Fig. 7: βII-spectrin reactions to mechanical perturbations highlight the interplay with actin.

a Cartoon representation of the cell-stretching device implemented in this study. b MEFs transfected with GFP-βII-spectrin (green) and LifeAct-RFP (magenta) seeded on the fibronectin-coated silicone membrane and stretched biaxially (sequential 5% step-size increase, up to 30%, representative images of n = 16 cells in 3 independent experiments). After each step, the cell under investigation was recentered and refocused; representative images by EPI-fluorescence microscopy are shown (white asterisk indicates transfected MEFs with high intensities excluded from the analysis, scale bar: 20 μm). Double white asterisks highlight a lamellipodia blocked during the stretching. In the dashed boxes 1–2, representative cell-edge behavior observed among independent experiments, highlighting peculiar condensation of βII-spectrin at curvatures not enriched by actin (arrowheads) at maximal stretch (30%). n = 9 cells in 2 independent experiments. c Cell compression setup and the applied step-increase protocol (d) are schematized. e GFP-βII-spectrin (green) and RFP-actin (magenta) expressing MEFs are imaged by live TIRFM during the entire compressive protocol. Four relevant time points are shown: precompression, early and late compression, and during the release phase (scale bar: 10 μm, n = 13 cells in 4 independent experiments). Key details (precompression 1, early and late compression 2–3, and release 4) consistently observed between independent experiments are highlighted by dashed boxes and zoomed in (f). Reaction in correspondence of the nuclear edge, brought into the TIRF plane by the compressive stress (white arrowheads), is quantified by the kymograph analysis (c, bottom) over the yellow rectangle. Fluorescence intensities across the dashed line in (f) are plotted in the graph; clearance of βII-spectrin and the delayed actin polymerization is observed (asterisks and dashed lines in the graph). g Control cells expressing PM marker (red) and βII-spectrin (green) were subjected to the same compression/relaxation protocol. Insets focused on the cortex underneath the nucleus, where PM marker retains its continuity (scale bar: 10 μm, n = 2 independent experiments).