Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

Abstract

The dynamic control of E-cadherin is critical for establishing and maintaining cell-cell junctions in epithelial cells. The concentration of E-cadherin molecules at adherens junctions (AJs) is regulated by lateral movement of E-cadherin within the plasma membrane and endocytosis. Here we set out to study the interplay between these processes and their contribution to E-cadherin dynamics. Using photoactivation (PA) and fluorescence recovery after photobleaching (FRAP) we were able to monitor the fate of E-cadherin molecules within the plasma membrane. Our results suggest that the motility of E-cadherin within, and away from, the cell surface are not exclusive or independent mechanisms and there is a fine balance between the two which when perturbed can have dramatic effects on the regulation of AJs.

Figure 1

Characterization of A431 cells expressing GFP-E-cadherin, PAGFP-E-cadherin and PAGFP-Farn2Palm. Expression and cellular localization of GFP-E-cadherin, PAGFP-E-cadherin and PAGFP-Farn2Palm in stable pools of A431 were confirmed by immunoblot of E-cadherin and GFP (A) and immunofluorescence of GFP (B). (C) Montage showing cellular localization of GFP-E-cadherin at different levels in the z-axis of the cells. The asterisk (*) indicates the focal plane chosen for E-cadherin dynamics analyses. Blue, DAPI staining. Scale bars, 20 µm.

Figure 2

Study of E-cadherin dynamics using photoactivation. (A) Representative images captured pre-activation and following activation showing basal fluorescence of PAGFP-E-cadherin and PAGFP-Farn2Palm when illuminated with 488 laser. Fluorescence intensity heat maps have been applied to facilitate visualization. Scale bars, 20 µm. (B) Representative still images of PAGFP-E-cadherin (top parts) and PAGFP-Farn2Palm (bottom parts) at the plasma membrane captured pre-activation and following activation. Fluorescence intensity heat maps have been applied to facilitate visualization. Scale bars, 5 µm. (C) Fluorescence decay curves following photoactivation of PAGFP-E-cadherin (red) and PAGFP-Farn2Palm (blue). (D) Quantification of t_1/2 following photoactivation of PAGFP-E-cadherin and PAGFP-Farn2Palm. Values represent the mean from at least 25 cells. Error bars, s.e.m.

Figure 3

Monitoring E-cadherin dynamics at the plasma membrane. (A) Integrated fluorescence intensity measurements over the plasma membrane at activation (full line) and t_1/2 time points (dash line) were fitted to Gaussian curves for PAGFP-E-cadherin (top) and PAGFP-Farn2Palm (bottom). (B) Quantification of the speed of lateral movement of PAGFP-E-cadherin and PAGFP-Farn2Palm within the membrane. (C) Integrated fluorescence intensity measurements over the plasma membrane at bleaching (full line) and t_1/2 time points (dash line) were fitted to Gaussian curves for GFP-E-cadherin (top) and GFP-Farn2Palm (bottom). (D) Quantification of the speed of lateral movement of GFP-E-cadherin and GFP-Farn2Palm within the membrane. Values represent the mean from at least 25 cells. Error bars, s.e.m.

Figure 4

E-cadherin is mainly retained at the plasma membrane of confluent monolayers of A431 cells. (A) Segmentation of fluorescence signal using ImageJ and corresponding still images from photoactivation experiments at activation and t_1/2 time points for PAGFP-E-cadherin and PAGFP-Farn2Palm used for membrane retention analyses. (B) Quantification of the proportion of fluorescence intensity that is retained in the plasma membrane at t_1/2 following activation of PAGFP-E-cadherin and PAGFP-Farn2Palm. (t test, *p = 0.003). Values represent the mean from at least 25 cells. Error bars, s.e.m.

Figure 5

Deregulation of intracellular trafficking specifically alters the rate of movement of E-cadherin at the plasma membrane. (A) Quantification of biotinylated E-cadherin internalization over 10 min in A431 cells or A431 cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 30 min). (B) Fluorescence recovery curves following photobleaching of GFP-Farn2Palm (left part) and GFP-E-cadherin (right part) in control cells (blue) or cells treated with dynasore (red) or bafilomycin A1 (green), (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). (C) Quantification of t_1/2 following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1. (D) Quantification of the speed of lateral movement of PAGFP-E-cadherin within the membrane in cells treated with bafilomycin A1. Values represent the mean from at least 25 cells. Error bars, s.e.m.

Figure 6

The immobile fraction of E-cadherin at the plasma membrane is not perturbed by dynasore or bafilomycinA1. Quantification of immobile fraction following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). Values represent the mean from at least 25 cells. Error bars, s.e.m.

Figure 7

Inhibition of endocytosis strengthens cell-cell adhesion. (A) Representative images of fragments detached from a dispase-treated monolayer of control or dynasore (80 µM, 30 min) pre-treated A431 cells after mechanical stress. (B) Quantification of the number of single cells that disaggregate from a dispase-treated monolayer after mechanical stress. (C) Quantification of the number of single cells that disaggregate after mechanical stress from a dispase-treated monolayer of A431 or A431 GFP-E-cadherin cells, transfected with either control or E-cadherin siRNA. (D) Still images obtained from photobleaching experiments showing GFP-E-cadherin localization in A431 cells untreated or treated with dynasore (80 µM, 30 min). Scale bars, 20 µm. (E) Immunoblot showing surface and total levels of GFP- and endogenous E-cadherin (150 kDa and 120 kDa, respectively) in A431 cells untreated or treated with dynasore (80 µM, 30 min). (B and C) Values represent the mean from at least three independent experiments. Error bars, s.e.m.