Figure 6.

MSC-dependent calcium influx is required for Rho-mediated reinforcement of ZO-1. (A) Left: Cell view of an embryo expressing membrane probe (membrane-tagBFP, cyan) and active Rho probe (mCherry-2xrGBD, magenta). Yellow arrow indicates the 2-pixel-wide region used to generate the kymograph. Right: Kymograph shows that membrane protrusion increases with Rho flare. Green arrow indicates the start of membrane protrusion and Rho flare. (B) Schematic showing that GsMTx4 inhibits MSC-mediated calcium influx in response to changes in membrane tension induced by membrane stretch or curvature. (C and D) Time-lapse images (FIRE LUT) of BFP-ZO-1, calcium (GCaMP6m), and active Rho (mCherry-2xrGBD). Embryos were treated with water (vehicle; C) or 12.5 µM GsMTx4 (MSC inhibitor; D). MSC inhibition resulted in a decrease in calcium influx; short-duration, low-intensity Rho flares (yellow arrowheads; D); and decreased ZO-1 reinforcement at the site of ZO-1 loss (yellow boxes, enlarged below; D). Time 0 s represents the start of Rho flares. (E–G) Graphs of mean normalized intensity of calcium (GCaMP6m; E), active Rho (F), and ZO-1 (G) at the site of ZO-1 loss over time in vehicle- (water, solid lines) or MSC-inhibited (GsMTx4, dashed lines) embryos. Shaded region represents SEM. Vehicle: n = 13 flares, 6 embryos, 6 experiments; GsMTx4: n = 17 flares, 7 embryos, 6 experiments. (E′–G′) AUCs for calcium (E′) and active Rho (F′) were calculated from E and F, respectively. (G′) Scatter plot of change in ZO-1 intensity (G′) was calculated from G. I₁ and I₂ represent average intensity of ZO-1 from individual traces at times −25 to 25 s and 400–450 s, respectively. Error bars represent mean ± SEM; significance calculated using Mann–Whitney U test.