Fig. 2: βII-spectrin and actin dynamics during spreading.

a Schematic representation of the different phases occurring during fibroblasts spreading on fibronectin-coated surfaces. The morphological changes in terms of cell shape, actin cytoskeleton (magenta), focal adhesion formation (blue), and PM organization (black) are drawn. b Cells untreated and treated with blebbistatin are visualized by live TIRFM, and representative images at relevant time points are shown (green: GFP-βII-spectrin, magenta: RFP-actin, scale bar: 20 µm, n = 9 cells (untreated) and n = 5 cells (blebbistatin) from independent experiments were analyzed). Peculiar mechanisms are highlighted by white dashed boxes and zoomed-in panels 1–3. In 1 is shown a typical noncontractile phase (P1), while the contractile phase (P2) is shown in 2; in the absence of myosin-II-dependent contractility (blebbistatin) cell spreading is stalled. In 3, coalescent actin nodes contribute to the maturation of the actin cytoskeleton, while blebbistatin treatment impairs these dynamic processes. c Schematic representation of the radial segmentation of the cell edge (red, 3.2-µm thickness) and cell body (blue) performed during the time lapse. d–f Radial kymograph analysis of cell-edge behavior of MEFs untreated (d) and treated with blebbistatin (f) is presented. The upper kymographs represent the integrated intensities of the two proteins (1–3) black arrowheads indicate the specific frames highlighted in (b), while the bottom kymographs display the edge speed related to the cell centroid. In (e) and (g), fluctuations from the mean signal intensities are plotted (actin: magenta and βII spectrin: green) in function of speed (untreated: n = 9 cells, blebbistatin: n = 5 cells, data are presented as mean values ± SEM).