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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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Related in: MedlinePlus

Internalization of surface-biotinylated E-cadherin. (a) Confluent MDCK cells treated with (+) or without (−) cycloheximide (CHX) were surface-biotinylated at 0°C. Detergent-soluble, surface-biotinylated proteins were recovered on streptavidin beads and analyzed by SDS-PAGE. E-Cadherin and Na+K+ATPase in these fractions were detected by immunoblotting as depicted. Biotinylated cell surface E-cadherin was recovered from the cell extracts (lanes 1 and 2). Glutathione stripping (g.s.) immediately after biotinylation at 0°C completely removed biotin from surface E-cadherin (lane 3). Surface-biotinylated cells were then incubated at 37°C and after 1 h, total biotinylated E-cadherin was recovered (lanes 4 and 5). E-Cadherin sequestered in an internal pool was recovered after glutathione stripping showing that some of the surface E-cadherin was endocytosed (lanes 6 and 7). Na+K+ATPase was biotinylated (lanes 1 and 2) but was not found in an internal pool under the same conditions (lanes 6 and 7). (b) A time course of E-cadherin uptake. CHX-treated cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C for periods of 0–180 min (lanes 2–7). A constant amount of surface-biotinylated E-cadherin was sequestered from glutathione stripping at chase times from 5–180 min (lanes 3–7), consistent with a steady-state flux of E-cadherin into and out of this intracellular pool. (c) When the same experiments were carried out on MDCK cells incubated at 18°C, internalized E-cadherin now accumulated, resulting in increasing amounts of E-cadherin in the internal pool over 180 min (lanes 3–7). Fainter, lower molecular weight bands (lane 6 and 7) may be due to some degradation of internalized E-cadherin after prolonged incubations. (d) Relative amounts of internalized E-cadherin, quantitated by densitometry and expressed as a percentage of the total initial surface-biotinylated pool in cells incubated at 37°C or 18°C. Data are means ± SEM from three separate experiments.
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Figure 2: Internalization of surface-biotinylated E-cadherin. (a) Confluent MDCK cells treated with (+) or without (−) cycloheximide (CHX) were surface-biotinylated at 0°C. Detergent-soluble, surface-biotinylated proteins were recovered on streptavidin beads and analyzed by SDS-PAGE. E-Cadherin and Na+K+ATPase in these fractions were detected by immunoblotting as depicted. Biotinylated cell surface E-cadherin was recovered from the cell extracts (lanes 1 and 2). Glutathione stripping (g.s.) immediately after biotinylation at 0°C completely removed biotin from surface E-cadherin (lane 3). Surface-biotinylated cells were then incubated at 37°C and after 1 h, total biotinylated E-cadherin was recovered (lanes 4 and 5). E-Cadherin sequestered in an internal pool was recovered after glutathione stripping showing that some of the surface E-cadherin was endocytosed (lanes 6 and 7). Na+K+ATPase was biotinylated (lanes 1 and 2) but was not found in an internal pool under the same conditions (lanes 6 and 7). (b) A time course of E-cadherin uptake. CHX-treated cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C for periods of 0–180 min (lanes 2–7). A constant amount of surface-biotinylated E-cadherin was sequestered from glutathione stripping at chase times from 5–180 min (lanes 3–7), consistent with a steady-state flux of E-cadherin into and out of this intracellular pool. (c) When the same experiments were carried out on MDCK cells incubated at 18°C, internalized E-cadherin now accumulated, resulting in increasing amounts of E-cadherin in the internal pool over 180 min (lanes 3–7). Fainter, lower molecular weight bands (lane 6 and 7) may be due to some degradation of internalized E-cadherin after prolonged incubations. (d) Relative amounts of internalized E-cadherin, quantitated by densitometry and expressed as a percentage of the total initial surface-biotinylated pool in cells incubated at 37°C or 18°C. Data are means ± SEM from three separate experiments.

Mentions: To establish whether surface E-cadherin can be internalized we developed an assay to track the uptake of E-cadherin labeled by biotinylation on the basolateral surfaces of MDCK cells. As described in Materials and Methods, cells were surface-biotinylated at 0°C then returned to 37°C for 1 h to allow trafficking to resume. Cells were then incubated in several washes of glutathione solution at 0°C to remove covalently bound biotin groups from amines exposed on the cell surface. Biotinylated E-cadherin internalized at 37°C should be sequestered and, therefore, protected from glutathione stripping. Cycloheximide-treated cells were included in all experiments to eliminate the pool of newly synthesized E-cadherin from consideration. In control experiments, cells were surface-biotinylated for 1 h at 0°C, then immediately washed in glutathione at 0°C. No E-cadherin was recovered in the biotinylated fraction (Fig. 2 a, lane 3), confirming that under these conditions glutathione efficiently stripped all biotinyl groups from surface proteins. In contrast, after 1 h at 37°C a biotinylated pool of E-cadherin was detected in cells following glutathione stripping of surface proteins (Fig. 2 a, lanes 6 and 7). This pool represented ∼13% of the total E-cadherin biotinylated at the beginning of the experiment, indicating that upon return to physiological temperature, a small amount of surface E-cadherin was internalized and hence protected from glutathione stripping. In contrast, the basolateral membrane protein Na+K+ATPase did not undergo internalization under the same conditions, since a glutathione-resistant pool of Na+K+ATPase was not detected after incubating biotinylated cells at 37°C for 1 h (Fig. 2 a, lanes 6 and 7).


