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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 is detected in compartments of the late endocytic pathway during transport to the Golgi apparatus. (A and B)  HeLa cells were incubated with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections were stained with anti–B-fragment antibody (10-nm gold particles). The arrow in B indicates a region of high BSA-gold concentration (bulk fluid phase) that is devoid of B-fragment. (C) Cells treated as in A and B were then shifted for 15 min to 37°C, fixed, and then cryosections were stained with  anti–B-fragment antibody (15-nm gold particles) and anti–CI-MPR antibody (10-nm gold particles). Various endocytic structures are indicated by numbers: areas 1, BSA-gold–labeled EE; areas 2, CI-MPR- and BSA-gold–positive multivesicular LE; areas 3, CI-MPR–positive multilamellar LE. GA, Golgi apparatus. (D) Quantification of BSA-gold–positive structures containing or not B-fragment, after  the 19.5°C incubation (lanes 1 and 2) or after an additional shift to 37°C for 15 min (lanes 3 and 4). (Lanes 1 and 3) Percentage of BSA-gold–containing structures that also contain B-fragment; (lanes 2 and 4), BSA-gold–containing structures without B-fragment. Bars, 100 nm.
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Figure 5: B-fragment is detected in compartments of the late endocytic pathway during transport to the Golgi apparatus. (A and B) HeLa cells were incubated with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections were stained with anti–B-fragment antibody (10-nm gold particles). The arrow in B indicates a region of high BSA-gold concentration (bulk fluid phase) that is devoid of B-fragment. (C) Cells treated as in A and B were then shifted for 15 min to 37°C, fixed, and then cryosections were stained with anti–B-fragment antibody (15-nm gold particles) and anti–CI-MPR antibody (10-nm gold particles). Various endocytic structures are indicated by numbers: areas 1, BSA-gold–labeled EE; areas 2, CI-MPR- and BSA-gold–positive multivesicular LE; areas 3, CI-MPR–positive multilamellar LE. GA, Golgi apparatus. (D) Quantification of BSA-gold–positive structures containing or not B-fragment, after the 19.5°C incubation (lanes 1 and 2) or after an additional shift to 37°C for 15 min (lanes 3 and 4). (Lanes 1 and 3) Percentage of BSA-gold–containing structures that also contain B-fragment; (lanes 2 and 4), BSA-gold–containing structures without B-fragment. Bars, 100 nm.

Mentions: Evolution of colocalization between B-fragment and BSA-gold (see Fig. 5 D): membrane profiles that were labeled for BSA-gold (at least two gold particles) were identified by scanning over cryosections of cells that had internalized B-fragment and BSA-gold at 19.5°C, or that were shifted to 37°C for an additional 15 min. It was then determined whether these profiles were also labeled for B-fragment (100 membrane profiles were analyzed per condition).


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 is detected in compartments of the late endocytic pathway during transport to the Golgi apparatus. (A and B)  HeLa cells were incubated with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections were stained with anti–B-fragment antibody (10-nm gold particles). The arrow in B indicates a region of high BSA-gold concentration (bulk fluid phase) that is devoid of B-fragment. (C) Cells treated as in A and B were then shifted for 15 min to 37°C, fixed, and then cryosections were stained with  anti–B-fragment antibody (15-nm gold particles) and anti–CI-MPR antibody (10-nm gold particles). Various endocytic structures are indicated by numbers: areas 1, BSA-gold–labeled EE; areas 2, CI-MPR- and BSA-gold–positive multivesicular LE; areas 3, CI-MPR–positive multilamellar LE. GA, Golgi apparatus. (D) Quantification of BSA-gold–positive structures containing or not B-fragment, after  the 19.5°C incubation (lanes 1 and 2) or after an additional shift to 37°C for 15 min (lanes 3 and 4). (Lanes 1 and 3) Percentage of BSA-gold–containing structures that also contain B-fragment; (lanes 2 and 4), BSA-gold–containing structures without B-fragment. Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: B-fragment is detected in compartments of the late endocytic pathway during transport to the Golgi apparatus. (A and B) HeLa cells were incubated with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections were stained with anti–B-fragment antibody (10-nm gold particles). The arrow in B indicates a region of high BSA-gold concentration (bulk fluid phase) that is devoid of B-fragment. (C) Cells treated as in A and B were then shifted for 15 min to 37°C, fixed, and then cryosections were stained with anti–B-fragment antibody (15-nm gold particles) and anti–CI-MPR antibody (10-nm gold particles). Various endocytic structures are indicated by numbers: areas 1, BSA-gold–labeled EE; areas 2, CI-MPR- and BSA-gold–positive multivesicular LE; areas 3, CI-MPR–positive multilamellar LE. GA, Golgi apparatus. (D) Quantification of BSA-gold–positive structures containing or not B-fragment, after the 19.5°C incubation (lanes 1 and 2) or after an additional shift to 37°C for 15 min (lanes 3 and 4). (Lanes 1 and 3) Percentage of BSA-gold–containing structures that also contain B-fragment; (lanes 2 and 4), BSA-gold–containing structures without B-fragment. Bars, 100 nm.
Mentions: Evolution of colocalization between B-fragment and BSA-gold (see Fig. 5 D): membrane profiles that were labeled for BSA-gold (at least two gold particles) were identified by scanning over cryosections of cells that had internalized B-fragment and BSA-gold at 19.5°C, or that were shifted to 37°C for an additional 15 min. It was then determined whether these profiles were also labeled for B-fragment (100 membrane profiles were analyzed per condition).

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