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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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Related in: MedlinePlus

PC-I-containing distensions never dissociate from Golgi cisternae during intra-Golgi traffic. Human fibroblasts were subjected to the large ER accumulation–chase protocol (or to the large-pulse protocol) and fast frozen (A–F) or treated with NEM to inhibit vesicle fusion (G–I). (A–F) 9 min after the shift to 32°C, the cells were fixed by ultra-fast freezing, and then cryosubstituted and embedded into Epon 812. Thick (250 nm) sections of Golgi cisternae were cut, prepared for electron microscopic tomography, and virtual 2–3-nm slices (A–C) were extracted from the tomograms using the IMOD software (Ladinsky et al., 1999). The 3-D reconstruction and surface rendering of the cisternae (yellow) and distensions (red) were performed using the SURFdriver program. The same structure is shown in two orientations (D and F). The image in F was chosen to show a pore in the cisterna which generates the impression of discontinuity between distension and cisterna in one of the virtual sections (arrow). Serial thin (50 nm) sections of Golgi cisternae were cut and used to reconstruct the image in E. (G–I) NEM treatment. 7 min after releasing the temperature block, cells were placed on ice, and medium with (H and I) or without (G) NEM (100 μM) was added for 15 min. After washing on ice, cells were shifted to 40°C again for an additional 3 min, and then fixed and prepared for EM. Tangential section of a cisterna with surrounding vesicles in a control cell (G). Tangential section of a cisterna in a NEM-treated cell; vesicular profiles are much more numerous (three- to fourfold) than in controls (H). PC-I distension connected with a cisternae in a NEM-treated cell (I). *, PC-I–containing distensions. Bar: (A and C–E) 150 nm; (F) 100 nm; (G and H) 300 nm; (I) 200 nm.
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fig4: PC-I-containing distensions never dissociate from Golgi cisternae during intra-Golgi traffic. Human fibroblasts were subjected to the large ER accumulation–chase protocol (or to the large-pulse protocol) and fast frozen (A–F) or treated with NEM to inhibit vesicle fusion (G–I). (A–F) 9 min after the shift to 32°C, the cells were fixed by ultra-fast freezing, and then cryosubstituted and embedded into Epon 812. Thick (250 nm) sections of Golgi cisternae were cut, prepared for electron microscopic tomography, and virtual 2–3-nm slices (A–C) were extracted from the tomograms using the IMOD software (Ladinsky et al., 1999). The 3-D reconstruction and surface rendering of the cisternae (yellow) and distensions (red) were performed using the SURFdriver program. The same structure is shown in two orientations (D and F). The image in F was chosen to show a pore in the cisterna which generates the impression of discontinuity between distension and cisterna in one of the virtual sections (arrow). Serial thin (50 nm) sections of Golgi cisternae were cut and used to reconstruct the image in E. (G–I) NEM treatment. 7 min after releasing the temperature block, cells were placed on ice, and medium with (H and I) or without (G) NEM (100 μM) was added for 15 min. After washing on ice, cells were shifted to 40°C again for an additional 3 min, and then fixed and prepared for EM. Tangential section of a cisterna with surrounding vesicles in a control cell (G). Tangential section of a cisterna in a NEM-treated cell; vesicular profiles are much more numerous (three- to fourfold) than in controls (H). PC-I distension connected with a cisternae in a NEM-treated cell (I). *, PC-I–containing distensions. Bar: (A and C–E) 150 nm; (F) 100 nm; (G and H) 300 nm; (I) 200 nm.

Mentions: PC-I has been reported to move along the secretory system without dissociating from cisternae (Bonfanti et al., 1998). However, because another type of secretory aggregates has been seen in containers (called megavesicles, 300–400 nm in diameter) adjacent to, but physically separate from, Golgi cisternae (Volchuk et al., 2000), we decided to reverify our previous proposition using new approaches. In our previous experiments, we had used standard fixation techniques for EM. Chemical fixation, albeit validated in countless experiments, is always a potential source of artifacts. For instance, in theory, if PC-I–containing megavesicles exist, they might be made to fuse with neighboring cisternae by the fixatives. This would mask evidence for their existence. To overcome this problem, we used ultrafast cryofixation, a virtually artifact-free method (Erk et al., 1998), and a synchronization protocol ensuring a high flux of PC-I, and therefore a large number of PC-I aggregates moving through the Golgi stacks. Both the ER accumulation–chase and the large-pulse protocols were used. Under both conditions, 6–8 min after release of the temperature block, all of the stacks exhibited numerous PC-I aggregate–containing distensions. Cryofixed cells were serial sectioned to establish the 3-D structure of these aggregates. Out of 120 distensions observed, none was found to be disconnected from the Golgi cisternae, either in thick sections (250 nm) subjected to electron tomography (Koster et al., 1997; Ladinsky et al., 1999) or in traditional thin sections. Fig. 4, D–F shows representative 3-D reconstructions of PC-I–containing distensions illustrating the above conclusion.


