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Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Mallard F, Antony C, Tenza D, Salamero J, Goud B, Johannes L - J. Cell Biol. (1998)

Bottom Line: This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus.B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes.Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre National de la Recherche Scientifique UMR 144, Laboratoire Mécanismes Moléculaires du Transport Intracellulaire, F-75248 Paris Cedex 05, France.

ABSTRACT
Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

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Kinetics of B-fragment transport from  EE/RE to the Golgi apparatus revealed by immunoelectron microscopy in HeLa cells. (A)  B-fragment was internalized for 1 h at 19.5°C,  cells were fixed and prepared for cryosectionning  as described under Materials and Methods.  B-fragment (10-nm gold particles) was detected  in tubular and vesicular elements that were also  labeled for the TfR (15-nm gold particles). Cells  that had internalized B-fragment at 19.5°C were  then shifted for 2 min (B), 10 min (C–D), and 30  min (E) to 37°C. Cryosections were stained for  B-fragment (10-nm gold particles in B, C, and E;  15-nm gold particles in D) and MPR46 (15-nm  gold particles in B; 10-nm gold particles in D).  Cryosections that were doubled stained for  MPR46 (in B and D) showed that the B-fragment  entered the Golgi apparatus via the TGN. Bars,  100 nm.
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Figure 2: Kinetics of B-fragment transport from EE/RE to the Golgi apparatus revealed by immunoelectron microscopy in HeLa cells. (A) B-fragment was internalized for 1 h at 19.5°C, cells were fixed and prepared for cryosectionning as described under Materials and Methods. B-fragment (10-nm gold particles) was detected in tubular and vesicular elements that were also labeled for the TfR (15-nm gold particles). Cells that had internalized B-fragment at 19.5°C were then shifted for 2 min (B), 10 min (C–D), and 30 min (E) to 37°C. Cryosections were stained for B-fragment (10-nm gold particles in B, C, and E; 15-nm gold particles in D) and MPR46 (15-nm gold particles in B; 10-nm gold particles in D). Cryosections that were doubled stained for MPR46 (in B and D) showed that the B-fragment entered the Golgi apparatus via the TGN. Bars, 100 nm.

Mentions: We next set out to follow B-fragment transport from EE/ RE to the Golgi apparatus. HeLa cells were incubated at 19.5°C with B-fragment, transferred for increasing times to 37°C, fixed, and then processed for ultracryomicrotomy followed by labeling with specific antibodies (Fig. 2). As described above, B-fragment (Fig. 2 A; 10-nm gold particles) accumulated at 19.5°C in vesicular and tubular structures (50–100 nm) that also contained the TfR (15-nm gold particles). The kinetics of B-fragment transport from EE/ RE to the Golgi apparatus was followed by warming the cells to 37°C. To identify membranes of the TGN, cryosections were also stained for MPR46, which at steady state in HeLa cells is mostly localized to the TGN, in contrast to the CI-MPR (not shown; see below). After 2-min incubation at 37°C (Fig. 2 B), some of the B-fragment (10-nm gold particles) accumulated already in membranes of the TGN in colocalization with MPR46 (15-nm gold particles) (Fig. 2 B), suggesting that the B-fragment entered the Golgi apparatus at the level of the TGN, in agreement with our previous results on sulfation site–carrying B-fragment mutants (Johannes et al., 1997). At 10 min (Fig. 2 C), B-fragment labeling (10-nm gold particles) at the Golgi apparatus increased significantly. As shown in Fig. 2 D, double-labeling experiments with B-fragment (15-nm gold particles) and MPR46 (10-nm gold particles) again identified some of the B-fragment–positive membranes as the TGN. After 30 min at 37°C (Fig. 2 E), all cisternae of the Golgi apparatus were strongly labeled with B-fragment (10-nm gold particles).


Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Mallard F, Antony C, Tenza D, Salamero J, Goud B, Johannes L - J. Cell Biol. (1998)

Kinetics of B-fragment transport from  EE/RE to the Golgi apparatus revealed by immunoelectron microscopy in HeLa cells. (A)  B-fragment was internalized for 1 h at 19.5°C,  cells were fixed and prepared for cryosectionning  as described under Materials and Methods.  B-fragment (10-nm gold particles) was detected  in tubular and vesicular elements that were also  labeled for the TfR (15-nm gold particles). Cells  that had internalized B-fragment at 19.5°C were  then shifted for 2 min (B), 10 min (C–D), and 30  min (E) to 37°C. Cryosections were stained for  B-fragment (10-nm gold particles in B, C, and E;  15-nm gold particles in D) and MPR46 (15-nm  gold particles in B; 10-nm gold particles in D).  Cryosections that were doubled stained for  MPR46 (in B and D) showed that the B-fragment  entered the Golgi apparatus via the TGN. Bars,  100 nm.
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Related In: Results  -  Collection

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Figure 2: Kinetics of B-fragment transport from EE/RE to the Golgi apparatus revealed by immunoelectron microscopy in HeLa cells. (A) B-fragment was internalized for 1 h at 19.5°C, cells were fixed and prepared for cryosectionning as described under Materials and Methods. B-fragment (10-nm gold particles) was detected in tubular and vesicular elements that were also labeled for the TfR (15-nm gold particles). Cells that had internalized B-fragment at 19.5°C were then shifted for 2 min (B), 10 min (C–D), and 30 min (E) to 37°C. Cryosections were stained for B-fragment (10-nm gold particles in B, C, and E; 15-nm gold particles in D) and MPR46 (15-nm gold particles in B; 10-nm gold particles in D). Cryosections that were doubled stained for MPR46 (in B and D) showed that the B-fragment entered the Golgi apparatus via the TGN. Bars, 100 nm.
Mentions: We next set out to follow B-fragment transport from EE/ RE to the Golgi apparatus. HeLa cells were incubated at 19.5°C with B-fragment, transferred for increasing times to 37°C, fixed, and then processed for ultracryomicrotomy followed by labeling with specific antibodies (Fig. 2). As described above, B-fragment (Fig. 2 A; 10-nm gold particles) accumulated at 19.5°C in vesicular and tubular structures (50–100 nm) that also contained the TfR (15-nm gold particles). The kinetics of B-fragment transport from EE/ RE to the Golgi apparatus was followed by warming the cells to 37°C. To identify membranes of the TGN, cryosections were also stained for MPR46, which at steady state in HeLa cells is mostly localized to the TGN, in contrast to the CI-MPR (not shown; see below). After 2-min incubation at 37°C (Fig. 2 B), some of the B-fragment (10-nm gold particles) accumulated already in membranes of the TGN in colocalization with MPR46 (15-nm gold particles) (Fig. 2 B), suggesting that the B-fragment entered the Golgi apparatus at the level of the TGN, in agreement with our previous results on sulfation site–carrying B-fragment mutants (Johannes et al., 1997). At 10 min (Fig. 2 C), B-fragment labeling (10-nm gold particles) at the Golgi apparatus increased significantly. As shown in Fig. 2 D, double-labeling experiments with B-fragment (15-nm gold particles) and MPR46 (10-nm gold particles) again identified some of the B-fragment–positive membranes as the TGN. After 30 min at 37°C (Fig. 2 E), all cisternae of the Golgi apparatus were strongly labeled with B-fragment (10-nm gold particles).

Bottom Line: This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus.B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes.Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre National de la Recherche Scientifique UMR 144, Laboratoire Mécanismes Moléculaires du Transport Intracellulaire, F-75248 Paris Cedex 05, France.

ABSTRACT
Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

Show MeSH
Related in: MedlinePlus