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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

Trafficking of newly synthesized E-cadherin is not affected by the 19°C Golgi block. (A) SNAP-CLIP cells were subjected to block, incubated at 37°C for 30 min to begin synthesis of new sodium pump and E-cadherin, and placed at 19 or 14°C for 2 h to accumulate newly synthesized protein in intracellular compartments. CT-TMR is depicted in red, Alexa 488–SNAP is shown in green, and the Golgi marker Vti1a is shown in blue. Bar, 5 μm. (B) Samples were treated as described and either fixed immediately (14°C) or warmed to 19°C for the indicated times. Samples were labeled as described, and the steady-state pool of Na,K-ATPase was labeled with an antibody directed against the HA-epitope tag to illuminate the basolateral membrane (blue). Bar, 5 μm. (C) Samples treated as in B were stained with antibodies against the Golgi compartment (gm130, Vti1a, and Golgin-84), and Manders colocalization analysis was performed as in Figure 2 to assess the fraction of E-cadherin and Na,K-ATPase that colocalized with the Golgi markers under each condition. Data are representative of two similar experiments. Asterisk indicates statistical significance using unpaired Student’s t tests. NS, not significant.
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Figure 4: Trafficking of newly synthesized E-cadherin is not affected by the 19°C Golgi block. (A) SNAP-CLIP cells were subjected to block, incubated at 37°C for 30 min to begin synthesis of new sodium pump and E-cadherin, and placed at 19 or 14°C for 2 h to accumulate newly synthesized protein in intracellular compartments. CT-TMR is depicted in red, Alexa 488–SNAP is shown in green, and the Golgi marker Vti1a is shown in blue. Bar, 5 μm. (B) Samples were treated as described and either fixed immediately (14°C) or warmed to 19°C for the indicated times. Samples were labeled as described, and the steady-state pool of Na,K-ATPase was labeled with an antibody directed against the HA-epitope tag to illuminate the basolateral membrane (blue). Bar, 5 μm. (C) Samples treated as in B were stained with antibodies against the Golgi compartment (gm130, Vti1a, and Golgin-84), and Manders colocalization analysis was performed as in Figure 2 to assess the fraction of E-cadherin and Na,K-ATPase that colocalized with the Golgi markers under each condition. Data are representative of two similar experiments. Asterisk indicates statistical significance using unpaired Student’s t tests. NS, not significant.

Mentions: Our previous analysis of Na,K-ATPase surface delivery used the Golgi temperature block, which prevents trafficking of proteins out of the TGN (Matlin and Simons, 1983; Saraste and Kuismanen, 1984; Rindler et al., 1985). Using this protocol, we were able to synchronize release of the newly synthesized Na,K-ATPase and VSV-G proteins from the TGN, allowing us to analyze their post-Golgi trafficking steps with a high degree of temporal resolution (Farr et al., 2009). As can be seen in Figure 4A, combining our block chase protocol with a 2-h incubation at 19°C results in accumulation of the newly synthesized cohort of Na,K-ATPase within the TGN, where it colocalized with the Golgi marker Vti1a. Of interest, in the same population of cells, the newly synthesized cohort of E-cadherin was detected at the cell surface, suggesting that its trafficking is not susceptible to inhibition at 19°C. To rule out the trivial explanation that the water bath temperature might not accurately reflect the temperature of the medium bathing the cells, we directly analyzed medium temperatures using a thermocouple device and confirmed that the cells were maintained at 19°C throughout these experiments (unpublished data).


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)

Trafficking of newly synthesized E-cadherin is not affected by the 19°C Golgi block. (A) SNAP-CLIP cells were subjected to block, incubated at 37°C for 30 min to begin synthesis of new sodium pump and E-cadherin, and placed at 19 or 14°C for 2 h to accumulate newly synthesized protein in intracellular compartments. CT-TMR is depicted in red, Alexa 488–SNAP is shown in green, and the Golgi marker Vti1a is shown in blue. Bar, 5 μm. (B) Samples were treated as described and either fixed immediately (14°C) or warmed to 19°C for the indicated times. Samples were labeled as described, and the steady-state pool of Na,K-ATPase was labeled with an antibody directed against the HA-epitope tag to illuminate the basolateral membrane (blue). Bar, 5 μm. (C) Samples treated as in B were stained with antibodies against the Golgi compartment (gm130, Vti1a, and Golgin-84), and Manders colocalization analysis was performed as in Figure 2 to assess the fraction of E-cadherin and Na,K-ATPase that colocalized with the Golgi markers under each condition. Data are representative of two similar experiments. Asterisk indicates statistical significance using unpaired Student’s t tests. NS, not significant.
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Figure 4: Trafficking of newly synthesized E-cadherin is not affected by the 19°C Golgi block. (A) SNAP-CLIP cells were subjected to block, incubated at 37°C for 30 min to begin synthesis of new sodium pump and E-cadherin, and placed at 19 or 14°C for 2 h to accumulate newly synthesized protein in intracellular compartments. CT-TMR is depicted in red, Alexa 488–SNAP is shown in green, and the Golgi marker Vti1a is shown in blue. Bar, 5 μm. (B) Samples were treated as described and either fixed immediately (14°C) or warmed to 19°C for the indicated times. Samples were labeled as described, and the steady-state pool of Na,K-ATPase was labeled with an antibody directed against the HA-epitope tag to illuminate the basolateral membrane (blue). Bar, 5 μm. (C) Samples treated as in B were stained with antibodies against the Golgi compartment (gm130, Vti1a, and Golgin-84), and Manders colocalization analysis was performed as in Figure 2 to assess the fraction of E-cadherin and Na,K-ATPase that colocalized with the Golgi markers under each condition. Data are representative of two similar experiments. Asterisk indicates statistical significance using unpaired Student’s t tests. NS, not significant.
Mentions: Our previous analysis of Na,K-ATPase surface delivery used the Golgi temperature block, which prevents trafficking of proteins out of the TGN (Matlin and Simons, 1983; Saraste and Kuismanen, 1984; Rindler et al., 1985). Using this protocol, we were able to synchronize release of the newly synthesized Na,K-ATPase and VSV-G proteins from the TGN, allowing us to analyze their post-Golgi trafficking steps with a high degree of temporal resolution (Farr et al., 2009). As can be seen in Figure 4A, combining our block chase protocol with a 2-h incubation at 19°C results in accumulation of the newly synthesized cohort of Na,K-ATPase within the TGN, where it colocalized with the Golgi marker Vti1a. Of interest, in the same population of cells, the newly synthesized cohort of E-cadherin was detected at the cell surface, suggesting that its trafficking is not susceptible to inhibition at 19°C. To rule out the trivial explanation that the water bath temperature might not accurately reflect the temperature of the medium bathing the cells, we directly analyzed medium temperatures using a thermocouple device and confirmed that the cells were maintained at 19°C throughout these experiments (unpublished data).

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