Fig 2: SCV-IAM Fusion Involves an STX4-Containing SNARE Complex(A) Quantification of the infected cells containing hyper-replicative salmonellae by high-throughput microscopy of HeLa cells transfected with an siRNA pool against STX3, STX12, or STX4. Graph represents data from three independent experiments, normalized with respective values of scramble. p values were obtained after t test. Error-bars: ±SEM. See also Figure S3 for knockdown efficiencies.(B) Time-lapse microscopy of HeLa cells transfected with GFP-tagged STX4 (in green) and infected with fluorescent Salmonella (in red). Scale bars: 10 μm. Orange arrowheads designate large SCVs, pink arrowheads designate IAMs, and white arrowheads designate tight SCVs. See also Video S4A and Figure S1G.(C) Time-lapse microscopy of IAM and SCV formations and interactions using HeLa cells transfected with GFP-2xFYVE (in green), and with an siRNA pool against STX4. Cells were infected with fluorescent Salmonella (in red). Scale bars: 10 μm. See Video S3D.(D) Quantification of the percentage of SCVs fusing with IAM upon knockdown of STX4. The graph represents data from three independent experiments. p values were obtained after t test. Error-bars: ±SEM.(E) Quantification of the percentage of SCVs fusing with IAM upon transfection of HeLa and Caco-2 cells with GFP-STX4 or GFP-STX4ΔSNARE. The graph represents data from three independent experiments. p values were obtained after t test. Error-bars: ±SEM.(F) Model of the SCV size control determining Salmonella intracellular lifestyle mechanism. Salmonella effectors induce ruffles and IAM formation (blue parts). SNAREs are recruited to the IAMs. SNAP25 and STX4 participate in the fusion of the IAMs with the SCV, leading to the enlargement of the bacterial vacuole. Increase in the SCV size prevents its rupture and favors vacuolar lifestyle. The bacterial effector SopB triggers the formation of SVATs, resulting in the shrinking of the vacuole (yellow parts). The decrease in the SCV size promotes its rupture and favors a cytosolic lifestyle.

Fig 3: Rescue of brush-border defect in Munc18-2 KO organoids by lentiviral complementation with the WT MUNC18-2 protein but not the P477L patient variant. (A and B) Characterization of apical protein trafficking: (A) alkaline phosphatase activity and (B) Cd10 immunostaining on paraffin sections. White arrows show subapical accumulation of brush-border components. Magnified images (boxes) are shown in the bottom rows. (C) Confocal analysis of Stx3 staining shows rescue of the apical location after expression of the WT MUNC18-2 protein. Diffuse cytoplasmic localization in the Munc18-2 KO (white arrow) after expression of the patient variant. (D) Confocal z-projection of F-actin staining shows a planar view onto the brush border in crypts. White arrowheads mark a disrupted brush border. Magnified images (boxes) are shown in the bottom row. (E) TEM images show restored ultrastructural phenotype by expression of the WT human protein. No MVIs were observed under expansion conditions. (F–H) Quantification of microvilli (MV) (F) length, (G) width, and (H) density. Mean values ±SD in n = 70 ±7, 40 ±2, 40 ±2, 70 ±7, 60 ±4, and 60 ±4 MVs each (with number of independent organoids in brackets). P values (t test) compared with the untransduced WT are shown. Scale bars: (A and B) 20 μm, (C and D) 10 μm, and (E) 2 μm. All staining was confirmed in at least 2 independent experiments.

Fig 4: Confocal analysis of brush border and trafficking defects in Munc18-2 KO organoids. (A) Defective deposition of brush-border F-actin as shown by phalloidin staining (red). Zonula occludens-1 (ZO-1) co-staining (green) shows that tight junction–associated F-actin is not affected. Single confocal sections (with DAPI-stained nuclei in white). The magnified z-projected images (bottom) show a planar view onto the brush border. WT organoids show a continuous brush border (asterisks) that is interrupted in the Munc18-2 KO (white arrowheads). (B) Confocal analysis of F-actin (red) and Stx3 (green). Stx3 shows a diffuse subapical localization in the Munc18-2 KO (white arrow). Bottom: Magnified z-projected images. Scale bars: 10 μm. Staining was confirmed in 3 independent experiments.

Fig 5: Depletion of STX2, STX3, STX18, VAMP8 and SNAP29 reduce the release of sEVs. sEVs were isolated by sequential centrifugation and their concentration measured by NTA after depletion of (A) STX2, (B) STX3, (C) STX18, (D) VAMP8 and (E) SNAP29. Knockdown efficiency was measured by immunoblotting 3 days after transfection with siRNA (25 nM) against (F) STX2, (G) STX3, (H) STX18, (I) VAMP8 and (J) SNAP29. A–J Data shows mean ± SEM from 3–4 independent experiments. *P < 0.05 versus non-targeting control (non). K sEVs were isolated from MCF-7, MDA-MB-231 and Caco-2 cells by sequential centrifugation after depletion of SNAP29 by siRNA (25 nM). For MDA-MB-231 and Caco-2 cells, vesicles were collected for 24 h, starting 2 days after transfection. For MCF-7, vesicles were collected for 42–44 h, starting 1 day after transfection. Particles in the 100,000×g pellet were measured by NTA. Knockdown efficiency 3 days after transfection was measured by immunoblotting, using actin as control. Experiments were performed twice (Caco-2) or 3 times (MCF-7, MDA-MB-231) in duplicate