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The Golgi ribbon structure facilitates anterograde transport of large cargoes.

Lavieu G, Dunlop MH, Lerich A, Zheng H, Bottanelli F, Rothman JE - Mol. Biol. Cell (2014)

Bottom Line: Yet the purpose of this remarkable structure has been an enigma.In addition, insect cells that naturally harbor dispersed Golgi stacks have limited capacity to transport artificial oversized cargoes.These results imply that the ribbon structure is an essential requirement for transport of large cargoes in mammalian cells, and we suggest that this is because it enables the dilated rims of cisternae (containing the aggregates) to move across the stack as they transfer among adjacent stacks within the ribbon structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520 gregory.lavieu@yale.edu james.rothman@yale.edu.

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Nocodazole inhibits intra-Golgi transport of aggregates. (A) Confocal micrographs illustrate representative three-color imaged stacks containing aggregated or disaggregated cargoes. HT1080 cells stably expressing ssGFP-FM4-hGH were pretreated with nocodazole for 2 h. Nocodazole was maintained during the rest of the experiment. Cells were incubated at 16°C in the presence of the disaggregating drug (1 h, 30 min, 1 μM) to allow for positioning the cargo within the cis-Golgi (I). The disaggregating drug was removed (III) or not (II) for 30 min before the temperature was shifted to 20°C for 30 min. Cells were fixed and prepared for immunofluorescence against the cis-Golgi marker gpp130 (labeled with a red dye) and against the trans-Golgi marker p230 (labeled with a far-red dye). (B) Graph represents the Pearson's coefficient for each marker combination for each condition. For each condition, we analyzed between 70 and 150 ministacks. *, p values < 0.01. (C) Electron micrographs show the retention of aggregates within the cis face of nocodazole-induced stacks. After nocodazole pretreatment, HeLa cells expressing ssGFP-FM4-hGH were incubated at 16°C for 1 h, 30 min in presence of the disaggregating drug. The drug was then removed for 30 min to allow for reaggregation within the cis face of nocodazole-induced ministacks. The temperature was shifted to 20°C for 1 h, and cells were prepared for EM (left panel). As a positive control for cis→trans transport at 20°C, cells subjected to the same nocodazole treatment were incubated for 1 h with the disaggregating drug at 20°C, and reaggregation was triggered by drug removal for 30 min at 20°C to allow for visualization of the aggregates by conventional EM (right panel).
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Figure 2: Nocodazole inhibits intra-Golgi transport of aggregates. (A) Confocal micrographs illustrate representative three-color imaged stacks containing aggregated or disaggregated cargoes. HT1080 cells stably expressing ssGFP-FM4-hGH were pretreated with nocodazole for 2 h. Nocodazole was maintained during the rest of the experiment. Cells were incubated at 16°C in the presence of the disaggregating drug (1 h, 30 min, 1 μM) to allow for positioning the cargo within the cis-Golgi (I). The disaggregating drug was removed (III) or not (II) for 30 min before the temperature was shifted to 20°C for 30 min. Cells were fixed and prepared for immunofluorescence against the cis-Golgi marker gpp130 (labeled with a red dye) and against the trans-Golgi marker p230 (labeled with a far-red dye). (B) Graph represents the Pearson's coefficient for each marker combination for each condition. For each condition, we analyzed between 70 and 150 ministacks. *, p values < 0.01. (C) Electron micrographs show the retention of aggregates within the cis face of nocodazole-induced stacks. After nocodazole pretreatment, HeLa cells expressing ssGFP-FM4-hGH were incubated at 16°C for 1 h, 30 min in presence of the disaggregating drug. The drug was then removed for 30 min to allow for reaggregation within the cis face of nocodazole-induced ministacks. The temperature was shifted to 20°C for 1 h, and cells were prepared for EM (left panel). As a positive control for cis→trans transport at 20°C, cells subjected to the same nocodazole treatment were incubated for 1 h with the disaggregating drug at 20°C, and reaggregation was triggered by drug removal for 30 min at 20°C to allow for visualization of the aggregates by conventional EM (right panel).

Mentions: We then assessed the intracellular localization of the nonsecreted fraction of the reaggregated protein using confocal microscopy. Working with nocodazole-induced ministacks allows for discriminating cis- from trans-Golgi cisternae, even at the light microscopy level (Dejgaard et al., 2007). Within the very same cells, the localization of the green fluorescent protein (GFP)-tagged disaggregated or aggregated cargo was compared with a cis-Golgi marker (Gpp130 labeled with a red dye) and a trans-Golgi marker (p230 labeled with a far-red dye). As expected, cis- and trans-Golgi markers were easily distinguishable, as judged by the lack of overlay and the low Pearson's coefficient value (Figure 2, A and B). At 16°C, the disaggregated cargo was specifically colocalizing with the cis-Golgi marker (Figure 2, A and B, condition I). When the cells were subsequently incubated at 20°C, a large portion of the disaggregated cargo reached the trans-Golgi (Figure 2, A and B, condition II), whereas the reaggregated cargo remained associated with the cis-Golgi cisternae (Figure 2, A and B, condition III). We observed the same cis-Golgi retention of the aggregates when the chase was performed at 37°C (Supplemental Figure S1).


The Golgi ribbon structure facilitates anterograde transport of large cargoes.

