Fig. 1: Highly fluid cytoplasm is formed in the expanding membrane blebs.

a, b Membrane blebbing of DLD1 cells expressing GFP-PLCδ-PH. Bin widths of the histogram (b) are 0.02 μm/s and the data presented are the means ± the standard deviation (SD) of measurements from 10 independent cells over three independent experiments. c–f QDs dynamics during bleb expansion and retraction. c Example tracks superimposed in situ (left) and 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. See also Supplementary Movie 1. d Cumulative distances of QDs motion. e Mean square displacement (MSD) analysis of five representative trajectories per condition. f Diffusion coefficient (D, μm²/msec) was calculated from the slope of the fitted regression line derived by MSD analysis of (d). QDs dynamics in the cell body of vehicle- (control) or Latrunculin B (LatB)-treated cells are also shown. N = 100 particles from 10 blebs from 10 independent cells per condition. Individual data points are plotted with the means ± SD in d and f. ****P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). g Membrane blebbing of DLD1 cells expressing GFP-Mena. Black and white arrowheads show expanding and retracting blebs, respectively. Result shown is representative of five independent experiments. See also Supplementary Movie 2. h–k Kymograph analysis of GFP-Mena in bleb cytoplasm. h Representative kymographs of NucBlue (blue) and GFP-Mena (green) from three independent experiments. Bleb extension is shown on the vertical axis, and time is shown on the horizontal axis. i Schematic of the analysis in (j, k). Fluorescence intensities in “bleb”　(yellow) and “cell body” (white) cytoplasm were quantified and expressed as ratios. j Representative kymographs of GFP-Mena (left) and the control cytoplasm protein, RFP (right). k Fluorescence intensities of GFP-Mena and RFP were quantified as in i. Data presented are means ± SD based on the values from five independent experiments. a and g Indicated times are relative to the first image. Scale bar, 2 μm. Source data are provided as a Source Data file.