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Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin.

Farr GA, Hull M, Stoops EH, Bateson R, Caplan MJ - Mol. Biol. Cell (2015)

Bottom Line: These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase.Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19 °C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network.Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8026.

No MeSH data available.


Related in: MedlinePlus

The Na,K-ATPase and E-cadherin are blocked from reaching the cell surface in the presence of brefeldin A. SNAP-CLIP cells were blocked and pulsed as described in the legend to Figure 4 and incubated in the presence or absence of 5 μg/ml BFA for 2 h at 19°C. Samples were fixed and imaged as described. CT-TMR is depicted in red, and Alexa 488–SNAP is shown in green. The inset depicts areas in which the sodium pump and E-cadherin are segregated from one another. Bar, 5 μm.
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Figure 6: The Na,K-ATPase and E-cadherin are blocked from reaching the cell surface in the presence of brefeldin A. SNAP-CLIP cells were blocked and pulsed as described in the legend to Figure 4 and incubated in the presence or absence of 5 μg/ml BFA for 2 h at 19°C. Samples were fixed and imaged as described. CT-TMR is depicted in red, and Alexa 488–SNAP is shown in green. The inset depicts areas in which the sodium pump and E-cadherin are segregated from one another. Bar, 5 μm.

Mentions: In a variety of cell types, treatment with brefeldin A (BFA) blocks the progress of newly synthesized proteins through the secretory pathway by preventing anterograde trafficking between the ER and the Golgi, thus inducing the dissolution of the cis and medial Golgi compartments and the redistribution of their intrinsic proteins to the ER. In polarized MDCK cells, however, the effects of BFA are less pronounced. BFA does not cause dissolution of the Golgi stacks but instead induces tubulation of endocytic organelles (Hunziker et al., 1991). BFA induces missorting of MDCK cell apical cargoes to the basolateral surface at low concentrations, 1–3 μg/ml, whereas it inhibits surface delivery altogether at concentrations >10 μg/ml (Low et al., 1991, 1992). In particular, E-cadherin surface delivery is blocked at BFA concentrations >3 μg/ml (Low et al., 1992). To test whether BFA could inhibit delivery of E-cadherin to the cell surface under the 19°C Golgi block conditions, we performed a 19°C pulse- chase experiment in the presence or absence of 5 μg/ml BFA. As expected from our earlier results, in the absence of BFA, E-cadherin was delivered to the cell surface at 19°C, whereas the Na,K-ATPase was blocked from exiting the TGN (Figure 6, 19°C). Of interest, in the presence of BFA, E-cadherin was retained in intracellular compartments. Closer examination revealed that under these conditions, a significant fraction, but not all, of the staining associated with newly synthesized E-cadherin was colocalized with markers of the Golgi complex. In addition, a large number of punctate structures could be detected that were exclusively stained either for newly synthesized E-cadherin or for newly synthesized sodium pump (Figure 6, inset, and Supplemental Figure S4). We were unable to identify an organelle-specific marker that colocalized with the discrete Na,K-ATPase–labeled structures. These data suggest that, under 19°C/BFA incubation conditions, at least a portion of the pools of newly synthesized E-cadherin and Na,K-ATPase resides in distinct compartments.


Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin.

Farr GA, Hull M, Stoops EH, Bateson R, Caplan MJ - Mol. Biol. Cell (2015)

The Na,K-ATPase and E-cadherin are blocked from reaching the cell surface in the presence of brefeldin A. SNAP-CLIP cells were blocked and pulsed as described in the legend to Figure 4 and incubated in the presence or absence of 5 μg/ml BFA for 2 h at 19°C. Samples were fixed and imaged as described. CT-TMR is depicted in red, and Alexa 488–SNAP is shown in green. The inset depicts areas in which the sodium pump and E-cadherin are segregated from one another. Bar, 5 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: The Na,K-ATPase and E-cadherin are blocked from reaching the cell surface in the presence of brefeldin A. SNAP-CLIP cells were blocked and pulsed as described in the legend to Figure 4 and incubated in the presence or absence of 5 μg/ml BFA for 2 h at 19°C. Samples were fixed and imaged as described. CT-TMR is depicted in red, and Alexa 488–SNAP is shown in green. The inset depicts areas in which the sodium pump and E-cadherin are segregated from one another. Bar, 5 μm.
Mentions: In a variety of cell types, treatment with brefeldin A (BFA) blocks the progress of newly synthesized proteins through the secretory pathway by preventing anterograde trafficking between the ER and the Golgi, thus inducing the dissolution of the cis and medial Golgi compartments and the redistribution of their intrinsic proteins to the ER. In polarized MDCK cells, however, the effects of BFA are less pronounced. BFA does not cause dissolution of the Golgi stacks but instead induces tubulation of endocytic organelles (Hunziker et al., 1991). BFA induces missorting of MDCK cell apical cargoes to the basolateral surface at low concentrations, 1–3 μg/ml, whereas it inhibits surface delivery altogether at concentrations >10 μg/ml (Low et al., 1991, 1992). In particular, E-cadherin surface delivery is blocked at BFA concentrations >3 μg/ml (Low et al., 1992). To test whether BFA could inhibit delivery of E-cadherin to the cell surface under the 19°C Golgi block conditions, we performed a 19°C pulse- chase experiment in the presence or absence of 5 μg/ml BFA. As expected from our earlier results, in the absence of BFA, E-cadherin was delivered to the cell surface at 19°C, whereas the Na,K-ATPase was blocked from exiting the TGN (Figure 6, 19°C). Of interest, in the presence of BFA, E-cadherin was retained in intracellular compartments. Closer examination revealed that under these conditions, a significant fraction, but not all, of the staining associated with newly synthesized E-cadherin was colocalized with markers of the Golgi complex. In addition, a large number of punctate structures could be detected that were exclusively stained either for newly synthesized E-cadherin or for newly synthesized sodium pump (Figure 6, inset, and Supplemental Figure S4). We were unable to identify an organelle-specific marker that colocalized with the discrete Na,K-ATPase–labeled structures. These data suggest that, under 19°C/BFA incubation conditions, at least a portion of the pools of newly synthesized E-cadherin and Na,K-ATPase resides in distinct compartments.

Bottom Line: These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase.Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19 °C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network.Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8026.

No MeSH data available.


Related in: MedlinePlus