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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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Kinetics of B-fragment transport from EE/RE to the Golgi apparatus. (A) Confocal microscopy on living HeLa cells. Fluorophore-labeled B-fragment was internalized for 1 h into HeLa cells at 19.5°C, upon which the cells were transferred to the stage of a confocal microscope and incubated at 37°C. Digital images (four integration frames) were acquired at the indicated time points. Note that  after 4 min, B-fragment was detected in peripheral structures, and then later concentrated in the perinuclear region. (B) Images as  shown in A were quantified, and the fraction of average Golgi associated fluorescence over average total cell-associated fluorescence is  represented in function of incubation time at 37°C. The means (± SE) of eight experiments are shown. The curve was fitted to f(x) =  1 + 2.97[1 − exp(−0.036×)], r = 0.9979. (C) Sulfation analysis. B-(Sulf)2 was internalized into HeLa cells at 19.5°C, and the cells were  then shifted to 37°C. After 0, 15, 30, 60, and 90 min, radioactive sulfate was added for 15 min. Note that B-(Sulf)2 is at its peak concentration in the TGN during the 15–30 min interval. A representative of 2 experiments is shown. In each experiment, the data points were obtained in duplicate. (D) Cotransport of B-fragment and Tf in living cells. For the points 3 and 10 min at 37°C, fluorophore-coupled  B-fragment and fluorophore-coupled Tf were internalized as described in A. For the point 30 min at 37°C, B-fragment alone was internalized continuously at 37°C, cells were then fixed and stained for the TfR. Note that the B-fragment concentrated in the Golgi area  (large arrow at 10 min), while remaining cytoplasmic B-fragment–containing structures always were Tf (4 and 10 min) or TfR (30 min)  positive (small arrows at 30 min). Single optical slices were obtained by confocal microscopy.
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Figure 3: Kinetics of B-fragment transport from EE/RE to the Golgi apparatus. (A) Confocal microscopy on living HeLa cells. Fluorophore-labeled B-fragment was internalized for 1 h into HeLa cells at 19.5°C, upon which the cells were transferred to the stage of a confocal microscope and incubated at 37°C. Digital images (four integration frames) were acquired at the indicated time points. Note that after 4 min, B-fragment was detected in peripheral structures, and then later concentrated in the perinuclear region. (B) Images as shown in A were quantified, and the fraction of average Golgi associated fluorescence over average total cell-associated fluorescence is represented in function of incubation time at 37°C. The means (± SE) of eight experiments are shown. The curve was fitted to f(x) = 1 + 2.97[1 − exp(−0.036×)], r = 0.9979. (C) Sulfation analysis. B-(Sulf)2 was internalized into HeLa cells at 19.5°C, and the cells were then shifted to 37°C. After 0, 15, 30, 60, and 90 min, radioactive sulfate was added for 15 min. Note that B-(Sulf)2 is at its peak concentration in the TGN during the 15–30 min interval. A representative of 2 experiments is shown. In each experiment, the data points were obtained in duplicate. (D) Cotransport of B-fragment and Tf in living cells. For the points 3 and 10 min at 37°C, fluorophore-coupled B-fragment and fluorophore-coupled Tf were internalized as described in A. For the point 30 min at 37°C, B-fragment alone was internalized continuously at 37°C, cells were then fixed and stained for the TfR. Note that the B-fragment concentrated in the Golgi area (large arrow at 10 min), while remaining cytoplasmic B-fragment–containing structures always were Tf (4 and 10 min) or TfR (30 min) positive (small arrows at 30 min). Single optical slices were obtained by confocal microscopy.

Mentions: HeLa cells were plated on 42-mm glass cover slides. Cy3-labeled B-fragment was bound to these cells and then internalized at 19.5°C, as described above. The cover slide was rapidly transferred to a POC-chamber (Bachofer Laboratoriumsgeräte, Reutlingen, Germany) on the stage at the confocal microscope, and culture medium at 37°C was added. Acquisitions during incubation at 37°C were done at frequencies as indicated in the figure legends. Care was taken to minimize light exposure as it was noticed that high image acquisition frequencies and high laser light intensities prevented B-fragment transport to the Golgi apparatus and caused the appearance of the protein in unidentified structures (not shown). Per time point, four optical slices were taken. Throughout an 80-min experiment, offset in z-direction was minimal. However, sometimes it was necessary to readjust the microscope. For quantification as shown in Fig. 3 B, the average fluorescent intensity in the Golgi area was divided by the average fluorescent intensity over the whole cell. To determine the fraction of Golgi-associated fluorescent material in percent after 80 min at 37°C, the ratio between total fluorescent contents in the Golgi area over whole cell total fluorescent contents was calculated. Background correction factors were determined from cells that did not have internalized B-fragment. All measurements were done using NIH image software (National Institutes of Health, Bethesda, MD).


