Figure 8.

Lentiviral expression of LifeAct reporter in organoids to monitor MVIs. (A) Confocal images of LifeAct reporter expression and phalloidin staining. LifeAct-mCherry (red signal, top) reporter transgenic WT and Munc18-2 KO organoids were fixed and stained by phalloidin-Alexa488 (green, middle). Nuclei were stained by DAPI. Organoids were kept in VPA-containing medium (expansion condition) or for 3 days in enterocyte differentiation medium (VPA + IWP-2) to induce MVIs. Both lumen-containing and noncontaining F-actin⁺ structures were co-stained in differentiated Munc18-2 KO organoids (yellow signal in merged image, bottom). Note that treatment with VPA alone reduced the presence of secretory cells (Paneth and goblet cells) in which spontaneous aggregation of the LifeAct reporter otherwise caused background signals (not shown). Scale bars: 50 μm. (B) High-resolution confocal analysis of LifeAct reporter expression and phalloidin staining. Organoids after 3 days in enterocyte differentiation medium. On the white lines (4 μm), scan of signal intensity was performed on apical microvilli (MV) in WT and on MVIs in Munc18-2 KO organoids; basal membrane in a WT organoid was used as a control, microvillus-free membrane.