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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 3. — Optogenetic activation of junctional RhoA is not sufficient for local calcium increase. (A) Schematic of TULIP-mediated optogenetic activation of RhoA by the recruitment of a prGEF upon 405-nm laser stimulation. (B) Quantification of experiments in C. Mean normalized intensity of active Rho (mCherry-2xrGBD) and calcium (Halo-PKC β-C2, Janelia Fluor 549 HaloTag) upon junction-specific 405-nm laser stimulation (gray box, 60–240 s). Shaded region represents SEM. Active Rho: n = 10 junctions, 5 embryos, 3 experiments; calcium (Halo-C2): n = 12 junctions, 7 embryos, 3 experiments. (C) Left: Cell view of an embryo expressing active Rho probe (mCherry-2xrGBD, FIRE LUT) with ROI for light stimulation indicated by white dashed box. Zoomed image of prGEF (orange LUT) localization at ROI. Right: Time-lapse montage of active Rho probe within the ROI that is stimulated with a 405-nm laser (gray box indicates duration of stimulation). (C′) Left: Cell view of an embryo expressing calcium probe (Halo-PKC β-C2, Janelia Fluor 549 HaloTag, FIRE LUT) with ROI for light stimulation indicated by white dashed box. Zoomed image of prGEF (orange LUT) localization at ROI. Right: Time-lapse montage of calcium probe (Halo-C2) within the ROI that is stimulated with a 405-nm laser (gray box indicates duration of stimulation). (D) Time-lapse montage of the junction highlighted in C′ expressing calcium probe (Halo-PKC β-C2, Janelia Fluor 549 HaloTag, FIRE LUT). Laser injury of the junction resulted in a local calcium increase at the site of the injury. Dashed white circle represents the site of junction injury at time 70 s.