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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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B-fragment and anti-TGN38, but not  anti–CI-MPR, are found in the same structures.  (A) Fluorophore-labeled B-fragment (left) and  anti-TGN38 antibody (right) were bound to  HeLa C7 cells on ice, upon which the cells were  incubated at 37°C for 10 min (top) and 60 min  (bottom) in the continued presence of both proteins. The cells were then fixed and stained for  internalized anti-TGN38 antibody (right). (B)  Fluorophore-labeled B-fragment (left) and anti– CI-MPR antibody (right) were bound to HeLa  cells on ice, upon which the cells were incubated  at 37°C for 30 min (top) and 4 h (bottom) in the  continued presence of both proteins. The cells  were then fixed and stained for internalized anti– CI-MPR antibody (right).
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Figure 11: B-fragment and anti-TGN38, but not anti–CI-MPR, are found in the same structures. (A) Fluorophore-labeled B-fragment (left) and anti-TGN38 antibody (right) were bound to HeLa C7 cells on ice, upon which the cells were incubated at 37°C for 10 min (top) and 60 min (bottom) in the continued presence of both proteins. The cells were then fixed and stained for internalized anti-TGN38 antibody (right). (B) Fluorophore-labeled B-fragment (left) and anti– CI-MPR antibody (right) were bound to HeLa cells on ice, upon which the cells were incubated at 37°C for 30 min (top) and 4 h (bottom) in the continued presence of both proteins. The cells were then fixed and stained for internalized anti– CI-MPR antibody (right).

Mentions: To test whether a cellular protein could follow the same route as the B-fragment, B-fragment was incubated at 37°C with HeLa cells in the presence of antibodies directed either to the CI-MPR or to rat TGN38 (Fig. 11). The CI-MPR and TGN38 both cycle between endosomes and the TGN (Humphrey et al., 1993; Reaves et al., 1993; Chapman and Munro, 1994; Ponnambalam et al., 1994; Munier-Lehmann et al., 1996). The antibodies were added at concentrations at which fluid phase internalization was not detectable (see Materials and Methods). After increasing periods of time, the cells were washed, fixed, and then processed for immunofluorescence. At 10 min, colabeling was found for TGN38 and B-fragment in vesicular structures at the level of EE of rat TGN38-expressing HeLa cells (Fig. 11 A, top row). After 30 min (not shown) or 60 min at 37°C (Fig. 11 A, bottom row), both proteins arrived in the Golgi apparatus. They labeled overlapping structures with subtle differences that may have resulted from B-fragment transport into the cisternae of the Golgi apparatus, while TGN38 remained confined to the TGN. In accordance with the hypothesis that TGN38 and the B-fragment could both follow the same route from EE/RE to the Golgi apparatus it was also observed that after BFA treatment, both proteins were found in tubular elements (not shown).


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)

B-fragment and anti-TGN38, but not  anti–CI-MPR, are found in the same structures.  (A) Fluorophore-labeled B-fragment (left) and  anti-TGN38 antibody (right) were bound to  HeLa C7 cells on ice, upon which the cells were  incubated at 37°C for 10 min (top) and 60 min  (bottom) in the continued presence of both proteins. The cells were then fixed and stained for  internalized anti-TGN38 antibody (right). (B)  Fluorophore-labeled B-fragment (left) and anti– CI-MPR antibody (right) were bound to HeLa  cells on ice, upon which the cells were incubated  at 37°C for 30 min (top) and 4 h (bottom) in the  continued presence of both proteins. The cells  were then fixed and stained for internalized anti– CI-MPR antibody (right).
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Related In: Results  -  Collection

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Figure 11: B-fragment and anti-TGN38, but not anti–CI-MPR, are found in the same structures. (A) Fluorophore-labeled B-fragment (left) and anti-TGN38 antibody (right) were bound to HeLa C7 cells on ice, upon which the cells were incubated at 37°C for 10 min (top) and 60 min (bottom) in the continued presence of both proteins. The cells were then fixed and stained for internalized anti-TGN38 antibody (right). (B) Fluorophore-labeled B-fragment (left) and anti– CI-MPR antibody (right) were bound to HeLa cells on ice, upon which the cells were incubated at 37°C for 30 min (top) and 4 h (bottom) in the continued presence of both proteins. The cells were then fixed and stained for internalized anti– CI-MPR antibody (right).
Mentions: To test whether a cellular protein could follow the same route as the B-fragment, B-fragment was incubated at 37°C with HeLa cells in the presence of antibodies directed either to the CI-MPR or to rat TGN38 (Fig. 11). The CI-MPR and TGN38 both cycle between endosomes and the TGN (Humphrey et al., 1993; Reaves et al., 1993; Chapman and Munro, 1994; Ponnambalam et al., 1994; Munier-Lehmann et al., 1996). The antibodies were added at concentrations at which fluid phase internalization was not detectable (see Materials and Methods). After increasing periods of time, the cells were washed, fixed, and then processed for immunofluorescence. At 10 min, colabeling was found for TGN38 and B-fragment in vesicular structures at the level of EE of rat TGN38-expressing HeLa cells (Fig. 11 A, top row). After 30 min (not shown) or 60 min at 37°C (Fig. 11 A, bottom row), both proteins arrived in the Golgi apparatus. They labeled overlapping structures with subtle differences that may have resulted from B-fragment transport into the cisternae of the Golgi apparatus, while TGN38 remained confined to the TGN. In accordance with the hypothesis that TGN38 and the B-fragment could both follow the same route from EE/RE to the Golgi apparatus it was also observed that after BFA treatment, both proteins were found in tubular elements (not shown).

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