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. 2017 Aug 18;6:e24820. doi: 10.7554/eLife.24820

Figure 4. Bound Fat4-Ds1 complexes on the boundary are more stable than unbound Fat4 and Ds1.

(A) A filmstrip showing a fluorescence recovery after photobleaching (FRAP) experiment on a boundary exhibiting accumulation (yellow) of Fat4-citrine (green) and Ds1-mCherry (red). (see Video 2) (B) A kymograph showing the fluorescence recovery profile along the boundary outlined in blue in (A). The fluorescence level (gray scale) is shown as a function of the position along the boundary (x-axis), and the time after photobleaching (y-axis). (C) A filmstrip from FRAP-TIRF experiment on a cell that express Fat4-citrine (see Video 3). Arrow indicates the bleached area. (D) A kymograph showing the fluorescence recovery profile in the bleached area in (C). Almost full recovery of the bleached area is obtained after 20 s. Scale bars - 5 μm. (E–F) Distributions of Diffusion coefficients (E) and exchange rates (F) obtained from analysis of FRAP experiments as those shown in (A–D). * and *** correspond to p-value<0.05 and p-value<0.001, respectively, as estimated by t-test. The number of experiments for each sample are: unbound Fat4 n = 29, unbound Ds1 n = 36, unbound N-cadherin n = 21, Fat4-Ds1 complex n = 10, N-cadherin complex n = 11. Error bars correspond to SEM. Supplementary figure (Figure 4—figure supplement 1) shows the analysis for unbound Ds1-mcherry, unbound N-cadherin-GFP, and the bound N-cadherin complex.

Figure 4.

Figure 4—figure supplement 1. Unbound Ds1-mCherry and N-cadherin-GFP exhibit fast membrane dynamics.

Figure 4—figure supplement 1.

(A–D) Filmstrips and analysis of FRAP-TIRF experiments on Hek293 cells express Ds1-mCherry (A–B) and N-cadherin-GFP (C–D). Scale bar - 5 μm. For the N-cadherin experiments, Ds1-mCherry cells were transiently transfected with N-cadherin-GFP constructs. Frapped region is indicated by arrows. (B,D) Kymographs showing the fluorescence recovery profile in (A) and (C), respectively. The fluorescence level (gray scale) is shown as a function of the position along the bleached area (y-axis), and the time after photobleaching (x-axis). (E) A filmstrip showing a FRAP experiment on a boundary exhibiting accumulation of N-cadherin-GFP. The experiment was performed in a similar way to the FRAP experiments with Fat4-citrine and Ds1-mCherry accumulating boundaries (Figure 4A). (F) A kymograph showing the recovery profile in (E).