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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 is transported directly to and from REs as visualized by time-lapse microscopy. See Online supplemental material for original video images, available at http://www.jcb.org/cgi/content/full/jcb.200408165/DC1. (A) Individual frames from a movie of cells as prepared in Fig. 1 that were imaged immediately upon release of the TGN block by incubation at 31°C. Images were taken every 7 s for 30 min. Frame sequence illustrates the entry of VSV-G (green structures denoted by arrows; the asterisk marks starting point) into Tfn+ (red) RE structures. Note the increase of VSV-G in REs over the time course of these images, indicated by the increase in yellow (noted by “carrots” in the first and last frames). (B) Purple dots denote the path followed by VSV-G from the first frame (start) to the last frame in the sequence illustrated in A (end). (C) Sequence of VSV-G exit from REs, showing the apparent generation of a green VSV-G tubule and resulting vesicles from a yellow structure contained within the red RE region (arrows). Time course is the same as in A. (D) Purple dots denote the path of VSV-G exit from first frame (start) to last frame (end) in the sequence illustrated in panel C. (E) Image of cell periphery from a cell prepared as in A. Images were taken every 7 s. Arrows denote colocalization of VSV-G with Tfn, an association that was maintained >2.5 min, suggesting that the VSV-G and Tfn were contained within the same structure or reflected two distinct but tethered structures.
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fig2: VSV-G is transported directly to and from REs as visualized by time-lapse microscopy. See Online supplemental material for original video images, available at http://www.jcb.org/cgi/content/full/jcb.200408165/DC1. (A) Individual frames from a movie of cells as prepared in Fig. 1 that were imaged immediately upon release of the TGN block by incubation at 31°C. Images were taken every 7 s for 30 min. Frame sequence illustrates the entry of VSV-G (green structures denoted by arrows; the asterisk marks starting point) into Tfn+ (red) RE structures. Note the increase of VSV-G in REs over the time course of these images, indicated by the increase in yellow (noted by “carrots” in the first and last frames). (B) Purple dots denote the path followed by VSV-G from the first frame (start) to the last frame in the sequence illustrated in A (end). (C) Sequence of VSV-G exit from REs, showing the apparent generation of a green VSV-G tubule and resulting vesicles from a yellow structure contained within the red RE region (arrows). Time course is the same as in A. (D) Purple dots denote the path of VSV-G exit from first frame (start) to last frame (end) in the sequence illustrated in panel C. (E) Image of cell periphery from a cell prepared as in A. Images were taken every 7 s. Arrows denote colocalization of VSV-G with Tfn, an association that was maintained >2.5 min, suggesting that the VSV-G and Tfn were contained within the same structure or reflected two distinct but tethered structures.

Mentions: In static images taken from the video data, Fig. 2 A illustrates one example of VSV-G+ carriers (Fig. 2 A, green, arrows) moving toward and into a region containing a large Tfn+ structure (Fig. 2 A, red, carrot). VSV-G was first detected as a large vesicular structure (Fig. 2 A, asterisk) that became tubular, eventually breaking up into smaller vesicular structures, at least some of which clearly proceeded into the large Tfn+ structure (Fig. 2 A, last panel, carrot). Although this phenomenon of tubular and vesicular transport of VSV-G has been previously described in MDCK cells, such colocalization VSV-G with Tfn+ membranes was not readily observed (Keller et al., 2001). Fig. 2 B summarizes the pathway of the VSV-G carrier denoted by the arrow in Fig. 2 A as it progressed from the periphery (Fig. 2 B, purple dots). Over time, the Tfn+ structures increased in yellow fluorescence due to increased spatial colocalization with VSV-G (Fig. 2 A, compare carrots in first and last frames; Video 3). Similar data were obtained using cells expressing an apical variant of VSV-G (Keller et al., 2001), suggesting that both basolateral and apical plasma membrane proteins were targeted to the RE region (Video 5).


