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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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Drug effects on  B-fragment transport to the  Golgi apparatus. HeLa cells  were incubated with fluorophore-labeled B-fragment  for 1 h at 19.5°C, before being  shifted to 37°C (A) in the  absence (CTL) or presence  of 1 μM Bafi or 1 μM CytoD,  or (B) in the presence of 5 μg/ ml BFA. The cells were  then fixed and labeled with  CTR433 antibody (A) or anti-TfR antibody (B). (C) BFA  inhibits B-fragment transport  to dispersed Golgi cisternae  in Noc-treated HeLa cells.  HeLa cells were pretreated  for 1 h with 10 μM Noc. The  cells were then transferred on  ice and incubated with fluorophore-labeled B-fragment  for 30 min, washed, and then  shifted for 30 min to 37°C in  the absence (top row) or presence (bottom row) of 5 μg/ml  BFA and in the continued  presence of Noc. The cells  were then fixed and stained  for the Golgi marker  CTR433. Note that in the absence of BFA, B-fragment associated with the dispersed  cisternae of the Golgi apparatus (top), while in the presence of the drug, the  CTR433-positive cisternae  were devoid of B-fragment  (bottom). Four optical slices  were obtained by confocal  microscopy.
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Figure 6: Drug effects on B-fragment transport to the Golgi apparatus. HeLa cells were incubated with fluorophore-labeled B-fragment for 1 h at 19.5°C, before being shifted to 37°C (A) in the absence (CTL) or presence of 1 μM Bafi or 1 μM CytoD, or (B) in the presence of 5 μg/ ml BFA. The cells were then fixed and labeled with CTR433 antibody (A) or anti-TfR antibody (B). (C) BFA inhibits B-fragment transport to dispersed Golgi cisternae in Noc-treated HeLa cells. HeLa cells were pretreated for 1 h with 10 μM Noc. The cells were then transferred on ice and incubated with fluorophore-labeled B-fragment for 30 min, washed, and then shifted for 30 min to 37°C in the absence (top row) or presence (bottom row) of 5 μg/ml BFA and in the continued presence of Noc. The cells were then fixed and stained for the Golgi marker CTR433. Note that in the absence of BFA, B-fragment associated with the dispersed cisternae of the Golgi apparatus (top), while in the presence of the drug, the CTR433-positive cisternae were devoid of B-fragment (bottom). Four optical slices were obtained by confocal microscopy.

Mentions: To test the influence of drugs with established effects on the endocytic pathway on B-fragment transport to the Golgi apparatus, cells that had internalized Cy3-labeled B-fragment at 19.5°C were incubated for 1 h at 37°C in presence of the vacuolar proton pump inhibitor bafilomycin A1 (Bafi), the actin-depolymerizing drug cytochalasin D (CytoD), or the fungal metabolite brefeldin A (BFA) (Fig. 6). Neither CytoD nor Bafi prevented B-fragment appearance in the Golgi apparatus, stained with the medial Golgi marker CTR433 (Fig. 6 A), consistent with recently published observations (Schapiro et al., 1998). The same results were obtained when the cells were pretreated for up to 2 h with these drugs (not shown). In the presence of BFA, which in addition to its profound effects on the biosynthetic/secretory pathway (for review see Klausner et al., 1992) has somewhat more subtle effects on the endocytic membrane system (Hunziker et al., 1991; Lippincott-Schwartz et al., 1991; Wood et al., 1991; Strous et al., 1993; for review see Hunziker et al., 1992), B-fragment accumulated in tubular elements that also contained the TfR (Fig. 6 B). The protein remained associated with these tubules even when the cells were incubated at 37°C for up to 4 h in presence of the drug (not shown). In the presence of the microtubule-destabilizing agent nocodazole (Noc), B-fragment was still transported to the dispersed cisternae of the Golgi apparatus where it colocalized with the Golgi marker CTR433 (Fig. 6 C, top row; see also Johannes et al., 1997) and with the TGN marker protein TGN38 (not shown). Interestingly, when Noc-treated cells were also exposed to BFA, CTR433-positive (Fig. 6 C, bottom row) Golgi compartments that persisted under these conditions (Lippincott-Schwartz et al., 1990) were not labeled by B-fragment any more, suggesting that transport between the BFA-induced, B-fragment-, and TfR-positive membrane system and the Golgi remnants had ceased.


