Fig. 2.

Podocytes of Actn4^K256E/K256E mice exhibit more correlated actin filament orientation and intracellular stress. (A) Representative 63× images of actin filaments in a WT (Upper) and an Actn4^K256E/K256E (Lower) podocyte grown on a collagen-coated coverslip. (Scale bar: 20 µm.) Light yellow lines indicate the average of the angles of actin filament vectors within a region of 10 × 10 pixels (1 px = 0.19 μm). (B) Quantification of the distribution of actin filament orientation across WT and Actn4^K256E/K256E podocytes. This quantification was calculated by the autocorrelation function C(r) (y axis), which is a measure of the correlation, or uniformity, of actin filament orientation over increasing distances across the cell (x axis). The closer to a value of 1, the more correlated, or uniform, the actin filament orientation between any two given regions of 10 × 10 pixels. Data were obtained from nine WT and nine Actn4^K256E/K256E podocytes grown on a collagen-coated coverslip and plotted as median (lines) and interquartile ranges (shaded areas). At increasing distances across the cell, Actn4^K256E/K256E podocytes maintain more spatially correlated actin filament orientation than WT. The black triangles along the x axis indicate distances where the median autocorrelation between Actn4^K256E/K256E podocytes and WT are significantly different (P < 0.05). (C) Representative intracellular stress maps of WT (Upper) and Actn4^K256E/K256E (Upper) podocytes grown on 26-kPa substrates. For the intracellular stress maps, red indicates the greatest magnitude of mean principal stress. The black lines represent the local intracellular stress both in terms of tension (σ, corresponding to length of the line) and orientation (θ, corresponding to orientation of the line). (Scale bar: 50 µm.) (D) Quantification of the distribution of σ across WT and Actn4^K256E/K256E podocytes. As above, y axis plots the autocorrelation function C(r) of σ, and the x axis plots distance across the cell. At increasing distances across the cell, Actn4^K256E/K256E podocytes maintain more spatially correlated, or more uniformly distributed, σ than WT. The black asterisk (*) along the x axis indicate distances where the median C(r) of σ between Actn4^K256E/K256E podocytes and WT are significantly different (P < 0.05). (E) Quantification of the distribution of σ across WT and Actn4^K256E/K256E podocytes. As above, y axis plots the autocorrelation function C(r) of θ, and the x axis plots distance across the cell. At increasing distances across the cell, Actn4^K256E/K256E podocytes maintain more spatially correlated θ than WT. The black asterisk (*) along the x axis indicate distances where the median C(r) of θ between Actn4^K256E/K256E podocytes and WT are significantly different (P < 0.05). For both D and E, the distance over which the C(r) decays to a value of 0.5 was extrapolated as a singular value from the autocorrelation curves of each individual cell; this value is termed correlation length r_{1/2 σ} and r_{1/2 θ}. The greater the r_1/2 value, the greater the distance over which intracellular stress maintains uniformity in its tension (r_{1/2 σ}) and its orientation (r_{1/2 θ}).