Figure 1.
Epithelial paracellular leaks induce a local intracellular calcium increase. (A) Time-lapse images (Fire LUT) of FluoZin-3 dye, membrane calcium probe (tagBFP-PKC β-C2), and active Rho (mCherry-2xrGBD). Calcium increase (white arrows) follows a paracellular leak indicated by increase in FluoZin-3 fluorescence (yellow arrows) at the site of Rho flare (yellow arrowheads). Time 0 s represents start of Rho flare. (B) Quantification of experiments shown in A. Top: Schematic showing ROIs used to quantify Rho flares, calcium (C2), and FluoZin-3. Bottom: Graph of mean normalized intensity shows that the leak (FluoZin-3) precedes the increases in local calcium and active Rho. Shaded region represents SEM; n = 18 flares, 10 embryos, 6 experiments. (C) Top: Cell view of an embryo expressing membrane calcium probe (mNeon-PKCβ-C2, green) and active Rho probe (mCherry-2xrGBD, magenta). The yellow arrow indicates the 5-pixel-wide region used to generate the kymograph. Bottom: Kymograph shows that calcium increase originates at the junction and then spreads. (D) Time-lapse images (FIRE LUT) of BFP-ZO-1, membrane calcium probe (mNeon-PKC β-C2), and active Rho (mCherry-2xrGBD). Local calcium increase (white arrows) is spatially localized to the site of ZO-1 decrease (yellow boxed region) and Rho flare (yellow arrowheads).

Epithelial paracellular leaks induce a local intracellular calcium increase. (A) Time-lapse images (Fire LUT) of FluoZin-3 dye, membrane calcium probe (tagBFP-PKC β-C2), and active Rho (mCherry-2xrGBD). Calcium increase (white arrows) follows a paracellular leak indicated by increase in FluoZin-3 fluorescence (yellow arrows) at the site of Rho flare (yellow arrowheads). Time 0 s represents start of Rho flare. (B) Quantification of experiments shown in A. Top: Schematic showing ROIs used to quantify Rho flares, calcium (C2), and FluoZin-3. Bottom: Graph of mean normalized intensity shows that the leak (FluoZin-3) precedes the increases in local calcium and active Rho. Shaded region represents SEM; n = 18 flares, 10 embryos, 6 experiments. (C) Top: Cell view of an embryo expressing membrane calcium probe (mNeon-PKCβ-C2, green) and active Rho probe (mCherry-2xrGBD, magenta). The yellow arrow indicates the 5-pixel-wide region used to generate the kymograph. Bottom: Kymograph shows that calcium increase originates at the junction and then spreads. (D) Time-lapse images (FIRE LUT) of BFP-ZO-1, membrane calcium probe (mNeon-PKC β-C2), and active Rho (mCherry-2xrGBD). Local calcium increase (white arrows) is spatially localized to the site of ZO-1 decrease (yellow boxed region) and Rho flare (yellow arrowheads).

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