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. 2022 Mar 7;221(4):e202105107. doi: 10.1083/jcb.202105107

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© 2022 Varadarajan et al.

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Figure 7. — MSC-mediated calcium influx is critical for epithelial barrier function and local junction contraction. (A) Time-lapse images (orange hot LUT) of epithelial permeability tested using ZnUMBA (FluoZin-3). Embryos were treated with water (vehicle, top) or 12.5 µM GsMTx4 (MSC inhibitor, bottom). Time 0 s represents the start of the time-lapse movie. (B) Graph of mean normalized intensity of whole-field FluoZin-3 for vehicle (water) or GsMTx4-treated embryos. Blocking MSCs increased global FluoZin-3 intensity over time compared with vehicle. Shaded region represents SEM. Vehicle: n = 6 embryos, 4 experiments; GsMTx4: n = 6 embryos, 4 experiments. (C) Frequency of Rho flares in vehicle- (water) and GsMTx4-treated embryos. Frequencies from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using paired two-tailed t test; n = 5 embryos, 4 experiments. (D and E) Montage of representative junctions from A (FIRE LUT). Blocking MSCs with GsMTx4 causes repeated increases in local FluoZin-3 (white arrows), reduced ZO-1 (BFP-ZO-1) reinforcement (yellow boxes, enlarged below), and reduced duration of Rho flares (mCherry-2xrGBD, yellow arrowheads) at sites of ZO-1 loss. Repeated FluoZin-3 increase is indicated by double white arrows, and failure to activate Rho flare after FluoZin-3 increase is indicated by yellow arrow. (F and F′) Graph of change in junction length for junctions with Rho flares in vehicle- (water) and GsMTx4-treated embryos. Shaded region represents SEM. (F′) Scatter plot of change in junction length was calculated from F. L₁ and L₂ represent average length of junction from individual traces from times −20 to 20 s and 200–240 s, respectively. Error bars represent mean ± SEM; significance calculated using Mann–Whitney U test. Vehicle: n = 10 flares, 6 embryos, 6 experiments; GsMTx4: n = 16 flares, 7 embryos, 6 experiments. (G) Kymographs of BFP-ZO-1 (magenta) and active Rho (mChe-2xrGBD, green) from representative junctions projected vertex to vertex over time for vehicle- (water) and GsMTx4-treated embryos. Kymographs highlight the repeating, short-duration Rho flares, loss of local junction contraction, and reduced ZO-1 reinforcement upon GsMTx4 treatment. (H) Model showing mechanism by which mechanosensitive calcium signaling regulates epithelial barrier function. Top: 3D view of epithelial cells. Bottom: En face view of the junction highlighted showing our model of the local calcium and RhoA signaling following mechanical stimuli. Dotted arrow indicates a possible positive feedback mechanism where active RhoA-mediated membrane protrusion regulates MSC-dependent calcium influx at the site of ZO-1 repair, thus helping to sustain local Rho activation.