Fig. 3: Fibroblasts spreading between adhesive lines highlight βII-spectrin and actin complementarity.

a Representative images of MEFs immunostained for GFP-βII-spectrin (green), RFP-actin (magenta), and fibronectin (red), visualized by simultaneous live TIRF and EPI-fluorescence microscopy (scale bar: 20 µm). Time lapses lasted for 30 min; four representative frames are presented to describe the progression of the spreading. b Zooms related to the white dashed box presented in A across parallel fibronectin-coated lines and the passivated nonadhesive surface (in black). Normalized intensity profiles for both channels are presented to highlight the non-colocalization between βII-spectrin and actin during lamellipodia protrusion (first two frames) and subsequent retraction characterized by βII-spectrin-rich negative curvature. c Representative images of micropattern-induced polarization during spreading (scale bar: 20 µm). The zooms related to the white dashed box (2) and the morphological changes during the time lapse are presented in (d). Kymograph across the cell over the nonadhesive surface is drawn (yellow dashed box); white arrowheads highlight the beginning of βII-spectrin accumulation in the curvature regions. e Representative image of late-spreading phases, where the cell presents actin bridges over the nonadhesive substrate (GFP-βII-spectrin: green, RFP-actin: magenta, and fibronectin: red, visualized by live TIRFM, scale bar: 20 µm). f GFP-βII-spectrin accumulation at the nonadherent portion of the cell, remarkably followed the forward movement of the leading actin bridge. Images are representative of n = 3 independent experiments.