Figure S1.
Dynamics of calcium flashes and calcium waves in gastrula-stage Xenopus epithelia. (A) Live imaging of calcium (GCaMP6m, green) and ZO-1 (BFP-ZO-1, magenta) in the animal cap epithelium of gastrula-stage Xenopus embryo. Calcium flash is short-lived and restricted to cell 1 and cell 2 (orange numbers). Time 0 s represents the start of the calcium flash. (B) Graphs showing change in cellular apical perimeter and calcium intensity in four different cells shown in A over time. Cells experiencing calcium flash (cells 1 and 2) decrease in apical perimeter compared with control cells (cells 3 and 4). (C) Live imaging of calcium (GCaMP6m, green) and ZO-1 (BFP-ZO-1, magenta) in the animal cap epithelium of gastrula-stage Xenopus embryos. Traveling calcium wave starts at time 0 s in region 1, propagates through regions 2 and 3, and subsides in ∼120 s. (C′) Graph showing the increase in calcium intensity over time in regions marked in the first frame of C. (D) Live imaging of calcium (GCaMP6m, green) and ZO-1 (BFP-ZO-1, magenta) showing a local calcium increase (white arrows) in cells expressing a reduced amount of GCaMP6m. Time 0 s represents the start of the calcium increase. (E) Left: Cell view of embryo expressing calcium probe (GCaMP6m, green) and active Rho probe (mCherry-2xrGBD, magenta). 5-pixel-wide yellow arrow indicates the region used to generate the kymograph. Right: Kymograph shows that cytosolic calcium increases locally at the site of the Rho flare. Individual images are shown using FIRE LUT. (F) Correlation plot between intensity of calcium and intensity of active RhoA at the maximum size of Rho flares shows a significant positive correlation, with a Pearson’s correlation coefficient of r = 0.7247. Solid line represents the best-fit line, and the dotted lines represent the 95% confidence bands of the best-fit line. Significance calculated using two-tailed t test. n = 19 flares, 14 embryos, 11 experiments. (G and G′) Time-lapse images (FIRE LUT) of calcium probe (R-GECO1) and F-actin probe (Lifeact-GFP) with sustained calcium increase (G) or a calcium spike (G′) following laser-induced TJ injury. Yellow arrowhead indicates the site of junctional laser injury at time 0 s. (G) Montage and graph of normalized intensity shows successful activation of F-actin accumulation (magenta) and repair of laser-induced F-actin break following a local sustained calcium increase (green). (G′) Montage and graph of normalized intensity shows failure to activate F-actin accumulation and lack of repair following a calcium spike that is not sustained over time. White arrows indicate the site of failed repair.

Dynamics of calcium flashes and calcium waves in gastrula-stage Xenopus epithelia. (A) Live imaging of calcium (GCaMP6m, green) and ZO-1 (BFP-ZO-1, magenta) in the animal cap epithelium of gastrula-stage Xenopus embryo. Calcium flash is short-lived and restricted to cell 1 and cell 2 (orange numbers). Time 0 s represents the start of the calcium flash. (B) Graphs showing change in cellular apical perimeter and calcium intensity in four different cells shown in A over time. Cells experiencing calcium flash (cells 1 and 2) decrease in apical perimeter compared with control cells (cells 3 and 4). (C) Live imaging of calcium (GCaMP6m, green) and ZO-1 (BFP-ZO-1, magenta) in the animal cap epithelium of gastrula-stage Xenopus embryos. Traveling calcium wave starts at time 0 s in region 1, propagates through regions 2 and 3, and subsides in ∼120 s. (C′) Graph showing the increase in calcium intensity over time in regions marked in the first frame of C. (D) Live imaging of calcium (GCaMP6m, green) and ZO-1 (BFP-ZO-1, magenta) showing a local calcium increase (white arrows) in cells expressing a reduced amount of GCaMP6m. Time 0 s represents the start of the calcium increase. (E) Left: Cell view of embryo expressing calcium probe (GCaMP6m, green) and active Rho probe (mCherry-2xrGBD, magenta). 5-pixel-wide yellow arrow indicates the region used to generate the kymograph. Right: Kymograph shows that cytosolic calcium increases locally at the site of the Rho flare. Individual images are shown using FIRE LUT. (F) Correlation plot between intensity of calcium and intensity of active RhoA at the maximum size of Rho flares shows a significant positive correlation, with a Pearson’s correlation coefficient of r = 0.7247. Solid line represents the best-fit line, and the dotted lines represent the 95% confidence bands of the best-fit line. Significance calculated using two-tailed t test. n = 19 flares, 14 embryos, 11 experiments. (G and G′) Time-lapse images (FIRE LUT) of calcium probe (R-GECO1) and F-actin probe (Lifeact-GFP) with sustained calcium increase (G) or a calcium spike (G′) following laser-induced TJ injury. Yellow arrowhead indicates the site of junctional laser injury at time 0 s. (G) Montage and graph of normalized intensity shows successful activation of F-actin accumulation (magenta) and repair of laser-induced F-actin break following a local sustained calcium increase (green). (G′) Montage and graph of normalized intensity shows failure to activate F-actin accumulation and lack of repair following a calcium spike that is not sustained over time. White arrows indicate the site of failed repair.

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