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Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

Le TL, Yap AS, Stow JL - J. Cell Biol. (1999)

Bottom Line: The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment.The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway.We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, 4072 Queensland, Australia.

ABSTRACT
E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

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Recycling of E-cadherin. Cells were surface-biotinylated on ice, then incubated at 18°C for 2 h to allow endocytosis and accumulation of E-cadherin. After glutathione stripping cells were then returned to 37°C. At each chase time (0–15 min) cells were trypsinized to release cell surface proteins. Glutathione stripping immediately after biotinylation effectively removed all surface-biotinylated proteins (g.s., lane 2). Biotinylated proteins from both the cell-associated (a, top) and trypsin-released fractions (a, bottom) were recovered on streptavidin beads and analyzed by SDS-PAGE. Intact E-cadherin (120 kD) or its trypsin-cleaved, 82-kD ecto-domain (a) and TfR (b) were detected by immunoblotting with specific antibodies. (a) The top shows that internalized E-cadherin accumulated at 18°C (lane 3) gradually disappeared from the internal pool over 15 min (lanes 4–7). The bottom shows that the 82-kD ectodomain of biotinylated E-cadherin was initially detected after 5 min at 37°C (lane 5) and maximal amounts were detected after 15 min at 37°C (lane 7) showing that the internalized E-cadherin was recycling and reappearing on the cell surface. (b) Surface-biotinylated (lane 1) TfR was also internalized (lane 4) but disappeared from the internal pool (lanes 5–7) more rapidly under the same conditions. Results are representative of four experiments.
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Figure 3: Recycling of E-cadherin. Cells were surface-biotinylated on ice, then incubated at 18°C for 2 h to allow endocytosis and accumulation of E-cadherin. After glutathione stripping cells were then returned to 37°C. At each chase time (0–15 min) cells were trypsinized to release cell surface proteins. Glutathione stripping immediately after biotinylation effectively removed all surface-biotinylated proteins (g.s., lane 2). Biotinylated proteins from both the cell-associated (a, top) and trypsin-released fractions (a, bottom) were recovered on streptavidin beads and analyzed by SDS-PAGE. Intact E-cadherin (120 kD) or its trypsin-cleaved, 82-kD ecto-domain (a) and TfR (b) were detected by immunoblotting with specific antibodies. (a) The top shows that internalized E-cadherin accumulated at 18°C (lane 3) gradually disappeared from the internal pool over 15 min (lanes 4–7). The bottom shows that the 82-kD ectodomain of biotinylated E-cadherin was initially detected after 5 min at 37°C (lane 5) and maximal amounts were detected after 15 min at 37°C (lane 7) showing that the internalized E-cadherin was recycling and reappearing on the cell surface. (b) Surface-biotinylated (lane 1) TfR was also internalized (lane 4) but disappeared from the internal pool (lanes 5–7) more rapidly under the same conditions. Results are representative of four experiments.

Mentions: In light of the immunofluorescence observation that E-cadherin accumulates intracellularly at 18°C, surface biotinylation was also used to assay the effect of low temperature on the internalization and accumulation of E-cadherin. Whereas at 37°C there was a constant internalized pool of biotinylated E-cadherin (Fig. 2 b), at 18°C the internalized pool of E-cadherin showed progressive accumulation (Fig. 2c and Fig. d). After 20 min at 18°C, 35% of the surface-biotinylated E-cadherin was internalized (Fig. 2 c, lane 5) and by 2 h the majority (80%) of the surface-biotinylated E-cadherin had accumulated inside cells (Fig. 2 c, lane 6). After prolonged accumulation (3 h) some apparent degradation products of E-cadherin were noted on gels (Fig. 3, lane 7). The absence of such bands at 37°C (Fig. 2 b) further suggests that E-cadherin is normally recycled. Overall these results show that there is active internalization of E-cadherin from the cell surface and that its uptake is selective, since other basolateral cell surface proteins, such as Na+K+ATPase, are not undergoing the same process.


Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

Le TL, Yap AS, Stow JL - J. Cell Biol. (1999)

Recycling of E-cadherin. Cells were surface-biotinylated on ice, then incubated at 18°C for 2 h to allow endocytosis and accumulation of E-cadherin. After glutathione stripping cells were then returned to 37°C. At each chase time (0–15 min) cells were trypsinized to release cell surface proteins. Glutathione stripping immediately after biotinylation effectively removed all surface-biotinylated proteins (g.s., lane 2). Biotinylated proteins from both the cell-associated (a, top) and trypsin-released fractions (a, bottom) were recovered on streptavidin beads and analyzed by SDS-PAGE. Intact E-cadherin (120 kD) or its trypsin-cleaved, 82-kD ecto-domain (a) and TfR (b) were detected by immunoblotting with specific antibodies. (a) The top shows that internalized E-cadherin accumulated at 18°C (lane 3) gradually disappeared from the internal pool over 15 min (lanes 4–7). The bottom shows that the 82-kD ectodomain of biotinylated E-cadherin was initially detected after 5 min at 37°C (lane 5) and maximal amounts were detected after 15 min at 37°C (lane 7) showing that the internalized E-cadherin was recycling and reappearing on the cell surface. (b) Surface-biotinylated (lane 1) TfR was also internalized (lane 4) but disappeared from the internal pool (lanes 5–7) more rapidly under the same conditions. Results are representative of four experiments.
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Related In: Results  -  Collection

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Figure 3: Recycling of E-cadherin. Cells were surface-biotinylated on ice, then incubated at 18°C for 2 h to allow endocytosis and accumulation of E-cadherin. After glutathione stripping cells were then returned to 37°C. At each chase time (0–15 min) cells were trypsinized to release cell surface proteins. Glutathione stripping immediately after biotinylation effectively removed all surface-biotinylated proteins (g.s., lane 2). Biotinylated proteins from both the cell-associated (a, top) and trypsin-released fractions (a, bottom) were recovered on streptavidin beads and analyzed by SDS-PAGE. Intact E-cadherin (120 kD) or its trypsin-cleaved, 82-kD ecto-domain (a) and TfR (b) were detected by immunoblotting with specific antibodies. (a) The top shows that internalized E-cadherin accumulated at 18°C (lane 3) gradually disappeared from the internal pool over 15 min (lanes 4–7). The bottom shows that the 82-kD ectodomain of biotinylated E-cadherin was initially detected after 5 min at 37°C (lane 5) and maximal amounts were detected after 15 min at 37°C (lane 7) showing that the internalized E-cadherin was recycling and reappearing on the cell surface. (b) Surface-biotinylated (lane 1) TfR was also internalized (lane 4) but disappeared from the internal pool (lanes 5–7) more rapidly under the same conditions. Results are representative of four experiments.
Mentions: In light of the immunofluorescence observation that E-cadherin accumulates intracellularly at 18°C, surface biotinylation was also used to assay the effect of low temperature on the internalization and accumulation of E-cadherin. Whereas at 37°C there was a constant internalized pool of biotinylated E-cadherin (Fig. 2 b), at 18°C the internalized pool of E-cadherin showed progressive accumulation (Fig. 2c and Fig. d). After 20 min at 18°C, 35% of the surface-biotinylated E-cadherin was internalized (Fig. 2 c, lane 5) and by 2 h the majority (80%) of the surface-biotinylated E-cadherin had accumulated inside cells (Fig. 2 c, lane 6). After prolonged accumulation (3 h) some apparent degradation products of E-cadherin were noted on gels (Fig. 3, lane 7). The absence of such bands at 37°C (Fig. 2 b) further suggests that E-cadherin is normally recycled. Overall these results show that there is active internalization of E-cadherin from the cell surface and that its uptake is selective, since other basolateral cell surface proteins, such as Na+K+ATPase, are not undergoing the same process.

Bottom Line: The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment.The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway.We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, 4072 Queensland, Australia.

ABSTRACT
E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

Show MeSH
Related in: MedlinePlus