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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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Study of B-fragment colocalization with established markers of the endocytic pathway during  incubation at 19.5°C. The following proteins were incubated with HeLa cells at  19.5°C for 1 h: (A) Tf (green)  and B-fragment (red), a large  arrow indicates a region of  perinuclear staining; (B)  EGF (red) and B-fragment  (green), arrows point out regions where B-fragment and  EGF staining are juxtaposed;  (D) TF (green) and EGF  (red); (E) Dex3 (green) and  B-fragment (red), note vesicular (large arrows) and tail-like (small arrows) Dex3  staining. For marker concentrations see Materials and  Methods. Digital images  (four integration frames)  were acquired by confocal  microscopy. The right panel  represents the superposition  of the red and green images.  Insets show selected areas at  higher magnification. In C,  EGF (red) and B-fragment  (green) were internalized at  19.5°C, as in B. The cells  were then shifted to 37°C for  10 min before fixation. Note  that B-fragment and EGF-specific labeling did basically  not overlap.
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Figure 1: Study of B-fragment colocalization with established markers of the endocytic pathway during incubation at 19.5°C. The following proteins were incubated with HeLa cells at 19.5°C for 1 h: (A) Tf (green) and B-fragment (red), a large arrow indicates a region of perinuclear staining; (B) EGF (red) and B-fragment (green), arrows point out regions where B-fragment and EGF staining are juxtaposed; (D) TF (green) and EGF (red); (E) Dex3 (green) and B-fragment (red), note vesicular (large arrows) and tail-like (small arrows) Dex3 staining. For marker concentrations see Materials and Methods. Digital images (four integration frames) were acquired by confocal microscopy. The right panel represents the superposition of the red and green images. Insets show selected areas at higher magnification. In C, EGF (red) and B-fragment (green) were internalized at 19.5°C, as in B. The cells were then shifted to 37°C for 10 min before fixation. Note that B-fragment and EGF-specific labeling did basically not overlap.

Mentions: We have previously shown that in HeLa cells, Shiga toxin B-fragment is transported from the plasma membrane via endosomes, the TGN and the Golgi apparatus to the ER (Johannes et al., 1997). To analyze transport from endosomes to the Golgi apparatus in further detail, we developed a low temperature incubation protocol that allowed the synchronization of this transport step. B-fragment was incubated with HeLa cells at various temperatures, and it was found that at 19.5°C, the protein accumulated efficiently in endosomes, while its transport to the Golgi apparatus was blocked (Fig. 1). These data are consistent with earlier studies that showed that the plant toxin ricin is not transferred to the Golgi apparatus during low temperature incubations, and that under these experimental conditions Shiga toxin is much less toxic to HeLa cells than at physiological temperatures (Sandvig et al., 1989).


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)

Study of B-fragment colocalization with established markers of the endocytic pathway during  incubation at 19.5°C. The following proteins were incubated with HeLa cells at  19.5°C for 1 h: (A) Tf (green)  and B-fragment (red), a large  arrow indicates a region of  perinuclear staining; (B)  EGF (red) and B-fragment  (green), arrows point out regions where B-fragment and  EGF staining are juxtaposed;  (D) TF (green) and EGF  (red); (E) Dex3 (green) and  B-fragment (red), note vesicular (large arrows) and tail-like (small arrows) Dex3  staining. For marker concentrations see Materials and  Methods. Digital images  (four integration frames)  were acquired by confocal  microscopy. The right panel  represents the superposition  of the red and green images.  Insets show selected areas at  higher magnification. In C,  EGF (red) and B-fragment  (green) were internalized at  19.5°C, as in B. The cells  were then shifted to 37°C for  10 min before fixation. Note  that B-fragment and EGF-specific labeling did basically  not overlap.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132951&req=5

Figure 1: Study of B-fragment colocalization with established markers of the endocytic pathway during incubation at 19.5°C. The following proteins were incubated with HeLa cells at 19.5°C for 1 h: (A) Tf (green) and B-fragment (red), a large arrow indicates a region of perinuclear staining; (B) EGF (red) and B-fragment (green), arrows point out regions where B-fragment and EGF staining are juxtaposed; (D) TF (green) and EGF (red); (E) Dex3 (green) and B-fragment (red), note vesicular (large arrows) and tail-like (small arrows) Dex3 staining. For marker concentrations see Materials and Methods. Digital images (four integration frames) were acquired by confocal microscopy. The right panel represents the superposition of the red and green images. Insets show selected areas at higher magnification. In C, EGF (red) and B-fragment (green) were internalized at 19.5°C, as in B. The cells were then shifted to 37°C for 10 min before fixation. Note that B-fragment and EGF-specific labeling did basically not overlap.
Mentions: We have previously shown that in HeLa cells, Shiga toxin B-fragment is transported from the plasma membrane via endosomes, the TGN and the Golgi apparatus to the ER (Johannes et al., 1997). To analyze transport from endosomes to the Golgi apparatus in further detail, we developed a low temperature incubation protocol that allowed the synchronization of this transport step. B-fragment was incubated with HeLa cells at various temperatures, and it was found that at 19.5°C, the protein accumulated efficiently in endosomes, while its transport to the Golgi apparatus was blocked (Fig. 1). These data are consistent with earlier studies that showed that the plant toxin ricin is not transferred to the Golgi apparatus during low temperature incubations, and that under these experimental conditions Shiga toxin is much less toxic to HeLa cells than at physiological temperatures (Sandvig et al., 1989).

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