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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 membranes containing Tfn-HRP, but not free-HRP, inhibited the cell surface arrival of VSV-G. (A) Representative dot plot of flow cytometry results from cells prepared as in Fig. 5 (Tfn-HRP). Levels of surface VSV-G, performed on nonpermeabilized cells using the TKG antibody, were quantified on the y-axis whereas total VSV-G, as monitored by YFP fluorescence, was quantified on the x-axis. Cells in quadrant I were positive for only surface VSV-G, cells in quadrant II were positive for both surface and total VSV-G, cells in quadrant III were negative for both markers, and cells in quadrant IV were negative for surface VSV-G and positive for total (i.e., intracellular) VSV-G. Numbers in corners represent percentage of cells in that quadrant. (B) MDCKT cells expressing VSV-G-YFP were incubated with free-HRP followed by chase in HRP-free media to load HRP into lysosomes (Free-HRP). Control cells were incubated with DAB alone while the “inactivated” set was incubated with DAB plus H2O2 for 1 h in the dark. (C) Percentage MFI of surface VSV-G was measured in cells that were positive for total VSV-G (all cells in quadrants II and IV) and had Tfn-HRP– or free-HRP–containing compartments inactivated. MFI was normalized based on levels in control cells. (D) Average percentage of cells based on three experiments performed in triplicate with surface VSV-G after an uptake of Tfn-HRP or free-HRP under control (black) or inactivation (gray) conditions. Error bars represent the SD from cells with surface VSV-G from three different experiments.
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fig7: Inactivation of membranes containing Tfn-HRP, but not free-HRP, inhibited the cell surface arrival of VSV-G. (A) Representative dot plot of flow cytometry results from cells prepared as in Fig. 5 (Tfn-HRP). Levels of surface VSV-G, performed on nonpermeabilized cells using the TKG antibody, were quantified on the y-axis whereas total VSV-G, as monitored by YFP fluorescence, was quantified on the x-axis. Cells in quadrant I were positive for only surface VSV-G, cells in quadrant II were positive for both surface and total VSV-G, cells in quadrant III were negative for both markers, and cells in quadrant IV were negative for surface VSV-G and positive for total (i.e., intracellular) VSV-G. Numbers in corners represent percentage of cells in that quadrant. (B) MDCKT cells expressing VSV-G-YFP were incubated with free-HRP followed by chase in HRP-free media to load HRP into lysosomes (Free-HRP). Control cells were incubated with DAB alone while the “inactivated” set was incubated with DAB plus H2O2 for 1 h in the dark. (C) Percentage MFI of surface VSV-G was measured in cells that were positive for total VSV-G (all cells in quadrants II and IV) and had Tfn-HRP– or free-HRP–containing compartments inactivated. MFI was normalized based on levels in control cells. (D) Average percentage of cells based on three experiments performed in triplicate with surface VSV-G after an uptake of Tfn-HRP or free-HRP under control (black) or inactivation (gray) conditions. Error bars represent the SD from cells with surface VSV-G from three different experiments.

Mentions: As shown in Fig. 7 A (taken from a representative experiment performed in triplicate), under control conditions, where MDCKT cells have internalized Tfn-HRP but were not subject to the DAB-peroxidase reaction (omitting DAB, Tfn-HRP, or H2O2; described for Fig. 5, top; and Fig. 6 A), nearly all of the VSV-G-GFP–expressing cells successfully transported VSV-G to the cell surface (Fig. 7 A, control, top panels). The majority of the cells appeared in quadrant II, indicating that they were positive for both surface and total VSV-G-GFP. However, if the DAB reaction was performed to inactivate Tfn-HRP–containing RE compartments before the 31°C chase, <13% of the cells, whose total VSV-G expression level was similar to those of control cells, were able to transport VSV-G to the cell surface (Fig. 7 A, inactivated, quadrant II). Thus, >60% of the cells containing Tfn-HRP were negative for surface VSV-G expression after RE inactivation (Fig. 7 A, quadrant IV). The mean fluorescence intensity (MFI) of surface VSV-G was also measured for control and inactivated cells whose total VSV-G levels were high, i.e., all cells in quadrants II and IV. Statistical analysis revealed that the MFI of surface VSV-G of inactivated MDCKT cells was only 15% of that in control cells, suggesting that Tfn-HRP–mediated inactivation of RE inhibited the transport of VSV-G to the cell surface by 85% (Fig. 7 C, Tfn-HRP). Moreover, those inactivated cells that displayed surface VSV-G did so at levels that were reduced relative to controls (Fig. 7, compare quadrant II); this finding is consistent with a partial inhibition or a reversal of the block at long chase times, as suggested in Fig. 6.


