IFN-γ induces rapid granule release that is caused by PC extrusion. (A) Time-lapse analysis of PC degranulation. Granules were labeled with the fluorescent Zn2+ chelator ZP-1, followed by live microscopy (still images of Videos 1–3). Untreated controls (top row) or cultures stimulated with 5 ng/ml (middle row) or 20 ng/ml IFN-γ (bottom row). Time after addition of IFN-γ is indicated on top (hours:min). Blue arrowheads show unaffected PCs; white arrowheads mark PCs that release their granules during the next time interval, as labeled by asterisks. Note that the fluorescent signal decreases due to bleaching. Experiment was repeated twice with at least 5 organoids per condition. Bars, 50 µm. (B) Transmission electron microscopy analysis of control organoids (left) and cultures stimulated with IFN-γ for 6 h (middle) or 12 h (right). Magnified regions (red boxes) are shown in the bottom row. Blue arrows mark PC granules; residual small granules are marked with a white arrow. The label V shows a large apical vacuole; P marks extruded cell fragments with densely packed granules; cell membranes are marked with dashed lines. Representative images from 1 experiment with n > 10 organoids analyzed per condition. Bars, 5 µm. (C) Cell-fate analysis of PCs after degranulation using live reporters (still images of Video 4). BAC transgenic organoids expressing Lyz2-RFP in PC granules (red signal) and lentiviral Histone 2B (H2B)-Dendra in nuclei (green signal). Time after addition of 1 ng/ml IFN-γ is indicated on top (hours:min). White arrowheads mark individual PCs that extrude both granules and cell nucleus during the next time interval (asterisks). After 24 h the epithelium is devoid of Lyz2-RFP and only few labeled nuclei remain (black arrowheads). Note that long-lived PCs accumulate H2B-Dendra. Results were reproduced in two independent experiments. (D) Quantification of PC fate from time-lapse data in C. In untreated control cultures (top) and after treatment (1 ng/ml IFN-γ; bottom), the status of >100 PCs each was tracked for 36 h. PCs were scored as unaffected (red bars), and then degranulated with nucleus present (orange bars) or extruded (gray bars).
