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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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Distribution of E-cadherin in preconfluent MDCK cells. (a) Immunofluorescence staining of E-cadherin in preconfluent cultures treated with or without cycloheximide (+/− CHX). At steady state (−CHX), there is a small amount of cell surface staining, prominent perinuclear staining over the Golgi complex, and bright vesicular staining throughout the cytoplasm. When protein synthesis is blocked (+CHX), the Golgi staining disappears but the vesicular staining remains. Representative fields of cells were photographed with similar exposures. (b) Surface biotinylation of E-cadherin. Preconfluent and confluent cells were surface-biotinylated; biotinylated E-cadherin in detergent-soluble cell extracts was recovered by streptavidin affinity, biotinylated and unbiotinylated (supernatant) fractions were analyzed by SDS-PAGE and immunoblotting. All of the biotinylated fraction and 20% of the total unbiotinylated fraction were loaded. Unbiotinylated E-cadherin from preconfluent cells (lane 1) and confluent cells (lane 3) was compared to the amounts of surface-biotinylated E-cadherin in preconfluent cells (lane 2) and confluent cells (lane 4). There is an increased amount of biotinylated cell surface E-cadherin in confluent cells with a concomitant decrease in the unbiotinylated fraction, which includes the intracellular pool. (c) The relative amounts of E-cadherin in detergent-soluble (biotinylated and unbiotinylated) and detergent-insoluble (TX-insoluble) fractions were compared by immunoblotting and densitometry in preconfluent and confluent cells. Detergent-insoluble E-cadherin, which is likely to represent protein incorporated into cytoskeleton-stabilized junctional complexes, increases in confluent cells. Within the detergent-soluble pool, biotinylated cell surface E-cadherin increases (from ∼10% to ∼50%) in confluent monolayers. Data are means ± SEM from three separate experiments.
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Figure 5: Distribution of E-cadherin in preconfluent MDCK cells. (a) Immunofluorescence staining of E-cadherin in preconfluent cultures treated with or without cycloheximide (+/− CHX). At steady state (−CHX), there is a small amount of cell surface staining, prominent perinuclear staining over the Golgi complex, and bright vesicular staining throughout the cytoplasm. When protein synthesis is blocked (+CHX), the Golgi staining disappears but the vesicular staining remains. Representative fields of cells were photographed with similar exposures. (b) Surface biotinylation of E-cadherin. Preconfluent and confluent cells were surface-biotinylated; biotinylated E-cadherin in detergent-soluble cell extracts was recovered by streptavidin affinity, biotinylated and unbiotinylated (supernatant) fractions were analyzed by SDS-PAGE and immunoblotting. All of the biotinylated fraction and 20% of the total unbiotinylated fraction were loaded. Unbiotinylated E-cadherin from preconfluent cells (lane 1) and confluent cells (lane 3) was compared to the amounts of surface-biotinylated E-cadherin in preconfluent cells (lane 2) and confluent cells (lane 4). There is an increased amount of biotinylated cell surface E-cadherin in confluent cells with a concomitant decrease in the unbiotinylated fraction, which includes the intracellular pool. (c) The relative amounts of E-cadherin in detergent-soluble (biotinylated and unbiotinylated) and detergent-insoluble (TX-insoluble) fractions were compared by immunoblotting and densitometry in preconfluent and confluent cells. Detergent-insoluble E-cadherin, which is likely to represent protein incorporated into cytoskeleton-stabilized junctional complexes, increases in confluent cells. Within the detergent-soluble pool, biotinylated cell surface E-cadherin increases (from ∼10% to ∼50%) in confluent monolayers. Data are means ± SEM from three separate experiments.

Mentions: In light of reports that cell-cell contact may influence recruitment of E-cadherin to the cell surface, we compared the immunofluorescence localization of E-cadherin in MDCK cells grown and maintained at different densities. In confluent cell monolayers, E-cadherin staining was found predominantly at the cell surface, as shown in Fig. 1 a. In contrast, preconfluent cells which were not yet polarized and had not yet formed extensive adherens junctions showed relatively little E-cadherin staining at the cell surface but there was a concomitantly larger intracellular pool of labeled E-cadherin (Fig. 5 a). Some of the intracellular staining in the perinuclear Golgi region disappeared after cycloheximide treatment and is thus likely to represent newly synthesized E-cadherin in the biosynthetic pathway. There was also prominent vesicular staining of E-cadherin in the peripheries of preconfluent cells. As in confluent cells, staining of this vesicular pool was not altered by cycloheximide treatment suggesting that it represents E-cadherin in an endocytic pathway, a pool which is enhanced in preconfluent cells.


