Figure 2.
Intracellular calcium flashes precede Rho flares during local ZO-1 reinforcement. (A) Time-lapse images of calcium (GCaMP6m, green) and active Rho (mCherry-2xrGBD, magenta). Calcium flash occurs in cells adjacent to the junction with the Rho flare (yellow arrowheads). Time 0 s represents start of Rho flare. (B) Dot plot of calcium flash duration (full duration at half-maximum, FDHM). Error bars represent mean ± SEM; n = 10 calcium flashes, 10 embryos, 9 experiments. (C) Montage of the junction indicated by the blue box in A (FIRE LUT). A calcium flash (GCaMP6m) follows a local loss of ZO-1 (BFP-ZO-1, yellow boxed region, enlarged below) and precedes the Rho flare (mCherry-2xrGBD, yellow arrowheads). (D) Quantification of experiments shown in C. Top: Schematic showing ROIs used to quantify Rho flares, calcium (GCaMP6m), and ZO-1. Bottom: Graph showing that calcium intensity increase follows a decrease in ZO-1 and precedes an increase in active Rho. Bracket represents the time interval between the peak of calcium and active Rho. Shaded region represents SEM; n = 19 flares, 14 embryos, 11 experiments. (E) Time-lapse images (FIRE LUT) of calcium probe (R-GECO1) and F-actin probe (Lifeact-GFP) during laser-induced TJ injury. Local calcium increase (white dotted circle) precedes F-actin increase (white arrowhead) at the site of junctional laser injury (yellow arrowhead). Time 0 s represents time of junctional injury. (F) Quantification of experiments shown in E. Graph of mean normalized intensity shows that calcium increase follows the laser-induced junctional injury (vertical dotted line) and precedes an increase in F-actin at the site of junctional injury. Bracket represents the time interval between the peak of calcium and F-actin. Shaded region represents SEM; n = 35 flares, 15 embryos, 3 experiments.

Intracellular calcium flashes precede Rho flares during local ZO-1 reinforcement. (A) Time-lapse images of calcium (GCaMP6m, green) and active Rho (mCherry-2xrGBD, magenta). Calcium flash occurs in cells adjacent to the junction with the Rho flare (yellow arrowheads). Time 0 s represents start of Rho flare. (B) Dot plot of calcium flash duration (full duration at half-maximum, FDHM). Error bars represent mean ± SEM; n = 10 calcium flashes, 10 embryos, 9 experiments. (C) Montage of the junction indicated by the blue box in A (FIRE LUT). A calcium flash (GCaMP6m) follows a local loss of ZO-1 (BFP-ZO-1, yellow boxed region, enlarged below) and precedes the Rho flare (mCherry-2xrGBD, yellow arrowheads). (D) Quantification of experiments shown in C. Top: Schematic showing ROIs used to quantify Rho flares, calcium (GCaMP6m), and ZO-1. Bottom: Graph showing that calcium intensity increase follows a decrease in ZO-1 and precedes an increase in active Rho. Bracket represents the time interval between the peak of calcium and active Rho. Shaded region represents SEM; n = 19 flares, 14 embryos, 11 experiments. (E) Time-lapse images (FIRE LUT) of calcium probe (R-GECO1) and F-actin probe (Lifeact-GFP) during laser-induced TJ injury. Local calcium increase (white dotted circle) precedes F-actin increase (white arrowhead) at the site of junctional laser injury (yellow arrowhead). Time 0 s represents time of junctional injury. (F) Quantification of experiments shown in E. Graph of mean normalized intensity shows that calcium increase follows the laser-induced junctional injury (vertical dotted line) and precedes an increase in F-actin at the site of junctional injury. Bracket represents the time interval between the peak of calcium and F-actin. Shaded region represents SEM; n = 35 flares, 15 embryos, 3 experiments.

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