Fig. 7: Coordination between Ca²⁺ signals and F-actin dynamics.

AX2 cells co-expressing GCaMP6s and Lifeact14-mScarletI were cultured for 6 days under shaking conditions with 10 μM blebbistatin. After starvation with DB, imaging was performed using confocal microscopy (40× objective; 5 s intervals). a Representative fluorescence time-lapse images of GCaMP6s in a giant cell. Time after imaging onset is shown in the top-right corner. Scale bars: 20 μm. b Kymograph of Ca²⁺ signal intensity along the 60 μm region within the white rectangle indicated in (a). Vertical axis: time [sec]. c Time-lapse images of actin waves (Lifeact14-mScarletI channel) in the same cell. Scale bars: 20 μm. d Kymograph of actin wave dynamics within the same 60 μm region. Vertical axis: time [sec]. e Merged fluorescence images showing Ca²⁺ signals (GCaMP6s, green) and actin waves (Lifeact14-mScarletI, red). Scale bars: 20 μm. f Merged kymograph of Ca²⁺ signals and actin waves. g Cell outline overlaid with centroid trajectory. An arrow indicates the direction of cell movement. h Linear approximation of the centroid trajectory. i Analysis regions for the front and rear were defined based on linear fit of the centroid trajectory. The origin was placed at the centroid, and the front and rear were defined at ±25 μm along the fit line (slope = −0.04). Solid and dashed circles indicate the front and rear regions, respectively. Region diameter: 11 μm. j Time series of normalized Ca²⁺ signal intensities in the front and rear regions. Horizontal axis: time [sec]; vertical axis: normalized Ca²⁺ intensity. k Time series of normalized actin wave intensities in the front and rear regions. Horizontal axis: time [sec]; vertical axis: normalized intensity. l Correlation between Ca²⁺ signaling and actin dynamics. The traces in (j) and (k) are superimposed.