Fig. 7. Myosin II dynamics act as the dominant force for spectrin clustering.

A Live imaging by TIRF microscopy of MEFs transiently transfected with GFP-βII-spectrin (green) and mCherry-MLC (magenta) seeded on microfabricated adhesive lines (not stained, gray dashes) to force discrete cortical organizations (scale bar = 20 μm). Live images are representative of at least 3 independent experiments. Three different regions are highlighted and display representative cortical dynamics (1–3). Automated tracking of mCherry-MLC puncta with two different filtering parameters: in (B), all tracks are shown in a single representative frame, while in (C) only long-lived tracks (> 150 s) preferentially localized on stress fibers. D Pulsative myosin dynamics is shown at spectrin-rich cortical domains (5 × 5 μm zoom). GFP-βII-spectrin and mCherry-MLC clustering are highlighted by white and yellow arrowheads respectively. Kymograph qualitative analysis showed fenestration in the GFP-βII-spectrin signal (white arrowheads) in correspondence with the mCherry-MLC pulse (yellow arrowhead). The yellow bar highlighted an MLC pulse of ≈ 60 s. E Differential lifetime of MLC puncta presented in (B) (All MLC Tracking) and (C) (Long-lived MLC Tracking), data are presented as the center (median), box (25th to 75th IQR), whiskers (min and max) (n = 2200-6965 tracks in 5 independent cells, statistical analysis Mann-Whitney test ****p-value < 0.0001). F Top: initial configuration of the theoretical model where the spectrin mesh is attached to the stress fibers (black edges) through myosin linkers with cable elements (magenta lines, with empty circles at their ends). Myosin rods with less rigid cable elements are randomly distributed in the mesh. Focal adhesions are pulled together at a constant velocity during the first 300 s of the simulation, after which, they are held fixed. The total duration of the simulation is 600 s. Bottom: position of the short-actin filaments at the end of the simulation (equilibration), color-coded to represent the number of attached spectrin bundles per node. G Histograms denote the initial and final number of spectrin bundles per short-actin filament. H Boxplot of the lifetime of the myosin rods in the spectrin network for the two different simulation conditions (n = 5 single myosin, n = 11 dynamic myosin), data are presented as center (median), box (25th to 75th IQR), whiskers (min and max). I The phase diagram integrates the different experimental and theoretical parameters identified in this study to explain the contribution of the different players in the topological transition of the spectrin meshwork. Panel (I) was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.