Skip to main content
. 2019 Apr 17;132(8):jcs224378. doi: 10.1242/jcs.224378

Fig. 2.

Fig. 2.

Alignment is regulated by polarized Rho signaling. (A) Right panel: A GFP sensor for Rho-GTP enriched along aligning junctions (purple in graph) relative to orthogonal junctions (green in graph). Left panel: Rho-GTP signal intensity is displayed using the Fire lookup table in ImageJ (calibration bar shows low to high signal). n=34 interfaces, 7 embryos. (B) GFP::RokK116A enriched along aligning interfaces relative to orthogonal junctions. n=38 interfaces, 7 embryos. RokK116A avoids potential artifacts from overactivation (Simões et al., 2010). (C) Anti-Dia antibody staining was enriched along aligning junctions relative to orthogonal junctions. n=98 interfaces, 8 embryos. (D) rho loss-of-function mutants (rho72O/72F, red) had significant alignment defects compared to wild-type sibling controls (yellow). Anti-pTyr antibody staining was used to visualize cell outlines. Control: n=76 interfaces, 6 embryos; rho72O/72F: n=75 interfaces, 5 embryos. (E) Pharmacological inhibition of Rok with either Y-27632 or H-1152 caused significant decreases in alignment. Y-27632: n=21 interfaces, 5 embryos; H-1152: n=11 interfaces, 3 embryos; H2O: n=26 interfaces, 6 embryos. (F) Pharmacological inhibition of Dia disrupted alignment. White arrowheads indicate cell junctions that became convoluted after drug treatment. SMIFH2: n=27 interfaces from 6 embryos; DMSO: n=16 interfaces from 3 embryos. Par3::mCherry was used to mark AJs in A,B, magenta. Anti-phospho-Tyrosine (pTyr) antibody staining was used as a marker for cell outlines in C,D. Each line in graphs of A–C represents one interface and matches average aligning (purple) and orthogonal (green) junction measurements. Fluorescently tagged E-cadherin was used in E,F to visualize cell outlines before (yellow) and after (red) drug treatments. Each line in the graphs of E,F represents one interface and matches measurements before (yellow) and after drug treatment (red) or vehicle treatment (gray). Control injections with either H2O (for Rok inhibitors) or DMSO (for SMIFH2) did not significantly reduce alignment. ***P<0.0001; N.S., not significant; Mann–Whitney U-test, bars show mean±s.d. Scale bars: 4 μm.