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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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Endocytosis of β-catenin. Confluent MDCK cells treated with (+) or without (−) CHX were surface-biotinylated at 0°C, biotinylated proteins were collected with streptavidin beads, and the bound fraction was analyzed by SDS-PAGE and immunoblotting. Typically, E-cadherin was detected in this fraction, with β-catenin also present (lane 1). Glutathione stripping (g.s.) immediately after biotinylation completely removed biotin from surface E-cadherin; neither E-cadherin nor β-catenin was then collected on streptavidin beads (lane 2). Surface-biotinylated cells were then incubated at 18°C, and after 3 h E-cadherin sequestered in an internal pool after glutathione stripping (lanes 3 and 4) was recovered on streptavidin beads. A small amount of β-catenin was also recovered in the biotinylated internalized fraction suggesting that it too was endocytosed (lanes 3 and 4). The results are representative of three separate experiments.
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Figure 11: Endocytosis of β-catenin. Confluent MDCK cells treated with (+) or without (−) CHX were surface-biotinylated at 0°C, biotinylated proteins were collected with streptavidin beads, and the bound fraction was analyzed by SDS-PAGE and immunoblotting. Typically, E-cadherin was detected in this fraction, with β-catenin also present (lane 1). Glutathione stripping (g.s.) immediately after biotinylation completely removed biotin from surface E-cadherin; neither E-cadherin nor β-catenin was then collected on streptavidin beads (lane 2). Surface-biotinylated cells were then incubated at 18°C, and after 3 h E-cadherin sequestered in an internal pool after glutathione stripping (lanes 3 and 4) was recovered on streptavidin beads. A small amount of β-catenin was also recovered in the biotinylated internalized fraction suggesting that it too was endocytosed (lanes 3 and 4). The results are representative of three separate experiments.

Mentions: At the cell surface classical cadherins exist in macromolecular complexes with cytoplasmic catenins (Takeichi 1991, Takeichi 1995; Yap et al. 1997a). As a preliminary investigation into whether catenins are internalized we probed for β-catenin in surface-biotinylated fractions containing E-cadherin. β-Catenin was coisolated with surface-biotinylated E-cadherin (Fig. 11). Then we allowed surface-biotinylated E-cadherin to be internalized at 18°C, collected the internalized pool on streptavidin beads after surface stripping with glutathione, and probed for β-catenin (Fig. 11). As shown previously (Fig. 2 c), the majority of surface-biotinylated E-cadherin is internalized under these conditions (Fig. 11). Immunoblotting showed that β-catenin is also present in the internalized, biotinylated fraction under these conditions. Insofar as E-cadherin is the major surface protein known to associate with β-catenin in MDCK cells, we assume that β-catenin and E-cadherin are being internalized as a complex. Interestingly, only a relatively small amount of β-catenin is internalized compared to the initial surface-associated pool. Thus, internalization may result in altered stoichiometry of cadherin-catenin complexes. Further studies are now required to extend these observations.


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

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

Endocytosis of β-catenin. Confluent MDCK cells treated with (+) or without (−) CHX were surface-biotinylated at 0°C, biotinylated proteins were collected with streptavidin beads, and the bound fraction was analyzed by SDS-PAGE and immunoblotting. Typically, E-cadherin was detected in this fraction, with β-catenin also present (lane 1). Glutathione stripping (g.s.) immediately after biotinylation completely removed biotin from surface E-cadherin; neither E-cadherin nor β-catenin was then collected on streptavidin beads (lane 2). Surface-biotinylated cells were then incubated at 18°C, and after 3 h E-cadherin sequestered in an internal pool after glutathione stripping (lanes 3 and 4) was recovered on streptavidin beads. A small amount of β-catenin was also recovered in the biotinylated internalized fraction suggesting that it too was endocytosed (lanes 3 and 4). The results are representative of three separate experiments.
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Related In: Results  -  Collection

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Figure 11: Endocytosis of β-catenin. Confluent MDCK cells treated with (+) or without (−) CHX were surface-biotinylated at 0°C, biotinylated proteins were collected with streptavidin beads, and the bound fraction was analyzed by SDS-PAGE and immunoblotting. Typically, E-cadherin was detected in this fraction, with β-catenin also present (lane 1). Glutathione stripping (g.s.) immediately after biotinylation completely removed biotin from surface E-cadherin; neither E-cadherin nor β-catenin was then collected on streptavidin beads (lane 2). Surface-biotinylated cells were then incubated at 18°C, and after 3 h E-cadherin sequestered in an internal pool after glutathione stripping (lanes 3 and 4) was recovered on streptavidin beads. A small amount of β-catenin was also recovered in the biotinylated internalized fraction suggesting that it too was endocytosed (lanes 3 and 4). The results are representative of three separate experiments.
Mentions: At the cell surface classical cadherins exist in macromolecular complexes with cytoplasmic catenins (Takeichi 1991, Takeichi 1995; Yap et al. 1997a). As a preliminary investigation into whether catenins are internalized we probed for β-catenin in surface-biotinylated fractions containing E-cadherin. β-Catenin was coisolated with surface-biotinylated E-cadherin (Fig. 11). Then we allowed surface-biotinylated E-cadherin to be internalized at 18°C, collected the internalized pool on streptavidin beads after surface stripping with glutathione, and probed for β-catenin (Fig. 11). As shown previously (Fig. 2 c), the majority of surface-biotinylated E-cadherin is internalized under these conditions (Fig. 11). Immunoblotting showed that β-catenin is also present in the internalized, biotinylated fraction under these conditions. Insofar as E-cadherin is the major surface protein known to associate with β-catenin in MDCK cells, we assume that β-catenin and E-cadherin are being internalized as a complex. Interestingly, only a relatively small amount of β-catenin is internalized compared to the initial surface-associated pool. Thus, internalization may result in altered stoichiometry of cadherin-catenin complexes. Further studies are now required to extend these observations.

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