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)

PC-I-containing distensions never dissociate from Golgi cisternae during intra-Golgi traffic. Human fibroblasts were subjected to the large ER accumulation–chase protocol (or to the large-pulse protocol) and fast frozen (A–F) or treated with NEM to inhibit vesicle fusion (G–I). (A–F) 9 min after the shift to 32°C, the cells were fixed by ultra-fast freezing, and then cryosubstituted and embedded into Epon 812. Thick (250 nm) sections of Golgi cisternae were cut, prepared for electron microscopic tomography, and virtual 2–3-nm slices (A–C) were extracted from the tomograms using the IMOD software (Ladinsky et al., 1999). The 3-D reconstruction and surface rendering of the cisternae (yellow) and distensions (red) were performed using the SURFdriver program. The same structure is shown in two orientations (D and F). The image in F was chosen to show a pore in the cisterna which generates the impression of discontinuity between distension and cisterna in one of the virtual sections (arrow). Serial thin (50 nm) sections of Golgi cisternae were cut and used to reconstruct the image in E. (G–I) NEM treatment. 7 min after releasing the temperature block, cells were placed on ice, and medium with (H and I) or without (G) NEM (100 μM) was added for 15 min. After washing on ice, cells were shifted to 40°C again for an additional 3 min, and then fixed and prepared for EM. Tangential section of a cisterna with surrounding vesicles in a control cell (G). Tangential section of a cisterna in a NEM-treated cell; vesicular profiles are much more numerous (three- to fourfold) than in controls (H). PC-I distension connected with a cisternae in a NEM-treated cell (I). *, PC-I–containing distensions. Bar: (A and C–E) 150 nm; (F) 100 nm; (G and H) 300 nm; (I) 200 nm.
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fig4: PC-I-containing distensions never dissociate from Golgi cisternae during intra-Golgi traffic. Human fibroblasts were subjected to the large ER accumulation–chase protocol (or to the large-pulse protocol) and fast frozen (A–F) or treated with NEM to inhibit vesicle fusion (G–I). (A–F) 9 min after the shift to 32°C, the cells were fixed by ultra-fast freezing, and then cryosubstituted and embedded into Epon 812. Thick (250 nm) sections of Golgi cisternae were cut, prepared for electron microscopic tomography, and virtual 2–3-nm slices (A–C) were extracted from the tomograms using the IMOD software (Ladinsky et al., 1999). The 3-D reconstruction and surface rendering of the cisternae (yellow) and distensions (red) were performed using the SURFdriver program. The same structure is shown in two orientations (D and F). The image in F was chosen to show a pore in the cisterna which generates the impression of discontinuity between distension and cisterna in one of the virtual sections (arrow). Serial thin (50 nm) sections of Golgi cisternae were cut and used to reconstruct the image in E. (G–I) NEM treatment. 7 min after releasing the temperature block, cells were placed on ice, and medium with (H and I) or without (G) NEM (100 μM) was added for 15 min. After washing on ice, cells were shifted to 40°C again for an additional 3 min, and then fixed and prepared for EM. Tangential section of a cisterna with surrounding vesicles in a control cell (G). Tangential section of a cisterna in a NEM-treated cell; vesicular profiles are much more numerous (three- to fourfold) than in controls (H). PC-I distension connected with a cisternae in a NEM-treated cell (I). *, PC-I–containing distensions. Bar: (A and C–E) 150 nm; (F) 100 nm; (G and H) 300 nm; (I) 200 nm.
Mentions: PC-I has been reported to move along the secretory system without dissociating from cisternae (Bonfanti et al., 1998). However, because another type of secretory aggregates has been seen in containers (called megavesicles, 300–400 nm in diameter) adjacent to, but physically separate from, Golgi cisternae (Volchuk et al., 2000), we decided to reverify our previous proposition using new approaches. In our previous experiments, we had used standard fixation techniques for EM. Chemical fixation, albeit validated in countless experiments, is always a potential source of artifacts. For instance, in theory, if PC-I–containing megavesicles exist, they might be made to fuse with neighboring cisternae by the fixatives. This would mask evidence for their existence. To overcome this problem, we used ultrafast cryofixation, a virtually artifact-free method (Erk et al., 1998), and a synchronization protocol ensuring a high flux of PC-I, and therefore a large number of PC-I aggregates moving through the Golgi stacks. Both the ER accumulation–chase and the large-pulse protocols were used. Under both conditions, 6–8 min after release of the temperature block, all of the stacks exhibited numerous PC-I aggregate–containing distensions. Cryofixed cells were serial sectioned to establish the 3-D structure of these aggregates. Out of 120 distensions observed, none was found to be disconnected from the Golgi cisternae, either in thick sections (250 nm) subjected to electron tomography (Koster et al., 1997; Ladinsky et al., 1999) or in traditional thin sections. Fig. 4, D–F shows representative 3-D reconstructions of PC-I–containing distensions illustrating the above conclusion.

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