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)

Kinetics of B-fragment transport from EE/RE to the Golgi apparatus. (A) Confocal microscopy on living HeLa cells. Fluorophore-labeled B-fragment was internalized for 1 h into HeLa cells at 19.5°C, upon which the cells were transferred to the stage of a confocal microscope and incubated at 37°C. Digital images (four integration frames) were acquired at the indicated time points. Note that  after 4 min, B-fragment was detected in peripheral structures, and then later concentrated in the perinuclear region. (B) Images as  shown in A were quantified, and the fraction of average Golgi associated fluorescence over average total cell-associated fluorescence is  represented in function of incubation time at 37°C. The means (± SE) of eight experiments are shown. The curve was fitted to f(x) =  1 + 2.97[1 − exp(−0.036×)], r = 0.9979. (C) Sulfation analysis. B-(Sulf)2 was internalized into HeLa cells at 19.5°C, and the cells were  then shifted to 37°C. After 0, 15, 30, 60, and 90 min, radioactive sulfate was added for 15 min. Note that B-(Sulf)2 is at its peak concentration in the TGN during the 15–30 min interval. A representative of 2 experiments is shown. In each experiment, the data points were obtained in duplicate. (D) Cotransport of B-fragment and Tf in living cells. For the points 3 and 10 min at 37°C, fluorophore-coupled  B-fragment and fluorophore-coupled Tf were internalized as described in A. For the point 30 min at 37°C, B-fragment alone was internalized continuously at 37°C, cells were then fixed and stained for the TfR. Note that the B-fragment concentrated in the Golgi area  (large arrow at 10 min), while remaining cytoplasmic B-fragment–containing structures always were Tf (4 and 10 min) or TfR (30 min)  positive (small arrows at 30 min). Single optical slices were obtained by confocal microscopy.
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Figure 3: Kinetics of B-fragment transport from EE/RE to the Golgi apparatus. (A) Confocal microscopy on living HeLa cells. Fluorophore-labeled B-fragment was internalized for 1 h into HeLa cells at 19.5°C, upon which the cells were transferred to the stage of a confocal microscope and incubated at 37°C. Digital images (four integration frames) were acquired at the indicated time points. Note that after 4 min, B-fragment was detected in peripheral structures, and then later concentrated in the perinuclear region. (B) Images as shown in A were quantified, and the fraction of average Golgi associated fluorescence over average total cell-associated fluorescence is represented in function of incubation time at 37°C. The means (± SE) of eight experiments are shown. The curve was fitted to f(x) = 1 + 2.97[1 − exp(−0.036×)], r = 0.9979. (C) Sulfation analysis. B-(Sulf)2 was internalized into HeLa cells at 19.5°C, and the cells were then shifted to 37°C. After 0, 15, 30, 60, and 90 min, radioactive sulfate was added for 15 min. Note that B-(Sulf)2 is at its peak concentration in the TGN during the 15–30 min interval. A representative of 2 experiments is shown. In each experiment, the data points were obtained in duplicate. (D) Cotransport of B-fragment and Tf in living cells. For the points 3 and 10 min at 37°C, fluorophore-coupled B-fragment and fluorophore-coupled Tf were internalized as described in A. For the point 30 min at 37°C, B-fragment alone was internalized continuously at 37°C, cells were then fixed and stained for the TfR. Note that the B-fragment concentrated in the Golgi area (large arrow at 10 min), while remaining cytoplasmic B-fragment–containing structures always were Tf (4 and 10 min) or TfR (30 min) positive (small arrows at 30 min). Single optical slices were obtained by confocal microscopy.
Mentions: HeLa cells were plated on 42-mm glass cover slides. Cy3-labeled B-fragment was bound to these cells and then internalized at 19.5°C, as described above. The cover slide was rapidly transferred to a POC-chamber (Bachofer Laboratoriumsgeräte, Reutlingen, Germany) on the stage at the confocal microscope, and culture medium at 37°C was added. Acquisitions during incubation at 37°C were done at frequencies as indicated in the figure legends. Care was taken to minimize light exposure as it was noticed that high image acquisition frequencies and high laser light intensities prevented B-fragment transport to the Golgi apparatus and caused the appearance of the protein in unidentified structures (not shown). Per time point, four optical slices were taken. Throughout an 80-min experiment, offset in z-direction was minimal. However, sometimes it was necessary to readjust the microscope. For quantification as shown in Fig. 3 B, the average fluorescent intensity in the Golgi area was divided by the average fluorescent intensity over the whole cell. To determine the fraction of Golgi-associated fluorescent material in percent after 80 min at 37°C, the ratio between total fluorescent contents in the Golgi area over whole cell total fluorescent contents was calculated. Background correction factors were determined from cells that did not have internalized B-fragment. All measurements were done using NIH image software (National Institutes of Health, Bethesda, MD).

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