Fig. 3.

Membrane blebs induced by P2X7R activation contain TG2. (A) P2X7R signaling induces rapid membrane blebbing. Fluo-4-AM-loaded P2X7R cells were stimulated with BzATP while acquiring fluorescence and phase-contrast images by real-time microscopy to visualize morphological changes and Ca²⁺ signaling simultaneously (top). Membrane blebs are indicated by arrows. ATP stimulation of parental cells induces oscillating Ca²⁺ signals but no overt morphological changes (bottom). Scale bar: 25 µm. (B,C) TG2 redistributes into membrane blebs. To confirm export of tagged TG2, TG2- (wild-type, WT) or TG2–GFP-expressing P2X7R cells were stimulated with 100 µM BzATP for 10 min, chased for 30 min in agonist-free medium, followed by analysis of conditioned media and cell extracts for TG2 by western blotting (B). To localize GFP-tagged TG2 during BzATP stimulation, real-time confocal microscopy was employed. Genesis of a membrane bleb is depicted (arrows), with an optical section of GFP fluorescence overlaid onto phase-contrast images to correlate morphological changes with changes in TG2 distribution (C). Scale bar: 10 µm.