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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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A fraction of internalized B-fragment is recycled to the plasma membrane.  FITC-labeled B-fragment  was internalized into HeLa  cells at 19.5°C, the cells were  then incubated with anti-FITC antibody on ice for 30  min and shifted to 37°C in the  continued presence of the  anti-FITC antibody for the indicated periods. Fluorescence at each time point was determined  as described in Materials and Methods and compared with the 0  time point to determine quenching due to newly recycled B-fragment. The means (± SE) for three independent experiments are  shown.
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Figure 4: A fraction of internalized B-fragment is recycled to the plasma membrane. FITC-labeled B-fragment was internalized into HeLa cells at 19.5°C, the cells were then incubated with anti-FITC antibody on ice for 30 min and shifted to 37°C in the continued presence of the anti-FITC antibody for the indicated periods. Fluorescence at each time point was determined as described in Materials and Methods and compared with the 0 time point to determine quenching due to newly recycled B-fragment. The means (± SE) for three independent experiments are shown.

Mentions: The presence of B-fragment in EE/RE in colocalization with Tf prompted us to test whether internalized B-fragment could be recycled to the plasma membrane, as described for other toxins (Sandvig and Olsnes, 1979; Nambiar and Wu, 1995; Alami et al., 1998). FITC-tagged B-fragment was internalized into HeLa cells at 19.5°C for 45 min. The cells were then put on ice and B-fragment that was still exposed at the cell surface was reacted with an anti-FITC antibody that quenched FITC fluorescence upon interaction with the fluorophore. The cells were subsequently transferred to 37°C in the continued presence of the anti-FITC antibody in the extracellular medium. It was found that 25% of FITC fluorescence that was emitted by intracellular B-fragments was quenched during a 30-min incubation at 37°C (Fig. 4). This value did not increase any more upon 50 additional min at 37°C in the presence of the anti-FITC antibody. 25% or 80% of total quenching was already obtained after 5 or 15 min at 37°C, respectively, indicating that B-fragment recycling to the plasma membrane was rapid. It can be concluded that only ∼25% of internalized B-fragment was recycled to the plasma membrane during an incubation period (80 min; Fig. 4) sufficiently long to have B-fragment at its peak concentration in the Golgi apparatus (Fig. 3). It should be added that following B-fragment internalization at 19.5°C and subsequent incubation at 37°C for 5–30 min, surface biotinylation studies failed to detect a significant increase in plasma membrane–associated B-fragment, consistent with the hypothesis of little B-fragment being recycled to the plasma membrane (not shown). The observation of limited recycling of B-fragment is also consistent with earlier studies that showed that plasma membrane associated Shiga toxin became rapidly insensitive to the neutralizing activity of anti-toxin antibody (Sandvig et al., 1989).


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)

A fraction of internalized B-fragment is recycled to the plasma membrane.  FITC-labeled B-fragment  was internalized into HeLa  cells at 19.5°C, the cells were  then incubated with anti-FITC antibody on ice for 30  min and shifted to 37°C in the  continued presence of the  anti-FITC antibody for the indicated periods. Fluorescence at each time point was determined  as described in Materials and Methods and compared with the 0  time point to determine quenching due to newly recycled B-fragment. The means (± SE) for three independent experiments are  shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132951&req=5

Figure 4: A fraction of internalized B-fragment is recycled to the plasma membrane. FITC-labeled B-fragment was internalized into HeLa cells at 19.5°C, the cells were then incubated with anti-FITC antibody on ice for 30 min and shifted to 37°C in the continued presence of the anti-FITC antibody for the indicated periods. Fluorescence at each time point was determined as described in Materials and Methods and compared with the 0 time point to determine quenching due to newly recycled B-fragment. The means (± SE) for three independent experiments are shown.
Mentions: The presence of B-fragment in EE/RE in colocalization with Tf prompted us to test whether internalized B-fragment could be recycled to the plasma membrane, as described for other toxins (Sandvig and Olsnes, 1979; Nambiar and Wu, 1995; Alami et al., 1998). FITC-tagged B-fragment was internalized into HeLa cells at 19.5°C for 45 min. The cells were then put on ice and B-fragment that was still exposed at the cell surface was reacted with an anti-FITC antibody that quenched FITC fluorescence upon interaction with the fluorophore. The cells were subsequently transferred to 37°C in the continued presence of the anti-FITC antibody in the extracellular medium. It was found that 25% of FITC fluorescence that was emitted by intracellular B-fragments was quenched during a 30-min incubation at 37°C (Fig. 4). This value did not increase any more upon 50 additional min at 37°C in the presence of the anti-FITC antibody. 25% or 80% of total quenching was already obtained after 5 or 15 min at 37°C, respectively, indicating that B-fragment recycling to the plasma membrane was rapid. It can be concluded that only ∼25% of internalized B-fragment was recycled to the plasma membrane during an incubation period (80 min; Fig. 4) sufficiently long to have B-fragment at its peak concentration in the Golgi apparatus (Fig. 3). It should be added that following B-fragment internalization at 19.5°C and subsequent incubation at 37°C for 5–30 min, surface biotinylation studies failed to detect a significant increase in plasma membrane–associated B-fragment, consistent with the hypothesis of little B-fragment being recycled to the plasma membrane (not shown). The observation of limited recycling of B-fragment is also consistent with earlier studies that showed that plasma membrane associated Shiga toxin became rapidly insensitive to the neutralizing activity of anti-toxin antibody (Sandvig et al., 1989).

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