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 membranes containing Tfn-HRP, but not free-HRP, inhibited the cell surface arrival of VSV-G. (A) Representative dot plot of flow cytometry results from cells prepared as in Fig. 5 (Tfn-HRP). Levels of surface VSV-G, performed on nonpermeabilized cells using the TKG antibody, were quantified on the y-axis whereas total VSV-G, as monitored by YFP fluorescence, was quantified on the x-axis. Cells in quadrant I were positive for only surface VSV-G, cells in quadrant II were positive for both surface and total VSV-G, cells in quadrant III were negative for both markers, and cells in quadrant IV were negative for surface VSV-G and positive for total (i.e., intracellular) VSV-G. Numbers in corners represent percentage of cells in that quadrant. (B) MDCKT cells expressing VSV-G-YFP were incubated with free-HRP followed by chase in HRP-free media to load HRP into lysosomes (Free-HRP). Control cells were incubated with DAB alone while the “inactivated” set was incubated with DAB plus H2O2 for 1 h in the dark. (C) Percentage MFI of surface VSV-G was measured in cells that were positive for total VSV-G (all cells in quadrants II and IV) and had Tfn-HRP– or free-HRP–containing compartments inactivated. MFI was normalized based on levels in control cells. (D) Average percentage of cells based on three experiments performed in triplicate with surface VSV-G after an uptake of Tfn-HRP or free-HRP under control (black) or inactivation (gray) conditions. Error bars represent the SD from cells with surface VSV-G from three different experiments.
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fig7: Inactivation of membranes containing Tfn-HRP, but not free-HRP, inhibited the cell surface arrival of VSV-G. (A) Representative dot plot of flow cytometry results from cells prepared as in Fig. 5 (Tfn-HRP). Levels of surface VSV-G, performed on nonpermeabilized cells using the TKG antibody, were quantified on the y-axis whereas total VSV-G, as monitored by YFP fluorescence, was quantified on the x-axis. Cells in quadrant I were positive for only surface VSV-G, cells in quadrant II were positive for both surface and total VSV-G, cells in quadrant III were negative for both markers, and cells in quadrant IV were negative for surface VSV-G and positive for total (i.e., intracellular) VSV-G. Numbers in corners represent percentage of cells in that quadrant. (B) MDCKT cells expressing VSV-G-YFP were incubated with free-HRP followed by chase in HRP-free media to load HRP into lysosomes (Free-HRP). Control cells were incubated with DAB alone while the “inactivated” set was incubated with DAB plus H2O2 for 1 h in the dark. (C) Percentage MFI of surface VSV-G was measured in cells that were positive for total VSV-G (all cells in quadrants II and IV) and had Tfn-HRP– or free-HRP–containing compartments inactivated. MFI was normalized based on levels in control cells. (D) Average percentage of cells based on three experiments performed in triplicate with surface VSV-G after an uptake of Tfn-HRP or free-HRP under control (black) or inactivation (gray) conditions. Error bars represent the SD from cells with surface VSV-G from three different experiments.
Mentions: As shown in Fig. 7 A (taken from a representative experiment performed in triplicate), under control conditions, where MDCKT cells have internalized Tfn-HRP but were not subject to the DAB-peroxidase reaction (omitting DAB, Tfn-HRP, or H2O2; described for Fig. 5, top; and Fig. 6 A), nearly all of the VSV-G-GFP–expressing cells successfully transported VSV-G to the cell surface (Fig. 7 A, control, top panels). The majority of the cells appeared in quadrant II, indicating that they were positive for both surface and total VSV-G-GFP. However, if the DAB reaction was performed to inactivate Tfn-HRP–containing RE compartments before the 31°C chase, <13% of the cells, whose total VSV-G expression level was similar to those of control cells, were able to transport VSV-G to the cell surface (Fig. 7 A, inactivated, quadrant II). Thus, >60% of the cells containing Tfn-HRP were negative for surface VSV-G expression after RE inactivation (Fig. 7 A, quadrant IV). The mean fluorescence intensity (MFI) of surface VSV-G was also measured for control and inactivated cells whose total VSV-G levels were high, i.e., all cells in quadrants II and IV. Statistical analysis revealed that the MFI of surface VSV-G of inactivated MDCKT cells was only 15% of that in control cells, suggesting that Tfn-HRP–mediated inactivation of RE inhibited the transport of VSV-G to the cell surface by 85% (Fig. 7 C, Tfn-HRP). Moreover, those inactivated cells that displayed surface VSV-G did so at levels that were reduced relative to controls (Fig. 7, compare quadrant II); this finding is consistent with a partial inhibition or a reversal of the block at long chase times, as suggested in Fig. 6.

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