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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 localizes to Tfn-positive membranes after exit from the Golgi complex. (A) MDCKT cells were infected with ts045 VSV-G-YFP and induced to express transferrin receptor (TfnR). Cells were incubated 2 h at 20°C (last hour in media plus CHX) to accumulate a synchronous pulse of VSV-G (green) at the TGN, and allowed to take up Alexa 546-Tfn (red) into REs. Cells were fixed and imaged. Arrows, REs. (B) Cells in A were released from the TGN block by incubation at 31°C in media plus CHX for 10 min, and then fixed and imaged. Arrows denote colocalization of VSV-G and Tfn (yellow). (C and D) Three-dimensional reconstruction of confocal serial sections, X-Y plane and sagittal section through REs (arrow) of representative cells in A and B.
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fig1: VSV-G localizes to Tfn-positive membranes after exit from the Golgi complex. (A) MDCKT cells were infected with ts045 VSV-G-YFP and induced to express transferrin receptor (TfnR). Cells were incubated 2 h at 20°C (last hour in media plus CHX) to accumulate a synchronous pulse of VSV-G (green) at the TGN, and allowed to take up Alexa 546-Tfn (red) into REs. Cells were fixed and imaged. Arrows, REs. (B) Cells in A were released from the TGN block by incubation at 31°C in media plus CHX for 10 min, and then fixed and imaged. Arrows denote colocalization of VSV-G and Tfn (yellow). (C and D) Three-dimensional reconstruction of confocal serial sections, X-Y plane and sagittal section through REs (arrow) of representative cells in A and B.

Mentions: Initial experiments relied on immunofluorescence microscopy of fixed, coverslip-grown MDCKT cells. A recombinant adenovirus was used to express YFP-tagged ts045 VSV-G, with cells exposed to a pulse of virus and then incubated overnight at 40°C before shifting to 20°C in the presence of Alexa 546-Tfn. As shown in Fig. 1 A, VSV-G (Fig. 1, green) and Tfn (Fig. 1, red) did not colocalize during the 20°C block. Tfn was localized to a characteristic tight perinuclear cluster (Fig. 1 A, arrows), whereas VSV-G, also in the perinuclear region, was found in largely distinct structures that in many examples appeared to circumscribe the RE. These VSV-G–containing structures colocalized instead with markers of the Golgi such as GM130 (Fölsch et al., 2001) and furin (unpublished data). However, upon shifting to 31°C for 5–10 min and immediately processing the cells for immunofluorescence, VSV-G was observed to be spatially localized with Tfn (Fig. 1 B, arrows) in the Tfn-positive cluster previously devoid of VSV-G. The apparent colocalization of VSV-G and Tfn did not reflect a superimposition of distinct structures in different focal planes as it was evident throughout the entire volume of three-dimensional renderings (Fig. 1, C and D; and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200408165/DC1). Such overlap of VSV-G with Tfn was not observed using cells not expressing human TfnR, due to a much lower labeling efficiency of Tfn and thus inefficient ascertainment of Tfn-containing structures (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)

VSV-G localizes to Tfn-positive membranes after exit from the Golgi complex. (A) MDCKT cells were infected with ts045 VSV-G-YFP and induced to express transferrin receptor (TfnR). Cells were incubated 2 h at 20°C (last hour in media plus CHX) to accumulate a synchronous pulse of VSV-G (green) at the TGN, and allowed to take up Alexa 546-Tfn (red) into REs. Cells were fixed and imaged. Arrows, REs. (B) Cells in A were released from the TGN block by incubation at 31°C in media plus CHX for 10 min, and then fixed and imaged. Arrows denote colocalization of VSV-G and Tfn (yellow). (C and D) Three-dimensional reconstruction of confocal serial sections, X-Y plane and sagittal section through REs (arrow) of representative cells in A and B.
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Related In: Results  -  Collection

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fig1: VSV-G localizes to Tfn-positive membranes after exit from the Golgi complex. (A) MDCKT cells were infected with ts045 VSV-G-YFP and induced to express transferrin receptor (TfnR). Cells were incubated 2 h at 20°C (last hour in media plus CHX) to accumulate a synchronous pulse of VSV-G (green) at the TGN, and allowed to take up Alexa 546-Tfn (red) into REs. Cells were fixed and imaged. Arrows, REs. (B) Cells in A were released from the TGN block by incubation at 31°C in media plus CHX for 10 min, and then fixed and imaged. Arrows denote colocalization of VSV-G and Tfn (yellow). (C and D) Three-dimensional reconstruction of confocal serial sections, X-Y plane and sagittal section through REs (arrow) of representative cells in A and B.
Mentions: Initial experiments relied on immunofluorescence microscopy of fixed, coverslip-grown MDCKT cells. A recombinant adenovirus was used to express YFP-tagged ts045 VSV-G, with cells exposed to a pulse of virus and then incubated overnight at 40°C before shifting to 20°C in the presence of Alexa 546-Tfn. As shown in Fig. 1 A, VSV-G (Fig. 1, green) and Tfn (Fig. 1, red) did not colocalize during the 20°C block. Tfn was localized to a characteristic tight perinuclear cluster (Fig. 1 A, arrows), whereas VSV-G, also in the perinuclear region, was found in largely distinct structures that in many examples appeared to circumscribe the RE. These VSV-G–containing structures colocalized instead with markers of the Golgi such as GM130 (Fölsch et al., 2001) and furin (unpublished data). However, upon shifting to 31°C for 5–10 min and immediately processing the cells for immunofluorescence, VSV-G was observed to be spatially localized with Tfn (Fig. 1 B, arrows) in the Tfn-positive cluster previously devoid of VSV-G. The apparent colocalization of VSV-G and Tfn did not reflect a superimposition of distinct structures in different focal planes as it was evident throughout the entire volume of three-dimensional renderings (Fig. 1, C and D; and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200408165/DC1). Such overlap of VSV-G with Tfn was not observed using cells not expressing human TfnR, due to a much lower labeling efficiency of Tfn and thus inefficient ascertainment of Tfn-containing structures (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