Dynamic filopodial forces induce accumulation, damage, and plastic remodeling of 3D extracellular matrices
Fig 3
ECM recruitment by dynamic actin-driven processes.
(a) Representative confocal images of HUVECs suspended in 3D biopolymer networks of fibrin (3 mg/mL) fixed and stained after 4h treatment with several cytoskeletal drugs and inhibitors. Blue is DAPI, red is phalloidin, and cyan is the fluorescently labeled fibrin fibers. Scale bar is 20 μm. (b) Statistical comparison for all treatments with drugs, in terms of densification factor (N>15 cells per case; ** p<0.01with one-way ANOVA with post-hoc Tukey HSD Test). (c) Computational simulation of an ECM fiber network shows network morphology before (left) and after (right) the application of loading forces near the left boundary. Colors on fibers indicate tension level according to the color bar (-300 to 300pN). Yellow spots are crosslinks (places where fiber-fiber crosslinking can occur). See also S4 Video and S5 Video. (d) Overlay of the time evolution of fiber concentration profiles, normalized by the initial concentration, in the force-loading direction as loading forces are exerted from the left boundary. Loading forces mimic dynamic filopodia pulling from the loading boundary, such that fibers within 2μm of that boundary experience a force pulling them toward the boundary. As new fibers or fiber segments move within that distance, new loading forces are exerted on them. Different colored curves represent different normalized times of: 0 (lower blue, uniform), 0.03 (red), 0.37 (yellow), 0.7 (purple, dashed), 1.03 (green), 1.37 (light blue), 1.7 (magenta), 2.03 (blue), where time is normalized to the total time of force application (starting at right after 0 and ending at 1). Some relaxation occurs after applied forces end, but matrix remodeling here is not reversed. The simulation setup is 100pN loading per fiber segment in the loading region, 1x crosslink zero-force unbinding rate, 0.3x crosslink mechanosensitivity, and 1x crosslink density (see S1 Table for default values). (e) MDA-MB-231 cells expressing fluorescent F-actin (green, left) inside a 3D collagen matrix (white, middle) with a concentration of 1.5mg/mL display many dynamic actin protrusions (blue arrows). Overlay image of actin and collagen is on the right. Images are maximum intensity z-stack projections. The scale bar is 20μm. See also S3 Video. (f) Schematic of ECM recruitment by dynamic filopodia. Step 1: Filopodia attach to fibers in the vicinity of the cell (loading zone). Step 2: Filopodia contract, via actomyosin-based contractile forces, pulling attached fibers toward the cell and breaking force-sensitive crosslinks. Step 3: New filopodia form and attach to new fiber regions in the loading zone. Step 4: Contraction cycle repeats, further pulling ECM fibers toward the cell. This dynamic filopodial force loading condition is applied in our discrete network simulations.