels), and arrows point at a TNT connecting two cells. Scale bar: 20 µm. c Starved (24 h) U2OS cells stably expressing GFP-WIPI1 were stained with phalloidin-AF546 and DAPI and imaged by confocal LSM. A video was generated from the 3D reconstruction of confocal z-stacks (Supplementary Video 2), and a still image displaying GFP-WIPI1 puncta within a TNT is presented (left panel). Likewise, a video was generated from the 3D reconstruction of confocal z-stacks (Supplementary Video 3), and a still image revealing the presence of RFP-GFP-LC3 puncta within a TNT is shown (right panel). d U2OS GFP-WIPI1 cells were fed for 24 h with high (10%, fed) or low (2.5%, low serum) serum or starved as indicated. GFP-WIPI1 puncta-positive cells were counted (left panel), as well as the numbers of TNTs per 100 cells (TNT index, middle panel) and the numbers (%) of TNTs containing GFP-WIPI1 puncta (right panel). One-way ANOVA with Holm-Sidak post-hoc test, up to 952 cells from n = 4 for each condition. e U2OS cells were transfected with plasmids encoding GFP or GFP-WIPI1 in the concentrations of 0.1 μg or 0.25 μg for 48 h, then re-seeded onto coverslips and cultured for 24 h in fed conditions. After fixation, cells were stained with DAPI, and F-actin stained with phalloidin-AF546 and tubulin with anti-αTubulin/AF647. The TNT index was calculated from up to 1057 cells per condition (n = 4). Two-way ANOVA, Tukey’s post-hoc test. Scale bar: 20 μm. f U2OS Cas9 control and U2OS WIPI1 KO were seeded onto coverslips and cultured overnight (16 h) and then fed for 24 h with high (10%, fed) or low (2.5%, low serum) serum or starved as indicated. Cells were stained with DAPI, WGA-AF488, phalloidin-AF546, anti-αTubulin/AF647 and the TNT index determined from up to 1440 cells per condition (n = 4, two-way ANOVA, Tukey’s post-hoc test). Representative image panels are shown in Supplementary Fig. 8c, d. g Airyscan superresolution live time-series microscopy (~30 min., Supplementary Video 4) of U2OS cells stably expressing GFP-WIPI1 was conducted, and merged GFP and brightfield channels are displayed (left image). Using the plug-in MTrackJ in Fiji, GFP-WIPI1 puncta were tracked within a TNT connecting two cells, and GFP-WIPI1 tracks of different puncta are indicated in distinct colours whereby the movement direction is indicated with colour-corresponding arrows (Supplementary Video 5). Scale bar: 20 μm. h U2OS GFP-WIPI1, i U2OS GFP-WIPI2 or j U2OS GFP-LC3 donor cells were cocultured (ratio 1:1) in fed conditions for 24 h with recipient U2OS cells either expressing wild-type ATG16L1 (WT) or not (U2OS ATG16L1 KO), along with ATG16L1 KO cells reconstituted for WT ATG16L1 (ATG16L1 KO + ATG16L1 WT). GFP puncta fluorescence from GFP-WIPI1, GFP-WIPI2 or GFP-LC3 within recipient MLS-EGFP-mCherry ATG16L1 U2OS cell lines bearing labelled mitochondria was clearly distinguishable from mitochondria, which appeared as orange fluorescence in recipient cells and which lacked GFP only puncta. Based on this, the numbers of recipient cells harbouring GFP-WIPI1 (h), GFP-WIPI2B (i) or GFP-LC3 (j) puncta were counted (one-way ANOVA with Holm-Sidak post-hoc test, up to 650 cells from n = 3 for each condition). k Representative merged images are displayed from (h) to (j), and green fluorescent puncta (arrows) in recipient cells (white asterisks), marked by mitochondrial staining (orange), are indicated (arrows). White boxes display zoomed-in sections. All cells, donor and recipient cells were stained with phalloidin-AF647 (violet) and DAPI (blue). All recipient cell lines were able to take up autophagic membranes decorated with GFP-WIPI1, GFP-WIPI2B or GFP-LC3, derived from respective donor cell lines in coculture settings. Extended image presentation in Supplementary Fig. 8e. l, m Donor U2OS cells stably expressing GFP-LC3 were transfected with different concentrations (0.2, 0.4 or 0.6 μg) of either control (9E10) or WIPI1 (9E10-WIPI1) expression plasmid for 24 h and then co-cultured with recipient U2OS cells stably expressing NLS-Scarlet for an additional 24 h. Subsequently, cells were stained with phalloidin-AF647 and DAPI. Representative images are shown (left panels). The percentage of recipient cells containing GFP-LC3 puncta derived from donor cells was calculated by manual counting (right panels, up to 700 cells per condition, two-way ANOVA with Tukey’s post-hoc test, mean ± SD, n = 3). Scale bar: 20 µm. Supplementary material is available (Supplementary Fig. 8; Supplementary Videos 2–5; Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.