Figure 2. Temporal reorganization of actin filaments and focal adhesions in cells cultured on circular micropatterned islands.

Single representative (black-and-white images, top row) and average (heat maps, bottom row, 25 cells; scale from 0 to 250, arbitrary intensity units) images of (A) actin filament (from fluorescent phalloidin staining) and (B) focal adhesion (vinculin, FA) distributions in B16F1 cells cultured on fibronectin-coated, circular microislands (diameter, 40 μm). Scale bar = 20 μm. (C) Scheme of circular cell. Polarization index (P) for f-actin is computed as distance (d) between the cell’s geometric center (GC) and center of mass (CoM) divided by cell’s radius. Front is defined as f-actin/FA rich region; back is the other end. (D) Quantification of the peripheral intensity levels (within 5-μm-thick peripheral region delineated in the 2h average image in A by the white annular rings). Red = actin; green = FA (vinculin). (E,F) Quantification of polarized actin and focal adhesion distributions (using images from A and B) by means of the polarization index P and Front/Back ratio F/B, respectively (cf. Experimental Procedures for details). Top and bottom of the box show 75^th and 25^th quartiles, respectively; whiskers indicate minimum and maximum values excluding outliers; circles show outliers, middle line is the median and cross is the mean. An asterisk (*) indicates statistically significant difference when compared to 2h-timepoint; ‘Pound’ symbol (#) indicates significant difference when compared with 3h-timepoint; ‘And’ symbol (&) indicated significant difference when compared with 4h-timepoint (p<0.05). Actin bundles and FAs in circular cells progressively and concurrently undergo reorganization from polarized distribution resembling moving cells to uniform peripheral organization of actin edge-bundles and focal adhesions.