Differential activity of nonmuscle myosin IIA and IIB isoforms generates a dynamic actomyosin network in a concentration-dependent manner

Figures

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  Figure 1. NMIIA-generated forces break the linear actomyosin network faster than NMIIB. The simulations include linear periodic contractile networks composed of white actin filaments and blue crosslinkers two types of motors are simulated: NMIIA (A) and NMIIB (B). NMIIA and NMIIB motor ensembles shown in red, crosslinkers are shown in blue, actin is shown in white. The simulation time steps are 0.5 s apart and the total time of simulation is 400 s. C, the time to network rupture was observed in five different simulations, and the average rupture time was plotted for networks containing only NMIIA and NMIIB. Data represent mean ± SEM, n = 5. p values ∗∗∗p < 0.001. Statistical significance was assessed using unpaired two-tailed Student’s t tests with Welch’s correction. D, a network was simulated with 360 NMIIB molecules, with NMIIA filaments in increments of 60 until reaching 360 molecules. Time of simulation is indicated. NMIIA motor ensembles are shown in red, whereas NMIIB motor ensembles are shown in yellow when both motors are added. E, in another scenario, NMIIB was added incrementally by 60 molecules on top of a network already containing 360 NMIIA molecules. Time of simulation is…

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  Figure 2. Overexpression of NMIIA in COS7 cells increases breakage of actin bundles. A, COS7 cells were transfected with either Lifeact-GFP (green), which binds to filamentous actin, or NMII-mCherry along with Lifeact-GFP (green). Imaging was performed at 10 s intervals, with cells maintained at 37 °C and 5% CO2. Representative images at 0, 40 and 80 s of imaging and corresponding kymographs are shown for COS7, NMIIA overexpression, and NMIIB overexpression. White arrows highlight the bundle breakage. The portion of the actin bundles for which the kymographs have been generated is shown in red. The scale bar represents 5 μm. B, quantification of actin bundle severing events. Breakage events were manually quantified across 120 frames for each cell, and the average number of events was plotted (n = 3–9 cells per condition; ∗∗∗p < 0.001). Statistical significance was assessed using unpaired two-tailed Student’s t tests with Welch’s correction. C, actin bundle length values extracted using the filament sensor were quantified and bundle length distribution >5 μm was plotted as indicated. D and E, the half-time of eGFP fluorescence recovery was measured in cells cotransfected with…

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  Figure 3. NMIIA overexpression changes actin structures in COS7 cells. A, COS7 cells were transfected with either NMIIA, NMIIB, or NMIIA deltaIQ2. Immunofluorescence image showing actin (magenta) and the corresponding NMII isoform (red) was taken under a confocal microscope. The white arrow shows the location of actin arcs. The scale bar represents 10 μm. B, the number of arcs was manually counted from each image, and the mean value was plotted (n = 41–133 cells per condition). p values ∗∗p < 0.01, ∗∗∗p < 0.001. Statistical significance was assessed using Welch’s one-way ANOVA with Games–Howell post hoc tests. NMII, nonmuscle myosin II.

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  Figure 4. NMIIA alters cofilin distribution in COS7 cells. A, COS7 cells were transfected with either NMIIA, NMIIB, or NMIIA deltaIQ2. Representative immunofluorescence image showing actin (magenta) and NMII isoform (red). Cofilin is shown here in green. The scale bar represents 10 μm. B, cells were divided into three zones: peripheral, middle zone, and perinuclear, and the number of cofilin particles was counted in each zone, and their averages were plotted (n = 7–9 cells per condition). p values ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Statistical significance was assessed using Welch’s one-way ANOVA with Games–Howell post hoc tests. NMII, nonmuscle myosin II.

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  Figure 5. NMIIA increases cortical stiffness while maintaining cortex dynamics. A, schematic of the experiment exhibiting sorting of cells with different levels of myosin expression tagged with mCherry. On the right is a schematic explaining the force spectroscopy measurement protocol. B and C, typical force-elongation plots exhibiting a few measurements with overexpression of NMIIA and NMIIB, respectively. The average variation of these few measurements is shown in black, and the red lines are linear fits to the average variations. D, COS7 cells (control) and overexpressing NMIIA were analyzed by the force spectroscopy protocol. Cells with medium and high NMIIA expression exhibited two force constant values (prior to and after yielding), indicating that the cells expressing NMIIA have two elastic regimes, suggestive of a dynamic cortical network that withstands longer tether elongations. In the case of NMIIA medium and high overexpression, the force constant measured after yielding transition is indicated in gray. Number of experiments performed in each condition: COS7—seven cells, 17 measurements; NMIIA (low)—six cells, 14 measurements; NMIIA (medium)—six cell, 14 measurements; and NMIIA…

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  Figure 6. Dynamics of actin with increasing expression of NMIIA. COS7 cells–expressing EGFP-actin and mCherry-NMIIA were sorted based on NMIIA fluorescence levels. Photobleaching of EGFP-actin followed by an analysis of its recovery kinetics was performed. A, a representative montage of COS7 cells–expressing EGFP-actin and mCherry-NMIIA shows the prebleach and postbleach recovery of EGFP-actin fluorescence (green). Bleaching was performed for a 20 × 20 μm area toward the periphery of the cells. The scale bar represents 10 μm. B, a representative fluorescence recovery curve of EGFP-actin for each indicated condition. C, half-life of recovery (t1/2) and (D) percent mobile fraction of EGFP-actin was estimated for cells with low, medium, and high NMIIA expression levels. (n = 9–13 cells per condition). ∗p < 0.05. Statistical significance was assessed using Welch’s one-way ANOVA with Games–Howell post hoc tests. COS7 cells–expressing EGFP-actin and mCherry-NMIIB were sorted based on NMIIB fluorescence levels. Photobleaching of EGFP-actin was followed by an analysis of its recovery kinetics. E, a representative fluorescence recovery curve of EGFP-actin for each indicated condition. F, half-life…

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  Figure 7. The gradient of NMIIA enhances actin breakages in COS7 cells. COS7 cells were transfected with either Lifeact-GFP alone or cotransfected with NMIIA-mCherry. Following transfection, cells were flow-sorted into populations with low, medium, and high NMIIA expression levels. These sorted cells were then plated on bottom-coverslip dishes and adhered to the surface overnight. After a 24-h incubation period, time-lapse imaging was performed at 10-s intervals, with the cells maintained under physiological conditions at 37 °C and 5% CO2. A, representative images at 0, 40 and 80 s of imaging and corresponding kymographs are shown for NMIIA low, medium, and high expressing cells. White arrows highlight the actin bundle breakage events. The portion of the actin bundles for which the kymographs have been generated is shown in red. The scale bar represents 5 μm. The last panel shows NMIIA-overexpressing cells after 30 min of Blebbistatin treatment. B, quantification of actin bundle severing events. Breakage events were manually observed across 120 frames in all the cells, and their average was plotted. (n = 3 cells per condition), ∗p < 0.05. C, actin bundle length values extracted using the…