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. 2024 Jun 25;34(8):572–585. doi: 10.1038/s41422-024-00992-7

Fig. 2. SNAREs mediate the fusion of intraluminal vesicles with the migrasome membrane.

Fig. 2

a SIM images of L929 cells stably expressing GFP-VAMP2 and T4-mCherry. Scale bar, 200 nm. b TEM images of L929 cells stably expressing APEX2-GFP-VAMP2 and reacted with DAB. Scale bar, 200 nm. c L929 cells stably expressing GFP-VAMP2 and T4-BFP, treated with or without 10 μM BAPTA-AM for 10 h, were stained with VAMP3 antibody and then visualized. Scale bar, 20 μm. Lower panels, enlarged ROI. Scale bar, 2 µm. Right panels, statistical analysis of the number of GFP-VAMP2 and VAMP3 puncta in migrasomes per cell. Data are means ± SEM. n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***P < 0.001. d, e Immunostaining of endogenous syntaxin4 (d) or SNAP23 (e) in L929-T4-mCherry cells. Scale bar, 20 µm. The right panels show enlarged migrasomes. Scale bar, 1 μm. f L929 cells were cultured in FN-precoated dishes for 10 h, and were then treated with Sulfo-NHS-SS-Biotin to biotinylated membrane proteins. Biotin-labeled membrane proteins were subsequently isolated from cell bodies or migrasomes using NeutrAvidin Agarose, respectively. Equal amounts of total protein from cell bodies (C) or migrasomes (M) were then subjected to western blot analysis. Integrin α5 (Itg α5) and PIGK were used as migrasome markers in L929 cells. Representative densitometry analysis of western blot gray values is shown. Three independent experiments were conducted. g Immunostaining of endogenous VAMP2 in wild-type (WT) or SNAP23 KD L929-T4-mCherry cells. Scale bar, 20 µm. Right panels, enlarged ROI. Scale bar, 2 µm. Statistical analysis of the number of VAMP2 puncta in migrasomes per cell is shown as the means ± SEM. n > 100 cells from three independent experiments were analyzed using the two-tailed unpaired t-test. ***P < 0.001. h L929 cells stably expressing GFP-VAMP2 were subjected to time-lapse imaging. Time-lapse images were acquired at intervals of 30 s. Scale bar, 2 µm. i Confocal images of L929 cells stably expressing VAMP2-pHluorin and T4-mCherry. Scale bar, 20 µm. The right panel shows statistical analysis of the fluorescence intensity ratio. Each point represents the migrasome (all the migrasomes from an individual cell)/cell body fluorescence intensity ratio. n > 100 cells from three independent experiments.