Supplementary Figure 3: Quantification of cortex/cytoplasm ratio and cortical full width half maximum (FWHM) of labeled F-actin and myosin II in live cells.

(a) Confocal images of a MYH9-GFP Lifeact-mCherry expressing HeLa cell were analyzed with a self-written Igor macro (Igor Pro). The center of the cell (purple circle) was determined by fitting a circle to the edge of the MYH9-GFP signal and 60 points (yellow markers) were initially positioned 6° apart along the circle. Next, the 60 points were automatically repositioned to be at the cell periphery. (b) Fluorescence intensity profile (cyan) taken from the average signal along a 0.6 μm thick radial line (yellow) through a representative peripheral point (yellow marker). (c) The 60 fluorescence intensity profiles (thin traces) through the peripheral yellow markers were aligned at the cell periphery (0 μm) and superimposed. The thick green trace represents the average of all 60 fluorescent intensity profiles taken from one cell. (d) Determination of cortex/cytoplasm ratio (R) of the whole cell from the average fluorescence intensity profile. Within each average profile, the peak intensity (p) between −2 and 0.5 μm from the cell edge and the mean intensity (m) between −2 and −1 μm from the cell edge were extracted. The cortex/cytoplasm ratio (R) for the fluorescence image was determined as the ratio of p and m. (e) Determining the cortical FWHM of the average fluorescence intensity profile shown in (d). The half peak height (I) is the intensity halfway between p and m. Cortical FWHM is the width (in μm) of the signal at I. (f) The yellow markers from the MYH9-GFP image were copied into the Lifeact-mCherry fluorescence image. (g) Fluorescence intensity profile (cyan) taken from the average signal along a 0.6 μm thick radial line (yellow) through a representative peripheral point (yellow marker). (h) The 60 fluorescence intensity profiles (thin traces) through the peripheral yellow markers were superimposed and aligned at the cell periphery (0 μm). The thick green trace represents the average of all intensity profiles. (i) Determination of cortex/cytoplasm ratio (R) of the whole cell from average fluorescence intensity profile. Within each average profile, the peak intensity (p) between −2 and 0.5 μm from the cell edge and the mean intensity (m) between −2 and −1 μm from the cell edge were extracted. The cortex/cytoplasm ratio (R) for the fluorescence image was determined as the ratio of p and m. (j) Determining the cortical FWHM from the average fluorescence intensity profiles of a whole cell. The half peak height (I) is the intensity halfway between p and m. Cortical FWHM is the width (in μm) of the signal at I. (k) Superimpositions of the average F-actin (red) and myosin II (green) fluorescence intensity profiles, from c and h, of a mitotic cell. (i) Average intensity profile quantifying the radial distribution of F-actin (red) and myosin II (green) in a mitotic cell treated with 20 μg ml⁻¹ C3-toxin.