Figure 6.
4.1R+17b forms functionally connect to the reassembly of E-cadherin at the AJs. MDCK cells transfected with pEGFP (A), EGFP–4.1R80 (B), or EGFP–4.1R80+17b (C) were examined for junctional reassembly at the indicated period of time after calcium repletion in immunofluorescence-labeled analyses using an anti-E-cadherin antibody. Regions were selected that contained a mix of transfected and untransfected cells. A, no difference in reassembly of E-cadherin at the AJs in vector pEGFP-expressing and -nonexpressing cells. Reassembly of E-cadherin to the cell–cell contacts of both expressing and nonexpressing cells was detected 0.5 h after calcium repletion. E-cadherin continues to reassemble to AJs after 1 h and significantly accumulates at newly assembled AJs of all cells after 2 h of calcium repletion. Intense E-cadherin localizes at the AJs at 3 h and at any time greater than 3 h after calcium repletion. B, expression of EGFP–4.1R80 causes reduced E-cadherin at the cell–cell contacts of the expressing cells but not that of nonexpressing cells throughout the AJ reassembly process. Fluorescent labeling shows rapid formation of circumferential E-cadherin at the junction of the nonexpressing cells (arrows) but not that of expressing cells (arrowheads) 0.5 h after calcium repletion. The occurrence persists all the way through confluent monolayer maturation 8 h after calcium repletion. C, expression of EGFP–4.1R80+17b does not affect the reassembly of E-cadherin at the cell–cell contacts of the expressing cells when compared with that of the nonexpressing cells. E-cadherin appears at the cell–cell contacts of all cells 0.5 h after calcium repletion. Mature monolayer cells with equal intensity of E-cadherin at AJs of all cells 3 h after calcium repletion. Composite images were generated by superimposition of the EGFP (green) and E-cadherin (red) signals; areas of overlap appear yellow. Bar, 10 μm. D, quantification of percent of junction assembly after calcium switch. The outline of the cell was drawn as the length of the cell periphery, and the regions of cell–cell contact labeled by E-cadherin were drawn as labeled length. Fifty EGFP+ cells were measured for both cell periphery length and E-cadherin–labeled length. The percentage of junction assembled was obtained by the ratio of the E-cadherin–labeled length over the length of the cell periphery, and the data were analyzed using GraphPad Prism 8 software and expressed as mean ± S.E. of mean. Asterisks indicate statistical significance (***, p < 0.001).