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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

E-Cadherin distribution in confluent MDCK cells. (a) Immunofluorescence staining of E-cadherin is mostly localized to the lateral cell surfaces in confluent MDCK monolayers. Small amounts of intracellular, vesicular staining can also be seen scattered throughout the periphery of cells. (b) When cells were fixed and stained after incubation at 18°C, intracellular staining of E-cadherin was more intense. The vesicular staining relocated and was now clustered in a perinuclear position. (c and d) Preincubation of cells in cycloheximide (CHX) did not significantly alter the staining pattern; both cell surface and vesicular staining can still be seen. (e and f) Phalloidin staining of F-actin at the level of adhesion junctions shows that cell morphology was preserved in both control cultures and in monolayers incubated at 18°C. Representative fields of cells were photographed with similar exposures.
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Figure 1: E-Cadherin distribution in confluent MDCK cells. (a) Immunofluorescence staining of E-cadherin is mostly localized to the lateral cell surfaces in confluent MDCK monolayers. Small amounts of intracellular, vesicular staining can also be seen scattered throughout the periphery of cells. (b) When cells were fixed and stained after incubation at 18°C, intracellular staining of E-cadherin was more intense. The vesicular staining relocated and was now clustered in a perinuclear position. (c and d) Preincubation of cells in cycloheximide (CHX) did not significantly alter the staining pattern; both cell surface and vesicular staining can still be seen. (e and f) Phalloidin staining of F-actin at the level of adhesion junctions shows that cell morphology was preserved in both control cultures and in monolayers incubated at 18°C. Representative fields of cells were photographed with similar exposures.

Mentions: E-Cadherin in confluent MDCK cells was localized mostly on the lateral plasma membrane. However, small amounts of staining were also seen intracellularly in a punctate, vesicular pattern close to the cell surface (Fig. 1 a). Nonpermeabilized cells showed no comparable punctate staining (data not shown), confirming that this staining pattern was intracellular. To see whether the internal pool of E-cadherin is possibly a result of endocytosis from the cell surface, we incubated cells at 18°C for 2 h before fixation and staining. An 18°C temperature block has been shown to cause the accumulation of endocytosed proteins in early or sorting endosomes (Czekay et al. 1997). The 18°C temperature block did not adversely affect cell morphology or cell-cell contacts as shown by the F-actin staining pattern (Fig. 1e and Fig. f), nor was there a detectable change in the cell surface staining of E-cadherin at 18°C. However, the vesicular staining of E-cadherin was more pronounced in cells incubated at 18°C (Fig. 1 b). Notably, at 18°C the location of the vesicular E-cadherin staining within the cells was now more prominent in the perinuclear region rather than being at the cell periphery. E-Cadherin staining was largely unaltered in cells pretreated with cycloheximide to stop protein synthesis (Fig. 1 d), indicating that both the cell surface staining and the intracellular staining represent stable pools of E-cadherin. Thus, while the majority of E-cadherin is present on the lateral plasma membrane, the presence of intracellular staining suggests that a pool of E-cadherin may be internalized from the cell surface, where it accumulates at 18°C.


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

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

E-Cadherin distribution in confluent MDCK cells. (a) Immunofluorescence staining of E-cadherin is mostly localized to the lateral cell surfaces in confluent MDCK monolayers. Small amounts of intracellular, vesicular staining can also be seen scattered throughout the periphery of cells. (b) When cells were fixed and stained after incubation at 18°C, intracellular staining of E-cadherin was more intense. The vesicular staining relocated and was now clustered in a perinuclear position. (c and d) Preincubation of cells in cycloheximide (CHX) did not significantly alter the staining pattern; both cell surface and vesicular staining can still be seen. (e and f) Phalloidin staining of F-actin at the level of adhesion junctions shows that cell morphology was preserved in both control cultures and in monolayers incubated at 18°C. Representative fields of cells were photographed with similar exposures.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: E-Cadherin distribution in confluent MDCK cells. (a) Immunofluorescence staining of E-cadherin is mostly localized to the lateral cell surfaces in confluent MDCK monolayers. Small amounts of intracellular, vesicular staining can also be seen scattered throughout the periphery of cells. (b) When cells were fixed and stained after incubation at 18°C, intracellular staining of E-cadherin was more intense. The vesicular staining relocated and was now clustered in a perinuclear position. (c and d) Preincubation of cells in cycloheximide (CHX) did not significantly alter the staining pattern; both cell surface and vesicular staining can still be seen. (e and f) Phalloidin staining of F-actin at the level of adhesion junctions shows that cell morphology was preserved in both control cultures and in monolayers incubated at 18°C. Representative fields of cells were photographed with similar exposures.
Mentions: E-Cadherin in confluent MDCK cells was localized mostly on the lateral plasma membrane. However, small amounts of staining were also seen intracellularly in a punctate, vesicular pattern close to the cell surface (Fig. 1 a). Nonpermeabilized cells showed no comparable punctate staining (data not shown), confirming that this staining pattern was intracellular. To see whether the internal pool of E-cadherin is possibly a result of endocytosis from the cell surface, we incubated cells at 18°C for 2 h before fixation and staining. An 18°C temperature block has been shown to cause the accumulation of endocytosed proteins in early or sorting endosomes (Czekay et al. 1997). The 18°C temperature block did not adversely affect cell morphology or cell-cell contacts as shown by the F-actin staining pattern (Fig. 1e and Fig. f), nor was there a detectable change in the cell surface staining of E-cadherin at 18°C. However, the vesicular staining of E-cadherin was more pronounced in cells incubated at 18°C (Fig. 1 b). Notably, at 18°C the location of the vesicular E-cadherin staining within the cells was now more prominent in the perinuclear region rather than being at the cell periphery. E-Cadherin staining was largely unaltered in cells pretreated with cycloheximide to stop protein synthesis (Fig. 1 d), indicating that both the cell surface staining and the intracellular staining represent stable pools of E-cadherin. Thus, while the majority of E-cadherin is present on the lateral plasma membrane, the presence of intracellular staining suggests that a pool of E-cadherin may be internalized from the cell surface, where it accumulates at 18°C.

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