Fig. 3: Artificially increasing the cytoplasmic calcium ion concentration upregulates cytoplasmic fluidity and exaggerates membrane blebbing.

a Membrane blebbing of DLD1 cells expressing GCaMP6s and Lifeact-RFP treated with the calcium ionophore 4-bromo-A23187 (10 µM). Arrowheads show blebs fusing. Result shown is representative of three independent experiments. See also Supplementary Movie 4. b–e QDs dynamics in the cell body of ionophore-treated cells. b Centered trajectory maps showing the sum of the frame-to-frame distances over 600 msec (30 frames at 50 Hz; right). Red, yellow, and blue trajectories indicate diffusion velocities >3.3 μm/s, >1.6 μm/s and <3.3 μm/s, and <1.6 μm/s, respectively. c Cumulative distances of QDs motion. d Mean square displacement (MSD) analysis of five representative trajectories per condition. e Diffusion coefficient (D, μm²/msec) was calculated from the slope of the fitted regression line derived by MSD analysis of (c). N = 110 particles from 11 blebs from 10 independent cells per condition. c, e The number (f, N = 20 cells), area (g, N = 20 blebs) and retraction velocity (h, N = 10 blebs) of membrane blebs in vehicle-treated (control) and ionophore-treated (4-bromo-A23187) DLD1 cells over 10 min from three independent experiments. i Membrane blebbing of DLD1 cells expressing GCaMP6s and Lifeact-RFP treated with the SERCA inhibitor Thapsigargin (1 µM). Arrowhead shows increased GCaMP6s signal intensity in the bleb. Result shown is representative of three independent experiments. a and i Indicated times are relative to drug treatment. Scale bar, 10 μm. c, e-h Individual data points are plotted with the means ± SD. ****P < 0.0001 (Two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.