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Fig. 3 ZnUMBA reveals a temporary but weak resealing of the barrier following laser injury and prior to contraction-mediated junction repair. (A–A″) Junction injury was performed by exposing the area indicated by the white dotted circle (A′) to intense 405 nm laser light. The junction that was injured is indicated by white box in A. Following laser injury (A′, time 0), FZ3 fluorescence [shown using the Green Fire Blue lookup table (LUT) applied using FIJI; bottom bar] increases sharply at the site of injury and less intensely along the length of the junction. F-actin (Lifeact–mRFP, shown using the Fire LUT applied using FIJI; top bar) accumulates at the site of the injury, and the barrier breach is repaired. A″ shows side views (x–z) of the images shown in A′. White arrow points out that FZ3 signal at the site of the injury is more apical than the signal along the length of the junction. (B) Quantification of laser injury experiments. The mean pixel intensity of a 1 µm-wide line drawn over the injured junction from vertex to vertex was normalized to a reference junction from the same movie. Graph shows mean normalized intensity (left axis) or mean junction length change (right axis)±s.e.m. (n=25 junctions from nine embryos across three experiments). (C–E) Junction injury was performed as in A. FZ3 is shown in the Green Fire Blue LUT (left bar), as in A, and membrane (mCherry–farnesyl) is shown in the Cyan Hot LUT applied using FIJI (right bar). Intensity values are in arbitrary units. Lifeact–miRFP703 is not shown. Peak FZ3 intensity is marked with an asterisk, evidence of membrane reorganization is indicated by a white arrowhead and endocytic vesicles are indicated by a yellow arrowhead. Laser injury induced a bleb-like membrane protrusion in five of 18 injured junctions (C,D). In three of these cases, the membrane protrusion occurred after FZ3 intensity peaked (C), and in the other cases the FZ3 intensity peak coincided with the expansion of the membrane protrusion (D). In the remaining 13 of 18 junctions, laser injury induced membrane reorganization during the contraction phase of junction repair (E), consistent with bunching or folding of the membrane during contraction. Eight of the eighteen junctions showed evidence of endocytosis (yellow arrowhead), and six of eighteen junctions had multiple FZ3 peaks, consistent with a temporary but weak resealing of the TJ barrier. n=18 junctions from seven embryos across one experiment. (F) A speculative model of how TJ strands form a temporary but weak seal prior to reinforcement via contraction of the strand network. (1) An intact TJ strand network. (2) Laser injury induces breaks in TJ strands, allowing Zn2+ and FZ3 to mix. (3) The strand network is partially re-established by annealing or elongation of existing strands but is susceptible to future breaks because of their dynamic nature. In some cases, damaged areas of the strand network might be removed via endocytosis. (4) Actomyosin-mediated junction contraction establishes robust crosslinking of the TJ network, making it less susceptible to future breaks.