Figure 5. Localization of Galvanin coincides with reduced local membrane speed and front-rear polarization.

A. Example analysis showing the temporal movement, Galvanin-GFP fluorescence intensity, and cellular protrusion/retraction activity of a single cell. Imaging was performed every 5 seconds over 15 minutes, with cells exposed to an electric field (300 mV/mm) between 5 minutes and 10 minutes. One-minute intervals are overlaid. See also Supplemental Movie 2. B. Kymograph shows the normalized Galvanin-GFP intensity, quantified at the cell periphery (in a band of 2 μm thickness) and averaged across 41 cells. Cell contours are defined with 360 positions, with position 0 corresponding to the right-most position relative to the cell centroid. C. Kymograph quantifies membrane speed along the cell periphery, by comparing cell shape changes between 5 second intervals and correcting against bulk cell translocation. Values represent an average across the 41 cells considered in part B. D. Averaged fluorescence at the anodal side (positions +120 to +240) and cathodal side (positions −60 to +60) during the first 10 minutes. E. Averaged membrane speed at the anodal side (positions +120 to +240) and cathodal side (positions −60 to +60) during the first 10 minutes. Dashed lines in D and E indicate when the electric field was turned on. F. Cross-correlation analyses comparing the averaged fluorescence and membrane speeds (parts D-E) at the anodal and cathodal sides of the cell. Dashed lines indicate the average minimum lag (0.1 minutes for the anode side and 0 minutes for the cathode side). The shaded region represents correlation values that are below a 99% confidence interval (see Methods). G. Cross-correlation analysis was performed using the Galvanin-GFP fluorescence and membrane velocities averaged across all 46 individual cells. Histograms show the lag corresponding to the minimum correlation value. Dashed lines near zero indicate the average lag value (anode side: 0.4 minutes; cathode side: 0.2 minutes).