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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

Newly synthesized E-cadherin traffics to the cell surface faster than the Na,K-ATPase. SNAP-CLIP cells were subjected to block, incubated at 37°C for 20 min to begin synthesis of new cohorts of sodium pump and E-cadherin, and then chased in medium containing 150 μg/ml CHX for the indicated times. At each time point, cultures were lysed and labeled with SNAP 782 and CLIP 647. (A) Lysates were processed by SDS–PAGE and imaged using an Odyssey Imager. (B) Samples were treated as described; however, before lysis, filter-grown cultures were subjected to cell surface biotinylation, and lysates prepared from these cells were precipitated with streptavidin–agarose to recover proteins that were exposed at the cell surface. (C) Quantification of data in B normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Data represent mean (n = 3) and SEM from three independent experiments.
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Figure 3: Newly synthesized E-cadherin traffics to the cell surface faster than the Na,K-ATPase. SNAP-CLIP cells were subjected to block, incubated at 37°C for 20 min to begin synthesis of new cohorts of sodium pump and E-cadherin, and then chased in medium containing 150 μg/ml CHX for the indicated times. At each time point, cultures were lysed and labeled with SNAP 782 and CLIP 647. (A) Lysates were processed by SDS–PAGE and imaged using an Odyssey Imager. (B) Samples were treated as described; however, before lysis, filter-grown cultures were subjected to cell surface biotinylation, and lysates prepared from these cells were precipitated with streptavidin–agarose to recover proteins that were exposed at the cell surface. (C) Quantification of data in B normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Data represent mean (n = 3) and SEM from three independent experiments.

Mentions: Although the images presented in Figure 2C strongly suggest that newly synthesized E-cadherin is inserted into the lateral PM after a 20-min chase period, it is formally possible that a portion of this signal could be attributed to vesicles that are docked at the PM but have yet to fuse. To determine whether the newly synthesized E-cadherin and Na,K-ATPase proteins are indeed inserted into the basolateral plasma membrane, we combined the SNAP- and CLIP-labeling techniques with cell surface biotinylation. This experiment used the fact that the SNAP-tag is readily amenable to labeling in cell lysates (Morton et al., 2010). In this experiment, cells were incubated with the membrane-permeant SNAP- and CLIP-blocking reagents as described earlier, and the cells were then allowed to synthesize new protein for 20 min at 37°C before addition of cycloheximide (CHX) to block further protein synthesis. At each time point, cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed in Triton extraction buffer, and labeled with fluorescent reagents for SNAP and CLIP. The labeled lysates were separated by SDS–PAGE, and fluorescence signals associated with the Na,K-ATPase and E-cadherin were visualized using a fluorescent gel imaging system as described in Materials and Methods. As shown in Figure 3, we observed significant labeling of the Na,K-ATPase and E-cadherin in unblocked control lysates. Addition of membrane-permeant blocking reagents abolished this labeling, consistent with what we observe in the microscopy protocol used in the experiments depicted in Figure 2. Newly synthesized cohorts of fluorescently labeled E-cadherin and Na,K-ATPase were readily detected in lysates from cells that had been incubated for 20 min at 37°C (pulse) after the blocking step. The appearance of the higher–molecular weight precursor form of E-cadherin, which is cleaved during postbiosynthetic processing (Shore and Nelson, 1991), provided further evidence that our labeling strategy specifically detected newly synthesized cohorts (Figure 3A, Pulse). The intensity of the band corresponding to the E-cadherin precursor diminished within 20 min of CHX addition in association with a concomitant increase in the intensity of the band corresponding to mature E-cadherin (Figure 3A, chase).


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)

Newly synthesized E-cadherin traffics to the cell surface faster than the Na,K-ATPase. SNAP-CLIP cells were subjected to block, incubated at 37°C for 20 min to begin synthesis of new cohorts of sodium pump and E-cadherin, and then chased in medium containing 150 μg/ml CHX for the indicated times. At each time point, cultures were lysed and labeled with SNAP 782 and CLIP 647. (A) Lysates were processed by SDS–PAGE and imaged using an Odyssey Imager. (B) Samples were treated as described; however, before lysis, filter-grown cultures were subjected to cell surface biotinylation, and lysates prepared from these cells were precipitated with streptavidin–agarose to recover proteins that were exposed at the cell surface. (C) Quantification of data in B normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Data represent mean (n = 3) and SEM from three independent experiments.
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Figure 3: Newly synthesized E-cadherin traffics to the cell surface faster than the Na,K-ATPase. SNAP-CLIP cells were subjected to block, incubated at 37°C for 20 min to begin synthesis of new cohorts of sodium pump and E-cadherin, and then chased in medium containing 150 μg/ml CHX for the indicated times. At each time point, cultures were lysed and labeled with SNAP 782 and CLIP 647. (A) Lysates were processed by SDS–PAGE and imaged using an Odyssey Imager. (B) Samples were treated as described; however, before lysis, filter-grown cultures were subjected to cell surface biotinylation, and lysates prepared from these cells were precipitated with streptavidin–agarose to recover proteins that were exposed at the cell surface. (C) Quantification of data in B normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Data represent mean (n = 3) and SEM from three independent experiments.
Mentions: Although the images presented in Figure 2C strongly suggest that newly synthesized E-cadherin is inserted into the lateral PM after a 20-min chase period, it is formally possible that a portion of this signal could be attributed to vesicles that are docked at the PM but have yet to fuse. To determine whether the newly synthesized E-cadherin and Na,K-ATPase proteins are indeed inserted into the basolateral plasma membrane, we combined the SNAP- and CLIP-labeling techniques with cell surface biotinylation. This experiment used the fact that the SNAP-tag is readily amenable to labeling in cell lysates (Morton et al., 2010). In this experiment, cells were incubated with the membrane-permeant SNAP- and CLIP-blocking reagents as described earlier, and the cells were then allowed to synthesize new protein for 20 min at 37°C before addition of cycloheximide (CHX) to block further protein synthesis. At each time point, cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed in Triton extraction buffer, and labeled with fluorescent reagents for SNAP and CLIP. The labeled lysates were separated by SDS–PAGE, and fluorescence signals associated with the Na,K-ATPase and E-cadherin were visualized using a fluorescent gel imaging system as described in Materials and Methods. As shown in Figure 3, we observed significant labeling of the Na,K-ATPase and E-cadherin in unblocked control lysates. Addition of membrane-permeant blocking reagents abolished this labeling, consistent with what we observe in the microscopy protocol used in the experiments depicted in Figure 2. Newly synthesized cohorts of fluorescently labeled E-cadherin and Na,K-ATPase were readily detected in lysates from cells that had been incubated for 20 min at 37°C (pulse) after the blocking step. The appearance of the higher–molecular weight precursor form of E-cadherin, which is cleaved during postbiosynthetic processing (Shore and Nelson, 1991), provided further evidence that our labeling strategy specifically detected newly synthesized cohorts (Figure 3A, Pulse). The intensity of the band corresponding to the E-cadherin precursor diminished within 20 min of CHX addition in association with a concomitant increase in the intensity of the band corresponding to mature E-cadherin (Figure 3A, chase).

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