Limits...
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.

Show MeSH

Related in: MedlinePlus

Quantification of γ-adaptin–positive membrane profiles. (A) Distribution of B-fragment and Tf-HRP–specific gold  particles in γ-adaptin–positive membrane profiles. The columns  represent the fraction of B-fragment (lanes 1 and 2) in γ-adaptin– positive structures that were labeled (lane 2) or not labeled  (lane 1) for Tf-HRP, or the fraction of Tf-HRP (lanes 3 and 4)  in γ-adaptin–positive structures that were labeled (lane 4) or not  labeled (lane 3) for B-fragment. (B) Characterization of γ-adaptin–positive structures. Note that significantly more γ-adaptin– positive profiles were labeled for only B-fragment (left column),  compared with such structures labeled for only Tf-HRP (right  column). * and **, direct comparison shows that these conditions  are significantly different (P < 0.01; see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132951&req=5

Figure 10: Quantification of γ-adaptin–positive membrane profiles. (A) Distribution of B-fragment and Tf-HRP–specific gold particles in γ-adaptin–positive membrane profiles. The columns represent the fraction of B-fragment (lanes 1 and 2) in γ-adaptin– positive structures that were labeled (lane 2) or not labeled (lane 1) for Tf-HRP, or the fraction of Tf-HRP (lanes 3 and 4) in γ-adaptin–positive structures that were labeled (lane 4) or not labeled (lane 3) for B-fragment. (B) Characterization of γ-adaptin–positive structures. Note that significantly more γ-adaptin– positive profiles were labeled for only B-fragment (left column), compared with such structures labeled for only Tf-HRP (right column). * and **, direct comparison shows that these conditions are significantly different (P < 0.01; see Materials and Methods).

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)

Quantification of γ-adaptin–positive membrane profiles. (A) Distribution of B-fragment and Tf-HRP–specific gold  particles in γ-adaptin–positive membrane profiles. The columns  represent the fraction of B-fragment (lanes 1 and 2) in γ-adaptin– positive structures that were labeled (lane 2) or not labeled  (lane 1) for Tf-HRP, or the fraction of Tf-HRP (lanes 3 and 4)  in γ-adaptin–positive structures that were labeled (lane 4) or not  labeled (lane 3) for B-fragment. (B) Characterization of γ-adaptin–positive structures. Note that significantly more γ-adaptin– positive profiles were labeled for only B-fragment (left column),  compared with such structures labeled for only Tf-HRP (right  column). * and **, direct comparison shows that these conditions  are significantly different (P < 0.01; see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Quantification of γ-adaptin–positive membrane profiles. (A) Distribution of B-fragment and Tf-HRP–specific gold particles in γ-adaptin–positive membrane profiles. The columns represent the fraction of B-fragment (lanes 1 and 2) in γ-adaptin– positive structures that were labeled (lane 2) or not labeled (lane 1) for Tf-HRP, or the fraction of Tf-HRP (lanes 3 and 4) in γ-adaptin–positive structures that were labeled (lane 4) or not labeled (lane 3) for B-fragment. (B) Characterization of γ-adaptin–positive structures. Note that significantly more γ-adaptin– positive profiles were labeled for only B-fragment (left column), compared with such structures labeled for only Tf-HRP (right column). * and **, direct comparison shows that these conditions are significantly different (P < 0.01; see Materials and Methods).
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