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. 2020 May 12;219(6):e201906153. doi: 10.1083/jcb.201906153

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© 2020 Bartle et al.

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Figure 1. — Hyperadhesion does not protect against loss of Dsg3 order in low-Ca²⁺ media. (A) Representative images of HaCaT cells treated with either vehicle (DMSO; mock) or Gö6976 and maintained in normal-Ca²⁺ medium (left) or switched into low-Ca²⁺ medium (right) for 90 min and labeled with antibodies for Dsg3 and DP. Scale bar = 20 µm. (B) Quantification of adhesive strength by dispase fragmentation assay in HaCaT cells with the same treatment conditions as in A (n = 3; representative of four independent experiments; mean ± SD). (C) Schematic of the fluorescence polarization microscope. Samples are sequentially illuminated with four different angles of polarized excitation, and the total emission is collected. (D) Ordered cadherins (top) are differentially excited by distinct excitation polarizations, resulting in modulated intensity across the four images. Disordered cadherins (bottom) are equally excited regardless of excitation polarization, resulting in equal intensity. Intensity was plotted as a function of excitation polarization, and the amplitude was used to calculate pixel-by-pixel order factor (OF). (E and F) HaCaT cells were transfected with Dsg3-ΔEA-GFP, mock (E) or Gö6976 (F) treated and switched from normal to low-Ca²⁺ medium. Dsg3 intensity and OF images of representative cells before and 30 min after the switch to low calcium with region of interest (ROI) indicated and intensity and Dsg3-ΔEA-GFP OR in the ROI over a 30-min time course after the switch to low-Ca²⁺ medium. Scale bar = 5 µm; ROI scale bar = 1 µm. (G–I) Population analysis of cells mock treated in normal-Ca²⁺ (n = 6) or low Ca²⁺ (n = 11) and Gö6976 treated in normal-Ca²⁺ (n = 4) or low Ca²⁺ (n = 15). (G) Normalized integrated intensity at t = 30 min (mean ± SD). (H) OF plotted as a function of time (mean ± SD). (I) OF at t = 30 min (mean ± SD) compared by one-way ANOVA with multiple comparisons. Data were acquired from three independent experiments (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.001).