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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

Dual pulse-chase strategy for following simultaneous trafficking of Na,K-ATPase and E-cadherin. (A) Live SNAP-CLIP cells were preincubated with nonfluorescent blocking reagents (SNAP + CLIP block) or mock treated (No Block) for 30 min at 37°C, fixed, stained as described in Figure 1, and processed for immunofluorescence with an anti–myosin 1c antibody to label the lateral plasma membrane (blue). Bar, 10 μm. (B) The dual pulse-chase strategy to specifically detect biosynthetic trafficking. (C) SNAP-CLIP cells pretreated with blockers were washed and allowed to synthesize new protein for 30 min at 37°C (pulse). After the pulse period, samples were treated with CHX and incubated at 37°C for the indicated times before fixation and labeling as described. Bar, 5 μm. (D) The fraction of protein present at the plasma membrane (as defined by staining for myo-1C) in the experiments outlined in C as assessed with Manders colocalization analysis. Data are mean ± SD from three independent experiments.
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Figure 2: Dual pulse-chase strategy for following simultaneous trafficking of Na,K-ATPase and E-cadherin. (A) Live SNAP-CLIP cells were preincubated with nonfluorescent blocking reagents (SNAP + CLIP block) or mock treated (No Block) for 30 min at 37°C, fixed, stained as described in Figure 1, and processed for immunofluorescence with an anti–myosin 1c antibody to label the lateral plasma membrane (blue). Bar, 10 μm. (B) The dual pulse-chase strategy to specifically detect biosynthetic trafficking. (C) SNAP-CLIP cells pretreated with blockers were washed and allowed to synthesize new protein for 30 min at 37°C (pulse). After the pulse period, samples were treated with CHX and incubated at 37°C for the indicated times before fixation and labeling as described. Bar, 5 μm. (D) The fraction of protein present at the plasma membrane (as defined by staining for myo-1C) in the experiments outlined in C as assessed with Manders colocalization analysis. Data are mean ± SD from three independent experiments.

Mentions: The SNAP- and CLIP-tag systems permit temporally defined cohorts of proteins to be selectively labeled and detected. This is accomplished by using membrane-permeable nonfluorescent BG and BC reagents to block covalently and irreversibly the reactive sites on previously synthesized collections of SNAP- or CLIP-tagged proteins. This blocking step prevents the fluorescence labeling and detection of the steady-state pools of the proteins of interest, allowing instead for the specific labeling and imaging of subsequently synthesized cohorts. To test the specificity and effectiveness of the blocking reagents in MDCK cells, we treated our stable cell line independently with either the SNAP or the CLIP blocking reagent for 30 min before fixation and fluorescence labeling. As shown in Figure 1C, the blocking reagents for both the SNAP- and CLIP-tags were found to be very efficient and selective, allowing us to block detection of either the sodium pump or E-cadherin. Simultaneous incubation with both SNAP- and CLIP-tag blocking reagents was also found to inhibit detection of both proteins within the same culture (Figure 2A).


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)

Dual pulse-chase strategy for following simultaneous trafficking of Na,K-ATPase and E-cadherin. (A) Live SNAP-CLIP cells were preincubated with nonfluorescent blocking reagents (SNAP + CLIP block) or mock treated (No Block) for 30 min at 37°C, fixed, stained as described in Figure 1, and processed for immunofluorescence with an anti–myosin 1c antibody to label the lateral plasma membrane (blue). Bar, 10 μm. (B) The dual pulse-chase strategy to specifically detect biosynthetic trafficking. (C) SNAP-CLIP cells pretreated with blockers were washed and allowed to synthesize new protein for 30 min at 37°C (pulse). After the pulse period, samples were treated with CHX and incubated at 37°C for the indicated times before fixation and labeling as described. Bar, 5 μm. (D) The fraction of protein present at the plasma membrane (as defined by staining for myo-1C) in the experiments outlined in C as assessed with Manders colocalization analysis. Data are mean ± SD from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Dual pulse-chase strategy for following simultaneous trafficking of Na,K-ATPase and E-cadherin. (A) Live SNAP-CLIP cells were preincubated with nonfluorescent blocking reagents (SNAP + CLIP block) or mock treated (No Block) for 30 min at 37°C, fixed, stained as described in Figure 1, and processed for immunofluorescence with an anti–myosin 1c antibody to label the lateral plasma membrane (blue). Bar, 10 μm. (B) The dual pulse-chase strategy to specifically detect biosynthetic trafficking. (C) SNAP-CLIP cells pretreated with blockers were washed and allowed to synthesize new protein for 30 min at 37°C (pulse). After the pulse period, samples were treated with CHX and incubated at 37°C for the indicated times before fixation and labeling as described. Bar, 5 μm. (D) The fraction of protein present at the plasma membrane (as defined by staining for myo-1C) in the experiments outlined in C as assessed with Manders colocalization analysis. Data are mean ± SD from three independent experiments.
Mentions: The SNAP- and CLIP-tag systems permit temporally defined cohorts of proteins to be selectively labeled and detected. This is accomplished by using membrane-permeable nonfluorescent BG and BC reagents to block covalently and irreversibly the reactive sites on previously synthesized collections of SNAP- or CLIP-tagged proteins. This blocking step prevents the fluorescence labeling and detection of the steady-state pools of the proteins of interest, allowing instead for the specific labeling and imaging of subsequently synthesized cohorts. To test the specificity and effectiveness of the blocking reagents in MDCK cells, we treated our stable cell line independently with either the SNAP or the CLIP blocking reagent for 30 min before fixation and fluorescence labeling. As shown in Figure 1C, the blocking reagents for both the SNAP- and CLIP-tags were found to be very efficient and selective, allowing us to block detection of either the sodium pump or E-cadherin. Simultaneous incubation with both SNAP- and CLIP-tag blocking reagents was also found to inhibit detection of both proteins within the same culture (Figure 2A).

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