Endocytosis is required for E-cadherin redistribution at mature adherens junctions

Abstract

E-cadherin plays a key role at adherens junctions between epithelial cells, but the mechanisms controlling its assembly, maintenance, and dissociation from junctions remain poorly understood. In particular, it is not known to what extent the number of E-cadherins engaged at junctions is regulated by endocytosis, or by dissociation of adhesive bonds and redistribution within the membrane from a pool of diffusive cadherins. To determine whether cadherin levels at mature junctions are regulated by endocytosis or dissociation and membrane diffusion, the dynamics of E-cadherin were quantitatively analyzed by a new approach combining 2-photon fluorescence recovery after photobleaching (FRAP) and fast 3D wide-field fluorescence microscopy. Image analysis of fluorescence recovery indicates that most E-cadherin did not diffuse in the membrane along mature junctions, but followed a first order turn-over process that was rate-limited by endocytosis. In confluent cultures of MCF7 or MDCK cells, stably expressed EGFP-E-cadherin was rapidly recycled with spatially uniform kinetics (50 s in MCF7 and 4 min in MDCK). In addition, when endocytosis was pharmacologically blocked by dynasore or MiTMAB, no fluorescence recovery was observed, suggesting that no endocytosis-independent membrane redistribution was occurring. Our data show that membrane redistribution of E-cadherin molecules engaged in mature junctions requires endocytosis and subsequent exocytosis, and lead to the notion that E-cadherins engaged at junctions do not directly revert to free membrane diffusion. Our results point to the possibility that a direct mechanical coupling between endocytosis efficiency and cadherin-mediated forces at junctions could help to regulate intercellular adhesion and locally stabilize epithelia.

Fig. 1.

Frame for FRAP data interpretation. (A) Different types of mobility are considered for membrane E-cadherin: membrane diffusion (1) or reaction-limited exchange with cytosolic vesicles via endocytosis (2) or between 2 membrane populations (3). (B and C) Fluorescence profile after photobleaching with D_mb = 0.05 μm²·s⁻¹ (B) or exchange with k_off = 0.025 s⁻¹ (C), at t = 0 (red), t = 1 s (blue), t = 6 s (cyan), t = 15 s (green), t = 45 s (yellow), or t = 100 s (orange). Individual profiles were fitted to a Gaussian. (D) Evolution of σ as a function of time (cyan: diffusion, blue: exchange).

Fig. 2.

Two-photon FRAP experiments at mature adherens junctions of MDCK-E-cadherin-GFP cells. (A) Two-photon FRAP was performed in a 1 μm-radius ellipsoid spot (red arrow) at adherens junctions in MDCK cells stably expressing E-cadherin-GFP. Fluorescence recovery was analyzed in the surrounding region (green frame). (B) A kymograph representing the intensity along the junction (y axis) as a function of time (x axis) is represented. (C) Profile analysis of the recovery. 16 fluorescence profile mean curves along the junction, before bleaching (red circle) and at 0.6 s (blue square), 150 s (green diamond), 450 s (black x), and 1,050 s (pink +) after photobleaching are shown. All postbleach curves are fitted by Gaussian models (dashed lines). (D) The width of the gaussians at different times after photobleaching are compared with the reference width after photobleaching in fixed cells. (E) The fluorescence recovery curve (red circle, n = 45) is well fit (blue line) by a first-order exchange model with k_off = 0.0043 s⁻¹ (gray: k_off = 0.0043 ± 0.0011 s⁻¹). (Upper Left Inset) Shorter timescales (Δt = 100 ms, n = 31). (Right Lower Inset) Linear time axis representation. (F) Photobleaching spots of 0.5 (n = 45, red circle), 0.8 (n = 20, blue square) and 1.2 (n = 20, green diamond) μm width along the junction axis are well fitted by first-order exchange with similar coefficients of k_off = 0.0043, 0.0043 and 0.0039 s⁻¹ respectively. (Upper Left Inset) The latter fitting curves were normalized so that the photobleaching depth is the same for all conditions.

Fig. 3.

E-cadherin mobility in MDCK junctions is suppressed by blocking endocytosis with dynasore or MiTMAB. (A–C) After treatment with control medium (A, A′, and A″), dynasore 60 μM (B, B′, and B″) or MiTMAB 30 μM (C, C′, and C″) MDCK E-cadherin-GFP cells were incubated with FM 4–64 5 μg/mL for the indicated times to visualize newly formed endocytosis vesicles. Merge image of the green and red staining are shown, and colocalisation appears in yellow. (D) Fluorescence recovery curve of E-cadherin in cells treated for 15 min with 60-μm dynasore (green) or 30 μM MiTMAB (gray) compared with cells treated with DMSO vector (red) or after washing in DMEM + vector (blue) (n = 16). (Upper Left Inset) Short timescale (Δt = 100 ms) mean FRAP curve after a 15-min dynasore incubation (60 μM) (n = 10). (E) Fluorescence profiles in cells treated 15 min with dynasore, and observed 0.6 s (blue triangle), 43 s (green square), 160 s (black x), 340 s (pink +) or 510 s (cyan triangle) after photobleaching.

Fig. 4.

Two-photon FRAP experiments at mature adherens junctions of MCF7-E-cadherin-GFP cells. (A) Typical image of MCF7 cells stably expressing E-cadhering GFP. (B) Profile analysis of fluorescence recovery. 17 fluorescence profile mean curves before bleaching (red circle) and at 0.6 s (blue square), 15 s (green diamond), 45 s (black x), 105 s (pink +), and 195 s (cyan triangle) after photobleaching are shown. All postbleach curves are fit by Gaussian models (dashed lines). (C) The widths of the gaussians at different times after photobleaching are compared with the reference width after photobleaching in fixed cells. The error bars are larger than for MDCK cells (Fig. 2) due to a decreased signal/noise ratio in MCF7 cells. (D) The recovery curve (red circle, n = 25) is well fit by a first-order exchange model with k_off = 0.0021 ± 0.05 s⁻¹. (Upper Left Inset) Linear time axis representation. Photobleaching spots of 0.5 (n = 25, red circle), 0.8 (n = 16, blue triangle) and 1.2 (n = 15, green square) μm width along the junction axis are fitted by first-order exchange with similar coefficients: k_off = 0.021 ± 0.005, 0.018 ± 0.005 and 0.015 ± 0.005 s⁻¹ respectively.