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)

Drug effects on  B-fragment transport to the  Golgi apparatus. HeLa cells  were incubated with fluorophore-labeled B-fragment  for 1 h at 19.5°C, before being  shifted to 37°C (A) in the  absence (CTL) or presence  of 1 μM Bafi or 1 μM CytoD,  or (B) in the presence of 5 μg/ ml BFA. The cells were  then fixed and labeled with  CTR433 antibody (A) or anti-TfR antibody (B). (C) BFA  inhibits B-fragment transport  to dispersed Golgi cisternae  in Noc-treated HeLa cells.  HeLa cells were pretreated  for 1 h with 10 μM Noc. The  cells were then transferred on  ice and incubated with fluorophore-labeled B-fragment  for 30 min, washed, and then  shifted for 30 min to 37°C in  the absence (top row) or presence (bottom row) of 5 μg/ml  BFA and in the continued  presence of Noc. The cells  were then fixed and stained  for the Golgi marker  CTR433. Note that in the absence of BFA, B-fragment associated with the dispersed  cisternae of the Golgi apparatus (top), while in the presence of the drug, the  CTR433-positive cisternae  were devoid of B-fragment  (bottom). Four optical slices  were obtained by confocal  microscopy.
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Figure 6: Drug effects on B-fragment transport to the Golgi apparatus. HeLa cells were incubated with fluorophore-labeled B-fragment for 1 h at 19.5°C, before being shifted to 37°C (A) in the absence (CTL) or presence of 1 μM Bafi or 1 μM CytoD, or (B) in the presence of 5 μg/ ml BFA. The cells were then fixed and labeled with CTR433 antibody (A) or anti-TfR antibody (B). (C) BFA inhibits B-fragment transport to dispersed Golgi cisternae in Noc-treated HeLa cells. HeLa cells were pretreated for 1 h with 10 μM Noc. The cells were then transferred on ice and incubated with fluorophore-labeled B-fragment for 30 min, washed, and then shifted for 30 min to 37°C in the absence (top row) or presence (bottom row) of 5 μg/ml BFA and in the continued presence of Noc. The cells were then fixed and stained for the Golgi marker CTR433. Note that in the absence of BFA, B-fragment associated with the dispersed cisternae of the Golgi apparatus (top), while in the presence of the drug, the CTR433-positive cisternae were devoid of B-fragment (bottom). Four optical slices were obtained by confocal microscopy.
Mentions: To test the influence of drugs with established effects on the endocytic pathway on B-fragment transport to the Golgi apparatus, cells that had internalized Cy3-labeled B-fragment at 19.5°C were incubated for 1 h at 37°C in presence of the vacuolar proton pump inhibitor bafilomycin A1 (Bafi), the actin-depolymerizing drug cytochalasin D (CytoD), or the fungal metabolite brefeldin A (BFA) (Fig. 6). Neither CytoD nor Bafi prevented B-fragment appearance in the Golgi apparatus, stained with the medial Golgi marker CTR433 (Fig. 6 A), consistent with recently published observations (Schapiro et al., 1998). The same results were obtained when the cells were pretreated for up to 2 h with these drugs (not shown). In the presence of BFA, which in addition to its profound effects on the biosynthetic/secretory pathway (for review see Klausner et al., 1992) has somewhat more subtle effects on the endocytic membrane system (Hunziker et al., 1991; Lippincott-Schwartz et al., 1991; Wood et al., 1991; Strous et al., 1993; for review see Hunziker et al., 1992), B-fragment accumulated in tubular elements that also contained the TfR (Fig. 6 B). The protein remained associated with these tubules even when the cells were incubated at 37°C for up to 4 h in presence of the drug (not shown). In the presence of the microtubule-destabilizing agent nocodazole (Noc), B-fragment was still transported to the dispersed cisternae of the Golgi apparatus where it colocalized with the Golgi marker CTR433 (Fig. 6 C, top row; see also Johannes et al., 1997) and with the TGN marker protein TGN38 (not shown). Interestingly, when Noc-treated cells were also exposed to BFA, CTR433-positive (Fig. 6 C, bottom row) Golgi compartments that persisted under these conditions (Lippincott-Schwartz et al., 1990) were not labeled by B-fragment any more, suggesting that transport between the BFA-induced, B-fragment-, and TfR-positive membrane system and the Golgi remnants had ceased.

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