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Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Mironov AA, Beznoussenko GV, Nicoziani P, Martella O, Trucco A, Kweon HS, Di Giandomenico D, Polishchuk RS, Fusella A, Lupetti P, Berger EG, Geerts WJ, Koster AJ, Burger KN, Luini A - J. Cell Biol. (2001)

Bottom Line: Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae.Transport was followed using a combination of video and EM, providing high resolution in time and space.Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," 66030 Santa Maria Imbaro, Chieti, Italy.

ABSTRACT
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

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Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the nanogold gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.
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fig8: Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the nanogold gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.

Mentions: The amount of cargo reaching the Golgi per unit time in this experiment is probably much larger than during physiological traffic. To examine the behavior of the transport pathway under conditions closer to normal, we used the small-pulse protocol in which the accumulation of cargo in the IC is reduced but still sufficient to allow the formation of visible cargo-containing IC elements (scheme in Fig. 1). In spite of some fluorescence background due to cargo remaining in the ER, it was clear that, after release of the 15°C block, PC-I and VSVG colocalized and reached the Golgi simultaneously within 3–4 min, resided in the Golgi area for ∼8 min (transit time, legend to Fig. 2), and then left it, again simultaneously (Fig. 2, L–N). Thus, the movements of PC-I and VSVG could not be dissociated, even using this protocol. Notably, the cargoes in the Golgi area were discontinuously distributed in 10–30 discrete fluorescent spots of variable intensities (Fig. 3 and see Fig. 8). Also, interestingly, the transit time of the two cargoes through the Golgi area was shorter than when using the large-pulse protocol (8 vs. 20 min; quantification in Fig. 2, O and Q), suggesting that the transport behavior of the Golgi is influenced by the amount of cargo processed per unit time. This cargo effect is being analyzed separately.


Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Mironov AA, Beznoussenko GV, Nicoziani P, Martella O, Trucco A, Kweon HS, Di Giandomenico D, Polishchuk RS, Fusella A, Lupetti P, Berger EG, Geerts WJ, Koster AJ, Burger KN, Luini A - J. Cell Biol. (2001)

Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the nanogold gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.
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fig8: Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the nanogold gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.
Mentions: The amount of cargo reaching the Golgi per unit time in this experiment is probably much larger than during physiological traffic. To examine the behavior of the transport pathway under conditions closer to normal, we used the small-pulse protocol in which the accumulation of cargo in the IC is reduced but still sufficient to allow the formation of visible cargo-containing IC elements (scheme in Fig. 1). In spite of some fluorescence background due to cargo remaining in the ER, it was clear that, after release of the 15°C block, PC-I and VSVG colocalized and reached the Golgi simultaneously within 3–4 min, resided in the Golgi area for ∼8 min (transit time, legend to Fig. 2), and then left it, again simultaneously (Fig. 2, L–N). Thus, the movements of PC-I and VSVG could not be dissociated, even using this protocol. Notably, the cargoes in the Golgi area were discontinuously distributed in 10–30 discrete fluorescent spots of variable intensities (Fig. 3 and see Fig. 8). Also, interestingly, the transit time of the two cargoes through the Golgi area was shorter than when using the large-pulse protocol (8 vs. 20 min; quantification in Fig. 2, O and Q), suggesting that the transport behavior of the Golgi is influenced by the amount of cargo processed per unit time. This cargo effect is being analyzed separately.

Bottom Line: Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae.Transport was followed using a combination of video and EM, providing high resolution in time and space.Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," 66030 Santa Maria Imbaro, Chieti, Italy.

ABSTRACT
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

Show MeSH
Related in: MedlinePlus