Abstract
E-cadherin and nectins are major cell-cell adhesion molecules at adherens junctions (AJs) in epithelial cells. When Madin-Darby canine kidney (MDCK) cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at normal Ca(2+), E-cadherin and nectin-1 are concentrated at the cell-cell contact sites. When these cells are cultured at low Ca(2+), E-cadherin disappears from the cell-cell contact sites, but nectin-1 persists there. When these cells are re-cultured at normal Ca(2+), E-cadherin is recruited to the nectin-based cell-cell contact sites. We found here that this recruitment was dependent on protein synthesis, because a protein synthesis inhibitor, cycloheximide, prevented the accumulation of E-cadherin. When nectin-1-MDCK cells, precultured at low Ca(2+) in the presence of a proteasome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal), were re-cultured at normal Ca(2+), E-cadherin was recruited to the nectin-based cell-cell contact sites but the level of E-cadherin was reduced. Similar results were obtained when wild-type MDCK cells were used instead of nectin-1-MDCK cells. These results suggest that degradation of one or more protein factors and de novo synthesis of the same or different protein factor(s) are needed for the formation of the E-cadherin-based AJs. We biochemically identified the annexin II-S100A10 complex as such a candidate. Depletion of plasma membrane cholesterol, which abolished the localization of the annexin II-S100A10 complex at the plasma membrane, inhibited the re-concentration of E-cadherin at the nectin-based cell-cell contact sites in the Ca(2+) switch experiment. Knockdown of annexin II by RNA interference also inhibited the re-concentration of E-cadherin. These results indicate that the annexin II-S100A10 complex is involved in the formation of the E-cadherin-based AJs in MDCK cells.
Highlights
Cell-cell junctions have essential roles in various cellular functions, including morphogenesis, differentiation, proliferation, and migration [1,2,3,4,5]
When nectin-1MDCK cells are cultured at 2 M Ca2ϩ for 3 h, nectin-1 remains at the cell-cell contact sites, whereas E-cadherin disappears from these sites [41, 42]
When nectin-1MDCK cells were cultured at 2 M Ca2ϩ for 3 h in the presence of cycloheximide, nectin-1 remained at the cell-cell contact sites, whereas E-cadherin disappeared from these sites (Fig. 1, Ab and Ac)
Summary
Antibodies, Chemicals, and Expression Vectors—A rabbit anti-nectin-1 polyclonal antibody (pAb) was prepared as described [30]. The cells were washed with DMEM and cultured at 2 mM Ca2ϩ in DMEM without serum in the presence or absence of 10 M cycloheximide, 50 M ALLN, or 50 M ALLM for 2 h. Stage of the protein synthesis for the formation of the E-cadherin-based AJs. Left panels, nectin-1-MDCK cells were cultured at 2 M Ca2ϩ for 3 h in the presence of 10 M cycloheximide. The samples were subjected to SDS-PAGE, followed by Western blotting with the anti-nectin-1, anti-E-cadherin, and anti-ERK1/2 Abs. Isolation of the Plasma Membrane Fraction—After the Ca2ϩ switch, nectin-1-MDCK cells were washed with PBS, and sonicated in Buffer A (10 mM HEPES-NaOH at pH 7.5, 100 mM KCl, 1 mM MgCl2, and 25 mM NaHCO3) on ice for 15 s six times at 3-min intervals. SDS-PAGE was done as described by Laemmli [40]
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