Limits...
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.

Show MeSH

Related in: MedlinePlus

Endocytosis of E-cadherin in the presence of bafilomycin A1 (BAF). Cells were surface-biotinylated at 0°C and then incubated at 37°C in the presence (+) or absence (−) of 1 μM BAF. Surface-biotinylated E-cadherin (lane 1) was recovered from total cell extracts (lanes 3 and 4) and from the internal pool (lanes 5 and 6) sequestered from glutathione stripping. A greater amount of internalized biotinylated E-cadherin was recovered after BAF treatment (lane 6) in comparison to the normal amount of internal E-cadherin (lane 5), consistent with BAF-induced accumulation of E-cadherin. The result is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199726&req=5

Figure 4: Endocytosis of E-cadherin in the presence of bafilomycin A1 (BAF). Cells were surface-biotinylated at 0°C and then incubated at 37°C in the presence (+) or absence (−) of 1 μM BAF. Surface-biotinylated E-cadherin (lane 1) was recovered from total cell extracts (lanes 3 and 4) and from the internal pool (lanes 5 and 6) sequestered from glutathione stripping. A greater amount of internalized biotinylated E-cadherin was recovered after BAF treatment (lane 6) in comparison to the normal amount of internal E-cadherin (lane 5), consistent with BAF-induced accumulation of E-cadherin. The result is representative of three independent experiments.

Mentions: To further test the influence of recycling on the internal pool of E-cadherin, we treated MDCK cells with bafilomycin A1. Bafilomycin A1 inhibits recycling by blocking transport of endocytosed material back to the cell surface at a late endosomal stage (Johnson et al. 1993; Presley et al. 1997). Cells were surface biotinylated at 0°C then incubated in the presence or absence of bafilomycin A1 (1 μM) at 37°C, followed by glutathione stripping. In the presence of bafilomycin A1 there was a threefold increase in the glutathione-resistant pool of E-cadherin (Fig. 4, lane 6) in comparison to the normal amount of internalized E-cadherin (Fig. 4, lane 5). Bafilomycin A1 was therefore effective in accumulating the endocytosed E-cadherin, consistent with a block in the recycling of E-cadherin.


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

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

Endocytosis of E-cadherin in the presence of bafilomycin A1 (BAF). Cells were surface-biotinylated at 0°C and then incubated at 37°C in the presence (+) or absence (−) of 1 μM BAF. Surface-biotinylated E-cadherin (lane 1) was recovered from total cell extracts (lanes 3 and 4) and from the internal pool (lanes 5 and 6) sequestered from glutathione stripping. A greater amount of internalized biotinylated E-cadherin was recovered after BAF treatment (lane 6) in comparison to the normal amount of internal E-cadherin (lane 5), consistent with BAF-induced accumulation of E-cadherin. The result is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199726&req=5

Figure 4: Endocytosis of E-cadherin in the presence of bafilomycin A1 (BAF). Cells were surface-biotinylated at 0°C and then incubated at 37°C in the presence (+) or absence (−) of 1 μM BAF. Surface-biotinylated E-cadherin (lane 1) was recovered from total cell extracts (lanes 3 and 4) and from the internal pool (lanes 5 and 6) sequestered from glutathione stripping. A greater amount of internalized biotinylated E-cadherin was recovered after BAF treatment (lane 6) in comparison to the normal amount of internal E-cadherin (lane 5), consistent with BAF-induced accumulation of E-cadherin. The result is representative of three independent experiments.
Mentions: To further test the influence of recycling on the internal pool of E-cadherin, we treated MDCK cells with bafilomycin A1. Bafilomycin A1 inhibits recycling by blocking transport of endocytosed material back to the cell surface at a late endosomal stage (Johnson et al. 1993; Presley et al. 1997). Cells were surface biotinylated at 0°C then incubated in the presence or absence of bafilomycin A1 (1 μM) at 37°C, followed by glutathione stripping. In the presence of bafilomycin A1 there was a threefold increase in the glutathione-resistant pool of E-cadherin (Fig. 4, lane 6) in comparison to the normal amount of internalized E-cadherin (Fig. 4, lane 5). Bafilomycin A1 was therefore effective in accumulating the endocytosed E-cadherin, consistent with a block in the recycling of E-cadherin.

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