Figure S5.

Sustained Rho flares are required for robust F-actin accumulation and successful reinforcement of ZO-1. (A) Montage of a representative junction used to construct kymograph in Fig. 7 G (FIRE LUT). Blocking MSCs with 12.5 µM GsMTx4 causes repeated increases in active Rho at the site of reduced ZO-1 at time 231 s (first flare, white arrows) and 1,260 s (second flare, yellow arrows). Note that ZO-1 is partially reinforced following the first flare but breaks at the same site before activation of second flare. Time 0 s represents the start of kymograph in Fig. 7 G. (B and C) Time-lapse images (FIRE LUT) of ZO-1 (BFP-ZO-1), F-actin (Lifeact-GFP), and active Rho (mCherry-2xrGBD) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. Blocking MSCs with 12.5 µM GsMTx4 causes reduced F-actin accumulation (C, white arrows) at the site of Rho flares (C, yellow arrowheads) compared with vehicle control in B. Note the robust F-actin accumulation scales (C, white arrowhead) with a higher-intensity repeating Rho flare (C, yellow arrow). Time 0 s represents the start of Rho flare. (D) Schematic of proposed positive feedback mechanism where initial activation of RhoA mediates membrane protrusion to promote MSC-dependent calcium influx, which in turn sustains the RhoA activation for robust actomyosin-mediated junction contraction at the site of ZO-1 repair.