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Cell surface N-glycans influence the level of functional E-cadherin at the cell-cell border.

Hall MK, Weidner DA, Dayal S, Schwalbe RA - FEBS Open Bio (2014)

Bottom Line: While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border.Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions.We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.

ABSTRACT
E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

No MeSH data available.


Related in: MedlinePlus

Localization of E-cadherin at the cell–cell border in live cells treated with PNGase F. Representative TIRF (colored) and DIC (gray) microscopic images of EGFP tagged E-cadherin transfected Pro-5 cells treated without and with PNGase F prior to (A) and after (B) establishment of cell–cell contacts as indicated. Representative scale bar (5 μM) was identical for all images. White arrows identify cell–cell interface. Bar graphs represent the fluorescence intensity measurement at the cell–cell interface (Icell–cell) divided by that away from the cell–cell interface (Icell) of the cell membrane patch of E-cadherin expressed in Pro-5 cells treated before (C) and after (D) cell–cell contacts were established. At the 0.00001 level, the differences of the population means are significantly different by Student t-test (∗). Data was collected from at least 3 experiments and n denotes the number of measurements.
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f0015: Localization of E-cadherin at the cell–cell border in live cells treated with PNGase F. Representative TIRF (colored) and DIC (gray) microscopic images of EGFP tagged E-cadherin transfected Pro-5 cells treated without and with PNGase F prior to (A) and after (B) establishment of cell–cell contacts as indicated. Representative scale bar (5 μM) was identical for all images. White arrows identify cell–cell interface. Bar graphs represent the fluorescence intensity measurement at the cell–cell interface (Icell–cell) divided by that away from the cell–cell interface (Icell) of the cell membrane patch of E-cadherin expressed in Pro-5 cells treated before (C) and after (D) cell–cell contacts were established. At the 0.00001 level, the differences of the population means are significantly different by Student t-test (∗). Data was collected from at least 3 experiments and n denotes the number of measurements.

Mentions: TIRF and DIC microscopic techniques were employed to determine the level of EGFP tagged E-cadherin protein at the cell–cell border of live cells as we previously described [12,17]. Images of cells in similar planes were also obtained in the wide-field mode (not shown) to verify that E-cadherin was in or near the plasma membrane in the TIRF mode. Representative TIRF (RGB color mode) and DIC (grayscale mode) images of E-cadherin transfected cells treated without (−) and with (+) PNGase F before (Fig. 3A) and after (Fig. 3B) formation of cell–cell contacts are shown. In all cases, the fluorescence signal of the adherent membrane patch was more concentrated at the cell–cell border than free of this border. However, the fluorescence signal at the cell–cell border was weakened and that free of the cell–cell interface was strengthened in transfected Pro-5 cells treated with PNGase F relative to those cells treated without PNGase F. Both regions of interest were verified by the accompanied DIC images. To further quantify the levels of E-cadherin at and free of the cell–cell border, we determined the ratio of the fluorescence intensity signal at the cell–cell border (Icell–cell) of the membrane patch to that free of this border (Icell) [12]. When cells were treated with PNGase F, there was a 43% and 24% reduction in the amount of fluorescence signal at the cell–cell border for cells treated with PNGase prior to (Fig. 3C) and after (Fig. 3D) cell–cell contacts were established, respectively, relative to those cells without PNGase F treatment. Hence, our results indicate that the amount of N-glycans at the cell surface has a role in regulating the clustering of E-cadherin to the cell–cell interface, along with the retention of E-cadherin at this interface.


Cell surface N-glycans influence the level of functional E-cadherin at the cell-cell border.

Hall MK, Weidner DA, Dayal S, Schwalbe RA - FEBS Open Bio (2014)

Localization of E-cadherin at the cell–cell border in live cells treated with PNGase F. Representative TIRF (colored) and DIC (gray) microscopic images of EGFP tagged E-cadherin transfected Pro-5 cells treated without and with PNGase F prior to (A) and after (B) establishment of cell–cell contacts as indicated. Representative scale bar (5 μM) was identical for all images. White arrows identify cell–cell interface. Bar graphs represent the fluorescence intensity measurement at the cell–cell interface (Icell–cell) divided by that away from the cell–cell interface (Icell) of the cell membrane patch of E-cadherin expressed in Pro-5 cells treated before (C) and after (D) cell–cell contacts were established. At the 0.00001 level, the differences of the population means are significantly different by Student t-test (∗). Data was collected from at least 3 experiments and n denotes the number of measurements.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0015: Localization of E-cadherin at the cell–cell border in live cells treated with PNGase F. Representative TIRF (colored) and DIC (gray) microscopic images of EGFP tagged E-cadherin transfected Pro-5 cells treated without and with PNGase F prior to (A) and after (B) establishment of cell–cell contacts as indicated. Representative scale bar (5 μM) was identical for all images. White arrows identify cell–cell interface. Bar graphs represent the fluorescence intensity measurement at the cell–cell interface (Icell–cell) divided by that away from the cell–cell interface (Icell) of the cell membrane patch of E-cadherin expressed in Pro-5 cells treated before (C) and after (D) cell–cell contacts were established. At the 0.00001 level, the differences of the population means are significantly different by Student t-test (∗). Data was collected from at least 3 experiments and n denotes the number of measurements.
Mentions: TIRF and DIC microscopic techniques were employed to determine the level of EGFP tagged E-cadherin protein at the cell–cell border of live cells as we previously described [12,17]. Images of cells in similar planes were also obtained in the wide-field mode (not shown) to verify that E-cadherin was in or near the plasma membrane in the TIRF mode. Representative TIRF (RGB color mode) and DIC (grayscale mode) images of E-cadherin transfected cells treated without (−) and with (+) PNGase F before (Fig. 3A) and after (Fig. 3B) formation of cell–cell contacts are shown. In all cases, the fluorescence signal of the adherent membrane patch was more concentrated at the cell–cell border than free of this border. However, the fluorescence signal at the cell–cell border was weakened and that free of the cell–cell interface was strengthened in transfected Pro-5 cells treated with PNGase F relative to those cells treated without PNGase F. Both regions of interest were verified by the accompanied DIC images. To further quantify the levels of E-cadherin at and free of the cell–cell border, we determined the ratio of the fluorescence intensity signal at the cell–cell border (Icell–cell) of the membrane patch to that free of this border (Icell) [12]. When cells were treated with PNGase F, there was a 43% and 24% reduction in the amount of fluorescence signal at the cell–cell border for cells treated with PNGase prior to (Fig. 3C) and after (Fig. 3D) cell–cell contacts were established, respectively, relative to those cells without PNGase F treatment. Hence, our results indicate that the amount of N-glycans at the cell surface has a role in regulating the clustering of E-cadherin to the cell–cell interface, along with the retention of E-cadherin at this interface.

Bottom Line: While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border.Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions.We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.

ABSTRACT
E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

No MeSH data available.


Related in: MedlinePlus