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

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

Distribution of E-cadherin in preconfluent MDCK cells. (a) Immunofluorescence staining of E-cadherin in preconfluent cultures treated with or without cycloheximide (+/− CHX). At steady state (−CHX), there is a small amount of cell surface staining, prominent perinuclear staining over the Golgi complex, and bright vesicular staining throughout the cytoplasm. When protein synthesis is blocked (+CHX), the Golgi staining disappears but the vesicular staining remains. Representative fields of cells were photographed with similar exposures. (b) Surface biotinylation of E-cadherin. Preconfluent and confluent cells were surface-biotinylated; biotinylated E-cadherin in detergent-soluble cell extracts was recovered by streptavidin affinity, biotinylated and unbiotinylated (supernatant) fractions were analyzed by SDS-PAGE and immunoblotting. All of the biotinylated fraction and 20% of the total unbiotinylated fraction were loaded. Unbiotinylated E-cadherin from preconfluent cells (lane 1) and confluent cells (lane 3) was compared to the amounts of surface-biotinylated E-cadherin in preconfluent cells (lane 2) and confluent cells (lane 4). There is an increased amount of biotinylated cell surface E-cadherin in confluent cells with a concomitant decrease in the unbiotinylated fraction, which includes the intracellular pool. (c) The relative amounts of E-cadherin in detergent-soluble (biotinylated and unbiotinylated) and detergent-insoluble (TX-insoluble) fractions were compared by immunoblotting and densitometry in preconfluent and confluent cells. Detergent-insoluble E-cadherin, which is likely to represent protein incorporated into cytoskeleton-stabilized junctional complexes, increases in confluent cells. Within the detergent-soluble pool, biotinylated cell surface E-cadherin increases (from ∼10% to ∼50%) in confluent monolayers. Data are means ± SEM from three separate experiments.
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Related In: Results  -  Collection

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Figure 5: Distribution of E-cadherin in preconfluent MDCK cells. (a) Immunofluorescence staining of E-cadherin in preconfluent cultures treated with or without cycloheximide (+/− CHX). At steady state (−CHX), there is a small amount of cell surface staining, prominent perinuclear staining over the Golgi complex, and bright vesicular staining throughout the cytoplasm. When protein synthesis is blocked (+CHX), the Golgi staining disappears but the vesicular staining remains. Representative fields of cells were photographed with similar exposures. (b) Surface biotinylation of E-cadherin. Preconfluent and confluent cells were surface-biotinylated; biotinylated E-cadherin in detergent-soluble cell extracts was recovered by streptavidin affinity, biotinylated and unbiotinylated (supernatant) fractions were analyzed by SDS-PAGE and immunoblotting. All of the biotinylated fraction and 20% of the total unbiotinylated fraction were loaded. Unbiotinylated E-cadherin from preconfluent cells (lane 1) and confluent cells (lane 3) was compared to the amounts of surface-biotinylated E-cadherin in preconfluent cells (lane 2) and confluent cells (lane 4). There is an increased amount of biotinylated cell surface E-cadherin in confluent cells with a concomitant decrease in the unbiotinylated fraction, which includes the intracellular pool. (c) The relative amounts of E-cadherin in detergent-soluble (biotinylated and unbiotinylated) and detergent-insoluble (TX-insoluble) fractions were compared by immunoblotting and densitometry in preconfluent and confluent cells. Detergent-insoluble E-cadherin, which is likely to represent protein incorporated into cytoskeleton-stabilized junctional complexes, increases in confluent cells. Within the detergent-soluble pool, biotinylated cell surface E-cadherin increases (from ∼10% to ∼50%) in confluent monolayers. Data are means ± SEM from three separate experiments.
Mentions: In light of reports that cell-cell contact may influence recruitment of E-cadherin to the cell surface, we compared the immunofluorescence localization of E-cadherin in MDCK cells grown and maintained at different densities. In confluent cell monolayers, E-cadherin staining was found predominantly at the cell surface, as shown in Fig. 1 a. In contrast, preconfluent cells which were not yet polarized and had not yet formed extensive adherens junctions showed relatively little E-cadherin staining at the cell surface but there was a concomitantly larger intracellular pool of labeled E-cadherin (Fig. 5 a). Some of the intracellular staining in the perinuclear Golgi region disappeared after cycloheximide treatment and is thus likely to represent newly synthesized E-cadherin in the biosynthetic pathway. There was also prominent vesicular staining of E-cadherin in the peripheries of preconfluent cells. As in confluent cells, staining of this vesicular pool was not altered by cycloheximide treatment suggesting that it represents E-cadherin in an endocytic pathway, a pool which is enhanced in preconfluent cells.

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