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. Author manuscript; available in PMC: 2024 Sep 26.
Published in final edited form as: Nature. 2024 Aug 21;632(8027):1110–1117. doi: 10.1038/s41586-024-07835-w

Fig. 3. VDIMs are delivered to lysosomes for degradation.

Fig. 3

a, Localization of VDIMs in LAMP1+ lysosomes. Right: 3D reconstruction. Arrowheads indicate the VDIMs. b, Number LAMP1positive VDIMs in experiments as in (a) (n= 36 cells, 3 experiments). c, Schematic illustrating the loss of GFP fluorescence from lysosome-localized VDIMs in cells expressing MCU-GFP-mCherry. d, VDIMs in cells expressing MCU-GFP-mCherry. Arrowheads indicate the VDIMs that retain the mCherry fluorescence, while GFP fluorescence is quenched. Rainbow pseudo-colored GFP channel also shown. Right: Pixel intensity plot for dashed line. Arrow indicates the vesicle. e, Number of mCherry+/GFP- VDIMs in experiments as in (d) (n= 330 vesicles, 26 cells, 3 experiments). f, CLEM analysis of cells expressing LAMP1-GFP (green) and mito-BFP (cyan), labeled with Mitotracker (magenta). Scale bars, 10μm. Bottom: Higher magnifications of single-z plane from indicated regions. (i-ii) Lysosome localized VDIMs and presence of membrane whorls. (iii) Lysosome devoid of mitotracker-labeled membrane (arrowheads). Scale bars, 200nm. g, Live-cell imaging sequence showing VDIM formation (arrowheads) in cells expressing mito-BFP (blue). Lysosomes were labeled with dextran (green) and mitochondria with mitotracker (magenta). h, Mean intensity of mitotracker and mito-BFP in the lysosome over time from (g). i, Top: Representative data from two experiments showing the percentage of VDIMs forming at mitochondrial midpoint or the periphery in experiments as in (g) (n= 52 events, 10 cells). Bottom: Schematic illustrating that VDIM formation does not occur at preferential sites along the length of the mitochondria. j,k, Number of VDIMs in cells with impaired lysosomes. Cells were treated with (j) bafilomycin A1 (BafA1) (n= 60 cells, 3 experiments) or (k) chloroquine (CQ) (n= 60 cells, 3 experiments). Data shown are mean±SEM shown as large circles and individual data points from corresponding experiments shown in the same colors. Statistical analysis was performed using two-tailed Student’s unpaired t-test. P values calculated are shown. Unless stated otherwise, scale bars: 3μm.