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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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γ-Adaptin and clathrin colocalize with B-fragment and Tf-HRP at the ultrastructural level on coated membrane profiles of  EE/RE. (A–E) HeLa cells were incubated for 1 h with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections of these  cells were labeled with anti–B-fragment antibody (15-nm gold particles) and anti–γ-adaptin antibody (10-nm gold particles in A–C) or  anti-clathrin antibody (10-nm gold particles in D and E). In A–C, arrowheads indicate regions of colocalization between γ-adaptin and  B-fragment. In D and E, arrowheads point out regions of colocalization between clathrin and B-fragment. (F-I) Serum-starved HeLa  cells were incubated for 1 h with B-fragment and Tf-HRP at 19.5°C. Cryosections of these cells were labeled with anti–B-fragment antibody (15-nm gold particles), anti-HRP antibody (10-nm gold particles), and anti–γ-adaptin antibody (5-nm gold particles). Arrowheads  in F and G point to double- or triple-labeled profiles. (H and I) Magnification of selected structures. Bars, 100 nm.
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Figure 9: γ-Adaptin and clathrin colocalize with B-fragment and Tf-HRP at the ultrastructural level on coated membrane profiles of EE/RE. (A–E) HeLa cells were incubated for 1 h with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections of these cells were labeled with anti–B-fragment antibody (15-nm gold particles) and anti–γ-adaptin antibody (10-nm gold particles in A–C) or anti-clathrin antibody (10-nm gold particles in D and E). In A–C, arrowheads indicate regions of colocalization between γ-adaptin and B-fragment. In D and E, arrowheads point out regions of colocalization between clathrin and B-fragment. (F-I) Serum-starved HeLa cells were incubated for 1 h with B-fragment and Tf-HRP at 19.5°C. Cryosections of these cells were labeled with anti–B-fragment antibody (15-nm gold particles), anti-HRP antibody (10-nm gold particles), and anti–γ-adaptin antibody (5-nm gold particles). Arrowheads in F and G point to double- or triple-labeled profiles. (H and I) Magnification of selected structures. Bars, 100 nm.

Mentions: To determine the distribution of B-fragment and Tf-HRP–specific immunogold label in γ-adaptin–positive membrane profiles (see Fig. 10 A), 204 fields of observation (a total number of 2,512 and 1,990 B-fragment and Tf-HRP–specific gold particles, respectively, were counted) with 278 γ-adaptin–positive membrane profiles (at least two γ-adaptin–specific gold labels) that contained 273 and 294 B-fragment and Tf-HRP–specific gold particles, respectively, were analyzed on triple-labeled cryosections (as shown for example in Fig. 9, F–I). For statistical evaluation, B-fragment or Tf-HRP–specific gold particles in γ-adaptin–positive membrane profiles were taken as denominators. The chi-square test showed a significative difference (P < 0.001) between the fraction of B-fragment–specific gold particles in only B-fragment and γ-adaptin–positive membrane profiles (180 out of 273), when compared with the fraction of Tf-HRP–specific gold particles in only Tf-HRP and γ-adaptin–positive membrane profiles (122 out of 294).


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)

γ-Adaptin and clathrin colocalize with B-fragment and Tf-HRP at the ultrastructural level on coated membrane profiles of  EE/RE. (A–E) HeLa cells were incubated for 1 h with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections of these  cells were labeled with anti–B-fragment antibody (15-nm gold particles) and anti–γ-adaptin antibody (10-nm gold particles in A–C) or  anti-clathrin antibody (10-nm gold particles in D and E). In A–C, arrowheads indicate regions of colocalization between γ-adaptin and  B-fragment. In D and E, arrowheads point out regions of colocalization between clathrin and B-fragment. (F-I) Serum-starved HeLa  cells were incubated for 1 h with B-fragment and Tf-HRP at 19.5°C. Cryosections of these cells were labeled with anti–B-fragment antibody (15-nm gold particles), anti-HRP antibody (10-nm gold particles), and anti–γ-adaptin antibody (5-nm gold particles). Arrowheads  in F and G point to double- or triple-labeled profiles. (H and I) Magnification of selected structures. Bars, 100 nm.
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Figure 9: γ-Adaptin and clathrin colocalize with B-fragment and Tf-HRP at the ultrastructural level on coated membrane profiles of EE/RE. (A–E) HeLa cells were incubated for 1 h with B-fragment and BSA-gold (5-nm gold particles) at 19.5°C. Cryosections of these cells were labeled with anti–B-fragment antibody (15-nm gold particles) and anti–γ-adaptin antibody (10-nm gold particles in A–C) or anti-clathrin antibody (10-nm gold particles in D and E). In A–C, arrowheads indicate regions of colocalization between γ-adaptin and B-fragment. In D and E, arrowheads point out regions of colocalization between clathrin and B-fragment. (F-I) Serum-starved HeLa cells were incubated for 1 h with B-fragment and Tf-HRP at 19.5°C. Cryosections of these cells were labeled with anti–B-fragment antibody (15-nm gold particles), anti-HRP antibody (10-nm gold particles), and anti–γ-adaptin antibody (5-nm gold particles). Arrowheads in F and G point to double- or triple-labeled profiles. (H and I) Magnification of selected structures. Bars, 100 nm.
Mentions: To determine the distribution of B-fragment and Tf-HRP–specific immunogold label in γ-adaptin–positive membrane profiles (see Fig. 10 A), 204 fields of observation (a total number of 2,512 and 1,990 B-fragment and Tf-HRP–specific gold particles, respectively, were counted) with 278 γ-adaptin–positive membrane profiles (at least two γ-adaptin–specific gold labels) that contained 273 and 294 B-fragment and Tf-HRP–specific gold particles, respectively, were analyzed on triple-labeled cryosections (as shown for example in Fig. 9, F–I). For statistical evaluation, B-fragment or Tf-HRP–specific gold particles in γ-adaptin–positive membrane profiles were taken as denominators. The chi-square test showed a significative difference (P < 0.001) between the fraction of B-fragment–specific gold particles in only B-fragment and γ-adaptin–positive membrane profiles (180 out of 273), when compared with the fraction of Tf-HRP–specific gold particles in only Tf-HRP and γ-adaptin–positive membrane profiles (122 out of 294).

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