The nature and intensity of mechanical stimulation drive different dynamics of MRTF-A nuclear redistribution after actin remodeling in myoblasts

Figures

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  Fig 1. A. Examples of cells classified according to the intracellular localization of MRTF-A GFP, from left to right: mainly cytoplasmic MRTF-A, homogeneously distributed and mainly nuclear MRTF-A. B. Mean fraction of MRTF-A-GFP in the nucleus and C: Cell populations classified by state of main localization of MRTF-A-GFP, as a function of the mass of MRTF-A-GFP plasmid DNA used for transfection with Nanofectine. More DNA led to nuclear accumulation of MRTF-A due to its overexpression relative to that of the G-actin pool (0.75 μg: 75 cells, 1.0 μg: 260 cells, 2.0 μg: 113 cells, 2.5 μg: 126 cells). Cells were grown on PDMS substrates. The reference state for no transfection was obtained using anti-MRTF-A antibody. D. Typical immunoblots for analyzing actin content in C2C12 cells either transfected for MRTF-A-GFP (1 μg of plasmid for 110 000 cells) or co-transfected for MRTF-A-GFP and mCherry-actin (1 μg of each plasmid for 110 000 cells). Hsc70 was used as a loading control; and corresponding quantification of actin expression levels, normalized by Hsc70 (n = 3 independent experiments). E. Typical immunoblots for analyzing the F-/G-actin ratio in C2C12 cells transfected for MRTF-A-GFP (1…

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  Fig 2. Fibronectin-coated beads actuated by magnetic tweezers induced local actin reorganization and global re-localization of MRTF-A-GFP to the nucleus. A. Live images (20x objective, 0.45 NA) of a cell with adhesive fibronectin-coated microbeads before the application of force, and after six force application and release cycles, each step lasting 125 seconds, for a total of 25 min. The beads appear in black. By the end of the experiment, MRTF-A-GFP relocated to the nucleus. B. Final state of MRTF-A-GFP localization in cells subjected to a 1 nN force through adhesive fibronectin-coated microbeads. The force was applied six times for 125 seconds, and released for the same time, for a total of 25 min. Number of cells: 29 for the control, 23 with force, 4 for control with mCherry-actin, 11 with mCherry-actin and force, 10 for control with LifeAct-mCherry, 6 with LifeAct-mCherry and force. p values were calculated using Fisher’s exact test (*p < 0.05, **p < 0.01, ***p < 0.0001). C. Images of the actin cytoskeleton with LifeAct-mCherry before and at the end of the application of force (same experimental conditions as in A, except for objective: 60x, 1.42 NA). An actin ring is clearly…

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  Fig 3. Live monitoring of MRTF-A localization in stretched cells. A. Mean value of the proportion of MRTF-A-GFP in the nucleus of cells as a function of time, in cells stretched by 10% vs control (control: 125 cells, n = 5 independent experiments; 10% strain: 109 cells, n = 5 independent experiments). B. Repartitions of the cells among the three categories of MRTF-A-GFP localization: “C” for mainly cytoplasmic, “N” for mainly nuclear, and “H” for homogeneously distributed. The cells were followed over time under microscope (Olympus IX81, 20x long working-distance air objective, NA = 0.45), t = 0 corresponds to the beginning of stretching. Cells stretched by 10% vs control (control: 143 cells, n = 5 independent experiments; 10% strain: 145 cells, n = 5 independent experiments). Different phases of nuclear accumulation or expulsion of MRTF-A-GFP, (i) to (iv), are described in text. C. Cumulative rates of nuclear accumulation and expulsion events, counted as the number of events per observed cells. A nuclear accumulation event corresponds to the observation of a cell changing from C to H state or from C to N state or from H to N state. A nuclear expulsion event corresponds to the…

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  Fig 4. A. F-/G-actin ratio in populations of cells subjected to 10% or 30% strain and stained after fixation at different times of stretching with phalloidin Alexa 647 for F-actin and DNase-I Alexa 594 for G-actin, measured as the ratio between total fluorescence intensities in the far red to red channels. (10% Strain, n = 2 independent experiments, 0min: 214 cells, 10min: 223 cells, 20 min: 246 cells, 35min: 248 cells. 30% Strain, n = 2 independent experiments, 0min: 310 cells, 10min: 331 cells, 20min: 280 cells, 45min: 175 cells). B. Mean value of the proportion of MRTF-A-GFP in the nucleus of cells and median value of the SiR-actin intensity relative to the initial fluorescence level as a function of time, in cells stretched by 10% vs control (10% strain: n = 3 independent experiments, 93 cells; control: n = 2 independent experiments, 124 cells). C. Same measurements for cells stretched by 30% vs control (30% strain: n = 4 independent experiments; 108 cells. Control n = 2, 53 cells). D. Confocal microscopy images of myoblasts in which F-actin was labelled with SiR-actin after fixation. Top: un-stretched control cell; top left: basal layer showing aligned actin stress fibers (z = 0);…