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

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

Internalization of surface-biotinylated E-cadherin. (a) Confluent MDCK cells treated with (+) or without (−) cycloheximide (CHX) were surface-biotinylated at 0°C. Detergent-soluble, surface-biotinylated proteins were recovered on streptavidin beads and analyzed by SDS-PAGE. E-Cadherin and Na+K+ATPase in these fractions were detected by immunoblotting as depicted. Biotinylated cell surface E-cadherin was recovered from the cell extracts (lanes 1 and 2). Glutathione stripping (g.s.) immediately after biotinylation at 0°C completely removed biotin from surface E-cadherin (lane 3). Surface-biotinylated cells were then incubated at 37°C and after 1 h, total biotinylated E-cadherin was recovered (lanes 4 and 5). E-Cadherin sequestered in an internal pool was recovered after glutathione stripping showing that some of the surface E-cadherin was endocytosed (lanes 6 and 7). Na+K+ATPase was biotinylated (lanes 1 and 2) but was not found in an internal pool under the same conditions (lanes 6 and 7). (b) A time course of E-cadherin uptake. CHX-treated cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C for periods of 0–180 min (lanes 2–7). A constant amount of surface-biotinylated E-cadherin was sequestered from glutathione stripping at chase times from 5–180 min (lanes 3–7), consistent with a steady-state flux of E-cadherin into and out of this intracellular pool. (c) When the same experiments were carried out on MDCK cells incubated at 18°C, internalized E-cadherin now accumulated, resulting in increasing amounts of E-cadherin in the internal pool over 180 min (lanes 3–7). Fainter, lower molecular weight bands (lane 6 and 7) may be due to some degradation of internalized E-cadherin after prolonged incubations. (d) Relative amounts of internalized E-cadherin, quantitated by densitometry and expressed as a percentage of the total initial surface-biotinylated pool in cells incubated at 37°C or 18°C. Data are means ± SEM from three separate experiments.
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Related In: Results  -  Collection

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Figure 2: Internalization of surface-biotinylated E-cadherin. (a) Confluent MDCK cells treated with (+) or without (−) cycloheximide (CHX) were surface-biotinylated at 0°C. Detergent-soluble, surface-biotinylated proteins were recovered on streptavidin beads and analyzed by SDS-PAGE. E-Cadherin and Na+K+ATPase in these fractions were detected by immunoblotting as depicted. Biotinylated cell surface E-cadherin was recovered from the cell extracts (lanes 1 and 2). Glutathione stripping (g.s.) immediately after biotinylation at 0°C completely removed biotin from surface E-cadherin (lane 3). Surface-biotinylated cells were then incubated at 37°C and after 1 h, total biotinylated E-cadherin was recovered (lanes 4 and 5). E-Cadherin sequestered in an internal pool was recovered after glutathione stripping showing that some of the surface E-cadherin was endocytosed (lanes 6 and 7). Na+K+ATPase was biotinylated (lanes 1 and 2) but was not found in an internal pool under the same conditions (lanes 6 and 7). (b) A time course of E-cadherin uptake. CHX-treated cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C for periods of 0–180 min (lanes 2–7). A constant amount of surface-biotinylated E-cadherin was sequestered from glutathione stripping at chase times from 5–180 min (lanes 3–7), consistent with a steady-state flux of E-cadherin into and out of this intracellular pool. (c) When the same experiments were carried out on MDCK cells incubated at 18°C, internalized E-cadherin now accumulated, resulting in increasing amounts of E-cadherin in the internal pool over 180 min (lanes 3–7). Fainter, lower molecular weight bands (lane 6 and 7) may be due to some degradation of internalized E-cadherin after prolonged incubations. (d) Relative amounts of internalized E-cadherin, quantitated by densitometry and expressed as a percentage of the total initial surface-biotinylated pool in cells incubated at 37°C or 18°C. Data are means ± SEM from three separate experiments.
Mentions: To establish whether surface E-cadherin can be internalized we developed an assay to track the uptake of E-cadherin labeled by biotinylation on the basolateral surfaces of MDCK cells. As described in Materials and Methods, cells were surface-biotinylated at 0°C then returned to 37°C for 1 h to allow trafficking to resume. Cells were then incubated in several washes of glutathione solution at 0°C to remove covalently bound biotin groups from amines exposed on the cell surface. Biotinylated E-cadherin internalized at 37°C should be sequestered and, therefore, protected from glutathione stripping. Cycloheximide-treated cells were included in all experiments to eliminate the pool of newly synthesized E-cadherin from consideration. In control experiments, cells were surface-biotinylated for 1 h at 0°C, then immediately washed in glutathione at 0°C. No E-cadherin was recovered in the biotinylated fraction (Fig. 2 a, lane 3), confirming that under these conditions glutathione efficiently stripped all biotinyl groups from surface proteins. In contrast, after 1 h at 37°C a biotinylated pool of E-cadherin was detected in cells following glutathione stripping of surface proteins (Fig. 2 a, lanes 6 and 7). This pool represented ∼13% of the total E-cadherin biotinylated at the beginning of the experiment, indicating that upon return to physiological temperature, a small amount of surface E-cadherin was internalized and hence protected from glutathione stripping. In contrast, the basolateral membrane protein Na+K+ATPase did not undergo internalization under the same conditions, since a glutathione-resistant pool of Na+K+ATPase was not detected after incubating biotinylated cells at 37°C for 1 h (Fig. 2 a, lanes 6 and 7).

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