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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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VSV-G and Tfn colocalize in endosomes by immuno-EM. (A and B) Immuno-EM of MDCKT cells expressing VSV-G-YFP and having endocytosed Alexa 488-Tfn after 2-h incubation at 20°C. VSV-G (5 nm gold, arrows) was localized to the Golgi complex, peripheral vesicles, and, occasionally, on endosomes (arrow on “e”). Tfn (10 nm gold) was localized to endosomes (e). Bars: (A) 200 nm; (B) 100 nm. (C and D) Immuno-EM of MDCKT cells released from the 20°C TGN block by incubation at 31°C for 10 min. VSV-G (5 nm gold, arrows) and Tfn (10 nm gold) were localized to the same endosomal compartments (e). Bars, 100 nm. (E) Labeling density of VSV-G on Tfn+ endosomes. Density of VSV-G labeling increases fourfold during 31°C incubation. (F) Ratio of number of gold particles labeling VSV-G in endosomes over the number of gold particles labeling Tfn. Error bars represent the SD of labeling density from three grids.
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fig3: VSV-G and Tfn colocalize in endosomes by immuno-EM. (A and B) Immuno-EM of MDCKT cells expressing VSV-G-YFP and having endocytosed Alexa 488-Tfn after 2-h incubation at 20°C. VSV-G (5 nm gold, arrows) was localized to the Golgi complex, peripheral vesicles, and, occasionally, on endosomes (arrow on “e”). Tfn (10 nm gold) was localized to endosomes (e). Bars: (A) 200 nm; (B) 100 nm. (C and D) Immuno-EM of MDCKT cells released from the 20°C TGN block by incubation at 31°C for 10 min. VSV-G (5 nm gold, arrows) and Tfn (10 nm gold) were localized to the same endosomal compartments (e). Bars, 100 nm. (E) Labeling density of VSV-G on Tfn+ endosomes. Density of VSV-G labeling increases fourfold during 31°C incubation. (F) Ratio of number of gold particles labeling VSV-G in endosomes over the number of gold particles labeling Tfn. Error bars represent the SD of labeling density from three grids.

Mentions: As expected, in cells maintained at 20°C, VSV-G was found mainly in the Golgi complex, present throughout the cisternal elements and not just the TGN (Fig. 3, A and B, arrows denote VSV-G, 5 nm gold). Some staining was also observed in Golgi-associated vesicles and electron-lucent structures. These structures included endosomes that were positively labeled for Tfn (labeled e in Fig. 3, A and B; 10 nm gold), which had been internalized for 2 h at 20°C. Tfn was absent from the Golgi complex per se. Accordingly, the bulk of VSV-G was present in elements of the Golgi complex at 20°C, with only a small amount found in Tfn+ endosomes.


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)

VSV-G and Tfn colocalize in endosomes by immuno-EM. (A and B) Immuno-EM of MDCKT cells expressing VSV-G-YFP and having endocytosed Alexa 488-Tfn after 2-h incubation at 20°C. VSV-G (5 nm gold, arrows) was localized to the Golgi complex, peripheral vesicles, and, occasionally, on endosomes (arrow on “e”). Tfn (10 nm gold) was localized to endosomes (e). Bars: (A) 200 nm; (B) 100 nm. (C and D) Immuno-EM of MDCKT cells released from the 20°C TGN block by incubation at 31°C for 10 min. VSV-G (5 nm gold, arrows) and Tfn (10 nm gold) were localized to the same endosomal compartments (e). Bars, 100 nm. (E) Labeling density of VSV-G on Tfn+ endosomes. Density of VSV-G labeling increases fourfold during 31°C incubation. (F) Ratio of number of gold particles labeling VSV-G in endosomes over the number of gold particles labeling Tfn. Error bars represent the SD of labeling density from three grids.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172492&req=5

fig3: VSV-G and Tfn colocalize in endosomes by immuno-EM. (A and B) Immuno-EM of MDCKT cells expressing VSV-G-YFP and having endocytosed Alexa 488-Tfn after 2-h incubation at 20°C. VSV-G (5 nm gold, arrows) was localized to the Golgi complex, peripheral vesicles, and, occasionally, on endosomes (arrow on “e”). Tfn (10 nm gold) was localized to endosomes (e). Bars: (A) 200 nm; (B) 100 nm. (C and D) Immuno-EM of MDCKT cells released from the 20°C TGN block by incubation at 31°C for 10 min. VSV-G (5 nm gold, arrows) and Tfn (10 nm gold) were localized to the same endosomal compartments (e). Bars, 100 nm. (E) Labeling density of VSV-G on Tfn+ endosomes. Density of VSV-G labeling increases fourfold during 31°C incubation. (F) Ratio of number of gold particles labeling VSV-G in endosomes over the number of gold particles labeling Tfn. Error bars represent the SD of labeling density from three grids.
Mentions: As expected, in cells maintained at 20°C, VSV-G was found mainly in the Golgi complex, present throughout the cisternal elements and not just the TGN (Fig. 3, A and B, arrows denote VSV-G, 5 nm gold). Some staining was also observed in Golgi-associated vesicles and electron-lucent structures. These structures included endosomes that were positively labeled for Tfn (labeled e in Fig. 3, A and B; 10 nm gold), which had been internalized for 2 h at 20°C. Tfn was absent from the Golgi complex per se. Accordingly, the bulk of VSV-G was present in elements of the Golgi complex at 20°C, with only a small amount found in Tfn+ endosomes.

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