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 is transported directly to and from REs as visualized by time-lapse microscopy. See Online supplemental material for original video images, available at http://www.jcb.org/cgi/content/full/jcb.200408165/DC1. (A) Individual frames from a movie of cells as prepared in Fig. 1 that were imaged immediately upon release of the TGN block by incubation at 31°C. Images were taken every 7 s for 30 min. Frame sequence illustrates the entry of VSV-G (green structures denoted by arrows; the asterisk marks starting point) into Tfn+ (red) RE structures. Note the increase of VSV-G in REs over the time course of these images, indicated by the increase in yellow (noted by “carrots” in the first and last frames). (B) Purple dots denote the path followed by VSV-G from the first frame (start) to the last frame in the sequence illustrated in A (end). (C) Sequence of VSV-G exit from REs, showing the apparent generation of a green VSV-G tubule and resulting vesicles from a yellow structure contained within the red RE region (arrows). Time course is the same as in A. (D) Purple dots denote the path of VSV-G exit from first frame (start) to last frame (end) in the sequence illustrated in panel C. (E) Image of cell periphery from a cell prepared as in A. Images were taken every 7 s. Arrows denote colocalization of VSV-G with Tfn, an association that was maintained >2.5 min, suggesting that the VSV-G and Tfn were contained within the same structure or reflected two distinct but tethered structures.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172492&req=5

fig2: VSV-G is transported directly to and from REs as visualized by time-lapse microscopy. See Online supplemental material for original video images, available at http://www.jcb.org/cgi/content/full/jcb.200408165/DC1. (A) Individual frames from a movie of cells as prepared in Fig. 1 that were imaged immediately upon release of the TGN block by incubation at 31°C. Images were taken every 7 s for 30 min. Frame sequence illustrates the entry of VSV-G (green structures denoted by arrows; the asterisk marks starting point) into Tfn+ (red) RE structures. Note the increase of VSV-G in REs over the time course of these images, indicated by the increase in yellow (noted by “carrots” in the first and last frames). (B) Purple dots denote the path followed by VSV-G from the first frame (start) to the last frame in the sequence illustrated in A (end). (C) Sequence of VSV-G exit from REs, showing the apparent generation of a green VSV-G tubule and resulting vesicles from a yellow structure contained within the red RE region (arrows). Time course is the same as in A. (D) Purple dots denote the path of VSV-G exit from first frame (start) to last frame (end) in the sequence illustrated in panel C. (E) Image of cell periphery from a cell prepared as in A. Images were taken every 7 s. Arrows denote colocalization of VSV-G with Tfn, an association that was maintained >2.5 min, suggesting that the VSV-G and Tfn were contained within the same structure or reflected two distinct but tethered structures.
Mentions: In static images taken from the video data, Fig. 2 A illustrates one example of VSV-G+ carriers (Fig. 2 A, green, arrows) moving toward and into a region containing a large Tfn+ structure (Fig. 2 A, red, carrot). VSV-G was first detected as a large vesicular structure (Fig. 2 A, asterisk) that became tubular, eventually breaking up into smaller vesicular structures, at least some of which clearly proceeded into the large Tfn+ structure (Fig. 2 A, last panel, carrot). Although this phenomenon of tubular and vesicular transport of VSV-G has been previously described in MDCK cells, such colocalization VSV-G with Tfn+ membranes was not readily observed (Keller et al., 2001). Fig. 2 B summarizes the pathway of the VSV-G carrier denoted by the arrow in Fig. 2 A as it progressed from the periphery (Fig. 2 B, purple dots). Over time, the Tfn+ structures increased in yellow fluorescence due to increased spatial colocalization with VSV-G (Fig. 2 A, compare carrots in first and last frames; Video 3). Similar data were obtained using cells expressing an apical variant of VSV-G (Keller et al., 2001), suggesting that both basolateral and apical plasma membrane proteins were targeted to the RE region (Video 5).

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