Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles

Figures

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  Figure 1. Spatial DFT analysis of Ca++-Fluo-4 in live T cells before and after blebbistatin treatment. (a) An image of Ca++-Fluo-4-highlighted, untreated T cell. (b) An image of the Ca++-Fluo-4-highlighted cell after 10 μM blebbistatin treatment. (c) Line scans of the relative light intensities [the pixels intensity divided by the average region of interest (ROI) intensity] in a line of 30 pixels, as illustrated in the corresponding image. (d) The average spectrum of the relative spatial amplitudes (i.e., the spatial amplitudes in a cell after blebbistatin treatment, divided by the corresponding amplitudes in the same cell before blebbistatin treatment) in all cells (N = 31). (e) The average relative amplitudes at spatial length scale < 1.1 μm in comparison to the average relative amplitudes at spatial length scale > 1.1 μm. (f) The average relative pixels’ information entropy (the pixels’ information entropy in a cell after blebbistatin treatment, divided by the pixels’ information entropy in the same cell before blebbistatin treatment) in all cells (N = 31). The pixels’ information entropy was calculated in two ways: first for all the ROI pixels’ values, and second by dividing the ROI to…

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  Figure 2. Inhomogeneity in living cells imaged by TMRM fluorescence (N = 21) (a), or label-free bright-field (BF) microscopy (N = 62) (b) and its relation to 10 μM blebbistatin treatment. Inhomogeneity in living cells imaged by Ca++-Fluo-4 fluorescence (N = 31) and its relation to ATP depletion treatment (c). From top to bottom rows: a representative cell before treatment, the same cell after treatment (blebbistatin or ATP depletion), its line scans of relative intensities (the pixels intensity divided by the average ROI intensity) before and after treatment, the average (for all cells) relative spatial amplitudes (see Figure 1) and the comparison of the average relative spatial amplitudes in two ranges of length. Error bars are SEM and p-values are shown.

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  Figure 3. Temporal image correlation spectroscopy (TICS) in live cells highlighted with Ca++-Fluo-4 before and after blebbistatin treatment. (a) Multiple time-dependent images of a Ca++-Fluo-4-highlighted cell before blebbistatin treatment. (b) Multiple time-dependent images of a Ca++-Fluo-4-highlighted cell after 10 μM blebbistatin treatment. (c) The average TICS results for the cells (N = 31) before blebbistatin treatment and for the same cells after 10 μM blebbistatin treatment. (d) The average correlation slope in cells before blebbistatin treatment, in comparison to the average correlation slope in the same cells after blebbistatin treatment. Error bars are SEM and p-value is shown in (d).

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  Figure 4. Autocorrelation of pixel intensities in the ROIs of Ca++-Fluo-4-highlighted live cells before and after blebbistatin treatment. (a) Average autocorrelation values of the pixel intensities in the ROIs in the cells (N = 31) before blebbistatin treatment, and of the same cells after 10 μM blebbistatin treatment. (b) Average autocorrelation results of the first time-lag in the cells before blebbistatin treatment in comparison to the corresponding results in the same cells after blebbistatin treatment. Error bars are SEM, and p-value is shown in (b).

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  Figure 5. Simulation of coefficient of variation (CV) of concentration of particles and the fluctuations of those CV values across a 5-pixel interval under different fBM conditions. (a) Average simulated concentration of particles across the 5-pixel interval for different H values. Number of simulations was 10. Illustration of matched CV of concentrations for each spatial distribution of concentration across the interval due to the related H value is added. (b) The amplitude of fluctuations of those H-related CV values in 500 sequential simulations under each H condition. (c) The average amplitudes that were presented in (b) and their relation to H values. Error bars in (a,c) are SEM, and p-values are shown in (c).

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  Figure 6. Temporal fluctuations (measured by the coefficient of variation (CV)) of the spatial amplitude in 31 live cells highlighted with Ca++-Fluo-4 before and after blebbistatin treatment. (a) Average CV values of the temporal fluctuations of the spatial intensity amplitudes in the cells before blebbistatin treatment, and of the same cells after 10 μM blebbistatin treatment. (b) Average CV values of the spatial amplitudes in the range of 0.39–1.09 μm in the cells before and after blebbistatin treatment. Error bars in (a,b) are SEM and p-value is shown in (b).