Movie 2.

Ca²⁺ flux following control aerolysin challenge. HeLa cells were labeled with Fluo-4-AM (green) for 30 min in DMEM. The media was replaced with imaging buffer supplemented either with 2 mM Ca²⁺ or 2 mM EGTA, including TO-PRO-3 (blue). Cells were imaged by confocal microscopy at 1-3 frames/sec for ∼45 min at 37°C. Sublytic (A) aerolysin (62 HU/mL) in presence of 2 mM EGTA or (B) mass equivalent to aerolysin of mutant AeroY221G, or (C) PBS were added 5 min after imaging started. Triton-X-100 was added just prior to the end of imaging as a positive control for cell permeabilization. Images were bleachcorrected by histogram matching. Time displays min post toxin challenge. Scale bar = 10 μm.