Lavieu G, Dunlop MH, Lerich A, Zheng H, Bottanelli F, Rothman JE - Mol. Biol. Cell (2014)

Nocodazole inhibits intra-Golgi transport of aggregates. (A) Confocal micrographs illustrate representative three-color imaged stacks containing aggregated or disaggregated cargoes. HT1080 cells stably expressing ssGFP-FM4-hGH were pretreated with nocodazole for 2 h. Nocodazole was maintained during the rest of the experiment. Cells were incubated at 16°C in the presence of the disaggregating drug (1 h, 30 min, 1 μM) to allow for positioning the cargo within the cis-Golgi (I). The disaggregating drug was removed (III) or not (II) for 30 min before the temperature was shifted to 20°C for 30 min. Cells were fixed and prepared for immunofluorescence against the cis-Golgi marker gpp130 (labeled with a red dye) and against the trans-Golgi marker p230 (labeled with a far-red dye). (B) Graph represents the Pearson's coefficient for each marker combination for each condition. For each condition, we analyzed between 70 and 150 ministacks. *, p values < 0.01. (C) Electron micrographs show the retention of aggregates within the cis face of nocodazole-induced stacks. After nocodazole pretreatment, HeLa cells expressing ssGFP-FM4-hGH were incubated at 16°C for 1 h, 30 min in presence of the disaggregating drug. The drug was then removed for 30 min to allow for reaggregation within the cis face of nocodazole-induced ministacks. The temperature was shifted to 20°C for 1 h, and cells were prepared for EM (left panel). As a positive control for cis→trans transport at 20°C, cells subjected to the same nocodazole treatment were incubated for 1 h with the disaggregating drug at 20°C, and reaggregation was triggered by drug removal for 30 min at 20°C to allow for visualization of the aggregates by conventional EM (right panel).
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Related In: Results  -  Collection

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Figure 2: Nocodazole inhibits intra-Golgi transport of aggregates. (A) Confocal micrographs illustrate representative three-color imaged stacks containing aggregated or disaggregated cargoes. HT1080 cells stably expressing ssGFP-FM4-hGH were pretreated with nocodazole for 2 h. Nocodazole was maintained during the rest of the experiment. Cells were incubated at 16°C in the presence of the disaggregating drug (1 h, 30 min, 1 μM) to allow for positioning the cargo within the cis-Golgi (I). The disaggregating drug was removed (III) or not (II) for 30 min before the temperature was shifted to 20°C for 30 min. Cells were fixed and prepared for immunofluorescence against the cis-Golgi marker gpp130 (labeled with a red dye) and against the trans-Golgi marker p230 (labeled with a far-red dye). (B) Graph represents the Pearson's coefficient for each marker combination for each condition. For each condition, we analyzed between 70 and 150 ministacks. *, p values < 0.01. (C) Electron micrographs show the retention of aggregates within the cis face of nocodazole-induced stacks. After nocodazole pretreatment, HeLa cells expressing ssGFP-FM4-hGH were incubated at 16°C for 1 h, 30 min in presence of the disaggregating drug. The drug was then removed for 30 min to allow for reaggregation within the cis face of nocodazole-induced ministacks. The temperature was shifted to 20°C for 1 h, and cells were prepared for EM (left panel). As a positive control for cis→trans transport at 20°C, cells subjected to the same nocodazole treatment were incubated for 1 h with the disaggregating drug at 20°C, and reaggregation was triggered by drug removal for 30 min at 20°C to allow for visualization of the aggregates by conventional EM (right panel).
Mentions: We then assessed the intracellular localization of the nonsecreted fraction of the reaggregated protein using confocal microscopy. Working with nocodazole-induced ministacks allows for discriminating cis- from trans-Golgi cisternae, even at the light microscopy level (Dejgaard et al., 2007). Within the very same cells, the localization of the green fluorescent protein (GFP)-tagged disaggregated or aggregated cargo was compared with a cis-Golgi marker (Gpp130 labeled with a red dye) and a trans-Golgi marker (p230 labeled with a far-red dye). As expected, cis- and trans-Golgi markers were easily distinguishable, as judged by the lack of overlay and the low Pearson's coefficient value (Figure 2, A and B). At 16°C, the disaggregated cargo was specifically colocalizing with the cis-Golgi marker (Figure 2, A and B, condition I). When the cells were subsequently incubated at 20°C, a large portion of the disaggregated cargo reached the trans-Golgi (Figure 2, A and B, condition II), whereas the reaggregated cargo remained associated with the cis-Golgi cisternae (Figure 2, A and B, condition III). We observed the same cis-Golgi retention of the aggregates when the chase was performed at 37°C (Supplemental Figure S1).

Bottom Line: Yet the purpose of this remarkable structure has been an enigma.In addition, insect cells that naturally harbor dispersed Golgi stacks have limited capacity to transport artificial oversized cargoes.These results imply that the ribbon structure is an essential requirement for transport of large cargoes in mammalian cells, and we suggest that this is because it enables the dilated rims of cisternae (containing the aggregates) to move across the stack as they transfer among adjacent stacks within the ribbon structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520 gregory.lavieu@yale.edu james.rothman@yale.edu.

Show MeSH
Related in: MedlinePlus