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Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.

Ang AL, Taguchi T, Francis S, Fölsch H, Murrells LJ, Pypaert M, Warren G, Mellman I - J. Cell Biol. (2004)

Bottom Line: Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells.Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface.Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute of Cancer Research, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

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Inactivation of REs causes accumulation of VSV-G in the perinuclear region. MDCKT cells were infected to express VSV-G-YFP (third column, green) and incubated overnight at 40°C. Tfn-HRP (third column, blue) was internalized for 45 min at 40°C and was chased into REs by incubating cells in media without Tfn-HRP for 15 min at 40°C. Control cells were subject to only DAB while the samples were exposed to DAB and H2O2 for 1 h in the dark. Cells were washed in warm media and incubated at 31°C for 1.5 h in media/CHX to release VSV-G from the ER. Cells were washed in PBScmf, trypsinized, fixed, and processed for immunofluorescence. Cell surface VSV-G labeling (second column, red) using an antibody, TKG, against the ectodomain of VSV-G was performed on nonpermeabilized cells before permeabilization and internal labeling for HRP. Arrows denote accumulation of intracellular VSV-G in the perinuclear region.
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fig5: Inactivation of REs causes accumulation of VSV-G in the perinuclear region. MDCKT cells were infected to express VSV-G-YFP (third column, green) and incubated overnight at 40°C. Tfn-HRP (third column, blue) was internalized for 45 min at 40°C and was chased into REs by incubating cells in media without Tfn-HRP for 15 min at 40°C. Control cells were subject to only DAB while the samples were exposed to DAB and H2O2 for 1 h in the dark. Cells were washed in warm media and incubated at 31°C for 1.5 h in media/CHX to release VSV-G from the ER. Cells were washed in PBScmf, trypsinized, fixed, and processed for immunofluorescence. Cell surface VSV-G labeling (second column, red) using an antibody, TKG, against the ectodomain of VSV-G was performed on nonpermeabilized cells before permeabilization and internal labeling for HRP. Arrows denote accumulation of intracellular VSV-G in the perinuclear region.

Mentions: VSV-G–expressing MDCKT cells, incubated at 40°C to retain VSV-G in the ER, were allowed to internalize Tfn-HRP, and then were treated with DAB and H2O2 on ice for 1 h. The reaction was stopped and the cells incubated at 31°C to release VSV-G from the ER and allow its transport through the Golgi complex and on to the cell surface. The 20°C block was omitted for these experiments. Cycloheximide (CHX) was included during the chase to prevent new VSV-G synthesis. The cells were analyzed for the presence of VSV-G at the cell surface using a monoclonal antibody (TKG) against the ectodomain of VSV-G on nonpermeabilized cells. Under control conditions, i.e., adding DAB without H2O2, most of the VSV-G was transported to the plasma membrane after 1.5 h of chase, as there was strong labeling of surface VSV-G and relatively little GFP fluorescence intracellularly (Fig. 5, top; red, surface VSV-G; green, VSV-G; blue, Tfn). However, upon RE inactivation (cells treated with DAB plus H2O2), a considerable amount of VSV-G-GFP was now found in the perinuclear region (Fig. 5, arrows). The intracellular VSV-G-GFP at least partially colocalized with Tfn-HRP, suggesting that it had been trapped in inactivated REs. There appeared to be much less VSV-G at the cell surface, although some surface VSV-G was indicated by the lateral staining between cells (Fig. 5, second column, red). Further controls demonstrated that inhibition of VSV-G transport was not observed if cells had not been exposed to Tfn-HRP but were still treated with DAB plus H2O2 (unpublished data).


Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.

Ang AL, Taguchi T, Francis S, Fölsch H, Murrells LJ, Pypaert M, Warren G, Mellman I - J. Cell Biol. (2004)

Inactivation of REs causes accumulation of VSV-G in the perinuclear region. MDCKT cells were infected to express VSV-G-YFP (third column, green) and incubated overnight at 40°C. Tfn-HRP (third column, blue) was internalized for 45 min at 40°C and was chased into REs by incubating cells in media without Tfn-HRP for 15 min at 40°C. Control cells were subject to only DAB while the samples were exposed to DAB and H2O2 for 1 h in the dark. Cells were washed in warm media and incubated at 31°C for 1.5 h in media/CHX to release VSV-G from the ER. Cells were washed in PBScmf, trypsinized, fixed, and processed for immunofluorescence. Cell surface VSV-G labeling (second column, red) using an antibody, TKG, against the ectodomain of VSV-G was performed on nonpermeabilized cells before permeabilization and internal labeling for HRP. Arrows denote accumulation of intracellular VSV-G in the perinuclear region.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172492&req=5

fig5: Inactivation of REs causes accumulation of VSV-G in the perinuclear region. MDCKT cells were infected to express VSV-G-YFP (third column, green) and incubated overnight at 40°C. Tfn-HRP (third column, blue) was internalized for 45 min at 40°C and was chased into REs by incubating cells in media without Tfn-HRP for 15 min at 40°C. Control cells were subject to only DAB while the samples were exposed to DAB and H2O2 for 1 h in the dark. Cells were washed in warm media and incubated at 31°C for 1.5 h in media/CHX to release VSV-G from the ER. Cells were washed in PBScmf, trypsinized, fixed, and processed for immunofluorescence. Cell surface VSV-G labeling (second column, red) using an antibody, TKG, against the ectodomain of VSV-G was performed on nonpermeabilized cells before permeabilization and internal labeling for HRP. Arrows denote accumulation of intracellular VSV-G in the perinuclear region.
Mentions: VSV-G–expressing MDCKT cells, incubated at 40°C to retain VSV-G in the ER, were allowed to internalize Tfn-HRP, and then were treated with DAB and H2O2 on ice for 1 h. The reaction was stopped and the cells incubated at 31°C to release VSV-G from the ER and allow its transport through the Golgi complex and on to the cell surface. The 20°C block was omitted for these experiments. Cycloheximide (CHX) was included during the chase to prevent new VSV-G synthesis. The cells were analyzed for the presence of VSV-G at the cell surface using a monoclonal antibody (TKG) against the ectodomain of VSV-G on nonpermeabilized cells. Under control conditions, i.e., adding DAB without H2O2, most of the VSV-G was transported to the plasma membrane after 1.5 h of chase, as there was strong labeling of surface VSV-G and relatively little GFP fluorescence intracellularly (Fig. 5, top; red, surface VSV-G; green, VSV-G; blue, Tfn). However, upon RE inactivation (cells treated with DAB plus H2O2), a considerable amount of VSV-G-GFP was now found in the perinuclear region (Fig. 5, arrows). The intracellular VSV-G-GFP at least partially colocalized with Tfn-HRP, suggesting that it had been trapped in inactivated REs. There appeared to be much less VSV-G at the cell surface, although some surface VSV-G was indicated by the lateral staining between cells (Fig. 5, second column, red). Further controls demonstrated that inhibition of VSV-G transport was not observed if cells had not been exposed to Tfn-HRP but were still treated with DAB plus H2O2 (unpublished data).

Bottom Line: Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells.Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface.Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute of Cancer Research, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

Show MeSH
Related in: MedlinePlus