Fig. 4: The impact of microgravity on the size of focal adhesion protein domains at the cell-substrate interface.

Representative images of Paxillin and Zyxin structures in WM266-4 WT-3B6 controls (1 g, A) and post exposure to 4 h simulated microgravity (RPM, B) and WM266-4 E1KO-3C6 cells under normal gravity conditions (1 g, C) or post exposure to 4 h simulated microgravity (RPM, D). Single channel images are presented as inverted grey scale. An overlay image, (Paxillin in green and Zyxin in magenta) is presented, alongside an inset that corresponds to the marked region in the overview image. Scale bar = 10 µm or 5 µm, as indicated. E–G Quantification of adhesion structure sizes defined by Paxillin and Zyxin staining, after exposure to 4 h simulated microgravity. E The Paxillin defined FA domains did not significantly vary in size in response to 4 h simulated microgravity in WT-3B6 cells. However, in E1KO-3C6 cells, these structures are significantly larger after application of 4 h simulated microgravity (Kruskal-Wallis with uncorrected Dunn’s test; WT-3B6 control; n = 330, WT-3B6 microgravity, n = 368, ns p = 0.21; E1KO-3C6 control = 324, E1KO-3C6 microgravity = 199, **p = 0.005). F After 4 h simulated microgravity, the Zyxin-defined FA domains were significantly smaller in WT cells but significantly larger in E1KO cells (Kruskal-Wallis with uncorrected Dunn’s test; WT-3B6 control; n = 330, WT-3B6 microgravity; n = 368, *p = 0.012; E1KO-3C6 control = 362, E1KO-3C6 microgravity = 199, **p = 0.006,). G When the ratio of Zyxin to Paxillin area was determined for each FA structure, this ratio was significantly decreased in WM-WT cells post application of 4 h simulated microgravity, but remained unchanged in WM-E1KO cells (Kruskal-Wallis with uncorrected Dunn’s test; WT-3B6 control; n = 330, WT-3B6 microgravity; n = 368, **p = 0.001; E1KO-3C6 control = 363, E1KO-3C6 microgravity = 199, ns p = 0.45). H–J Quantification of adhesion structure sizes defined by Paxillin and Zyxin staining, after exposure to 16 h simulated microgravity. H The Paxillin defined FA domains did not significantly vary in size after application of 16 h simulated microgravity in WT or E1KO cells (Kruskal-Wallis with uncorrected Dunn’s test; WT-3B6 control; n = 246, WT-3B6 microgravity, n = 222, ns p = 0.29; E1KO-3C6 control = 232, E1KO-3C6 microgravity = 451, ns p = 0.45). I After 16 h simulated microgravity applied to WT cells, the Zyxin-defined FA domains not significantly different in comparison with 1 g controls. However, Zyxin-defined domains at the cell periphery were significantly larger in E1KO cells treated with simulated microgravity, in comparison with 1 g controls (Kruskal-Wallis with uncorrected Dunn’s test; WT-3B6 control; n = 240, WT-3B6 microgravity; n = 222, ns p = 0.16; E1KO-3C6 control n = 274, E1KO-3C6 microgravity n = 464, **p = 0.009). J When the ratio of Zyxin to Paxillin area was determined for each FA structure, this ratio remained unchanged in WT cells treated with 16 h microgravity, in comparison with 1 g controls. In contrast, the ratio of Zyxin to Paxillin area was significantly decreased in E1KO cells treated with 16 h of simulated microgravity, in comparison with 1 g controls (Kruskal-Wallis with uncorrected Dunn’s test; WT-3B6 control; n = 238, WT-3B6 microgravity; n = 222, ns p = 0.14; E1KO-3C6 control; n = 232, E1KO-3C6 microgravity; n = 451, ***p = 0.0002). Each data point denotes an individual focal adhesion, the line underlying individual data points corresponds to the sample median.