Fig 2: ANXA6 depletion blocks ionomycin-mediated exosome secretion and stalls multivesicular bodies (MVBs) at the cell periphery.(A) Immunoblot analysis of ANXA6 and GAPDH expression from GFP, ANXA6-I, and ANXA6-II shRNA CD63-nanoluciferase (Nluc) cells is shown. (B) Immunoblot analysis of CPNE3 and GAPDH expression from GFP, CPNE3-I, and CPNE3-II shRNA CD63-Nluc cells are shown. (C) Normalized exosome production derived from GFP, ANXA6-I, ANXA6-II, CPNE3-I, and CPNE3-II shRNA CD63-Nluc cells grown in conditioned medium for 24 hr are shown. (D) Normalized exosome production derived from GFP, ANXA6-I, ANXA6-II, CPNE3-I, and CPNE3-II shRNA CD63-Nluc cells treated with 5 µM ionomycin for 30 min are shown. Data plotted represent the means from three independent experiments, and error bars represent SDs. Statistical significance was performed using an ANOVA (*p<0.05, ****p<0.0001, and ns = not significant). (E) CD63 immunofluorescence and phalloidin staining of GFP or ANXA6-I shRNA CD63-Nluc cells after 30 min of DMSO or 5 µM ionomycin treatment are shown. White arrows indicate peripheral CD63 puncta. Scale bars: 10 µm. Figure 4—source data 1.Uncropped immunoblot images corresponding to Figure 4.

Fig 3: Schematic depicting the current model of Ca2+- and ANXA6-dependent exosome secretion.(A) Upon physiological mechanical stress or bacterial infection, plasma membrane lesions form. This results in the flow of Ca2+ from the extracellular space into the cytoplasm. (B) This influx of Ca2+ mediates the recruitment of ANXA6 to multivesicular bodies (MVBs), which are then transported on microtubules to a plasma membrane lesion. (C) The MVBs then dock at the plasma membrane (with ANXA6 serving as a putative membrane tether) and undergo fusion, resulting in plasma membrane repair and exosome secretion.

Fig 4: Biochemical reconstitution of Ca2+- and ANXA6-dependent exosome secretion in permeabilized cells.(A) Schematic illustrating the permeabilized cell exosome secretion assay. (B) Permeabilized cell exosome secretion reactions with or without SLO, ATP regeneration system (ATPr)/GTP, rat liver cytosol, and Ca2+ are indicated. (C) Permeabilized exosome secretion assays with or without HCT116 WT cytosol and Ca2+ are shown. (D) ATP requirements for the permeabilized exosome secretion assay. Reactions with or without nucleotide-depleted rat liver cytosol, Ca2+, and ATPr/GTP are indicated. (E) Requirement of ANXA6 in the permeabilized exosome secretion assay. Reactions with or without HCT116 WT cytosol, an anti-GFP rabbit IgG antibody, and an anti-ANXA6 rabbit IgG antibody are depicted. Data plotted represent the means from three independent experiments, and error bars represent SDs. Statistical significance was performed using an ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns = not significant).