Fig. 4: Jasplakinolide treatment drives spectrin clusters formation.

A Schematic pipeline and representative ExM image of MEFs seeded on microfabricated adhesive lines (4 μm adhesive cross-section, 12 μm non-adhesive surface) and immunolabelled for endogenous βII-spectrin (green) and β-actin (magenta). A large volumetric imaging was performed by tile scan approach, and a projection of multiple planes is shown (scale bar: 200 μm). B Two protrusions from independent cells are shown to highlight the differential positioning of βII-spectrin and β-actin, black arrowheads indicate clusters with incomplete periodic organization (scale bar: 20 μm). Images are representative of at least 3 independent experiments. C Live imaging by TIRF microscopy of MEFs transiently transfected with GFP-βII-spectrin (green) and RFP-actin (magenta), treated with Jasplakinolide 100 nM and Blebbistatin 10 μM for 3-4 h. Relevant frames are shown to highlight the differential effects on cell shape and protein clustering the two drugs induce (scale bare 20 μm). Cluster area normalized to the initial frames is calculated in response to each treatment and plotted over time (n = 10 independent cells, data are presented as mean ± SD, statistical analysis: one-way ANOVA of values at t = 100 min). D ExM images of MEFs treated for 3-4 h with 10 μM Blebbistatin and 100 nM Jasplakinolide, immunolabelled for βII-spectrin (scale bars: 20 μm). 30 μm² zooms are shown, corresponding to the yellow boxes, to highlight the differential effects of the drugs on spectrin organization. NND values are reported, as well as line scans related to the yellow lines in the zooms (1 and 2). Images are representative of at least 3 independent experiments. E, F Recovery curves resulting from the FRAP assay are shown for GFP-βII-spectrin and GFP-Actin, transiently transfected in MEFs and treated 3-4 h with the different cytoskeletal impairing drugs (Jasplakinolide 100 nM, Blebbistatin 10 μM). Mobile fractions are reported in the graphs (data are presented as mean ± SD, statistical analysis one-way ANOVA with multiple comparisons, ***p-value < 0.005, ****p-value < 0.0001, n = 25-28(GFP-βII-spectrin) and 19-25(RFP-actin) cells in 3 independent experiments). Half-time recovery rates resulting from the fitting of the raw data with a one-exponential equation are reported in the graphs (data are presented as mean ± SD, statistical analysis one-way ANOVA with multiple comparisons, *p-value < 0.05, **** p-value < 0.0001, n = 25-28(GFP-βII-spectrin) and 19-25(RFP-actin) cells in 3 independent experiments). Panel (A) was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.