Fig. 1.

In response to repeated stretch, the Actn4^K256E/K256E podocytes failed to recover their contraction, developed irreparable disruptions in their actin cytoskeleton, and detached at a higher rate. (A) Representative traction force maps for WT and Actn4^K256E/K256E podocytes. Different colors correspond with different magnitudes of traction, with red corresponding to the highest magnitude. Arrows indicate direction of traction. (Scale bar: 50 µm.) (B) Single-podocyte contraction dynamics in response to three transient stretches over time. A custom-designed stretch device was used to superimpose a 4-s, 10% transient stretch (followed by release) onto individual WT and Actn4^K256E/K256E podocytes adherent to a 26-kPa substrate. The y axis plots individual podocyte contractile forces, assessed by the contractile moment, normalized by their baseline value before stretch, while the x axis plots serial time points relative to the three transient stretches. Graphed values represent the median (lines) and interquartile ranges (shaded areas). Black triangles along the x axis indicate time points where there is a significant difference between WT and mutant (P < 0.01). Compared with WT podocytes (n = 13), Actn4^K256E/K256E podocytes (n = 13) showed decreasing contraction after three stretches, with a median recovery of only 26% of their initial contraction, whereas WT podocytes maintained over 78% of their baseline contraction at 9 min (final time point). Data were pooled from five separate independent experiments. (C) In response to stretch, Actn4^K256E/K256E podocytes showed breakages in their actin structure after stretch that failed to recover. Representative 20× images of actin filaments of actin of WT (Upper) and Actn4^K256E/K256E (Lower) podocytes before and after one stretch cycle. Actn4^K256E/K256E podocytes demonstrated visible cytoskeletal breakages after stretch that did not repair during the 3-min recovery period (red arrow), whereas WT podocytes demonstrated small areas of actin breakages that were largely repaired. (Scale bar: 20 µm.) Time-lapse images are shown for WT in Movie S1 and Actn4^K256E/K256E in Movie S2. (D) After repeated stretches, Actn4^K256E/K256E podocytes were more likely to detach from their substrates than WT when subjected to a spinning-disk detachment assay. Blue indicates the number of cells that remain adherent to their substrate after three stretches, and red indicates the number of cells that detached from their substrate after three stretches. The cell count was pooled from four separate independent experiments. Only 2 of 19 (11%) WT podocytes detached from their substrate after three stretches, whereas 11 of 23 (48%) mutant podocytes detached (odds ratio, 7.42; 95% CI, 1.27, 80.75; P = 0.017).