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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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S2 cells poorly secrete aggregates, which are retained within the cis-Golgi. (A) Confocal micrograph showing separated cis- and trans-Golgi within single stacks through the cytoplasm of S2 cells. S2 cells were fixed and prepared for immunofluorescence against Gm130 (green-labeled cis-Golgi marker) and G245 (red labeled trans-Golgi marker). Graph shows Pearson's coefficient value for each imaged single Golgi stack. Red line represents the average value over 50 analyzed stacks. (B) Transport kinetics of disaggregated cargo at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10°C for various time points in the presence of the disaggregating drug, before being fixed and prepared for immunofluorescence against Gm130 (cis-Golgi) or G245 (trans-Golgi) labeled with a red dye, the green signal emanating from the GFP-tagged cargo. Confocal micrographs show representative Golgi stacks at each time point. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of three independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (C) Immunoblot showing the secretion block at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10 or 20°C for 0 or 40 min in the presence of the disaggregating drug. Media and cell contents were analyzed by immunoblot. (D) Confocal micrographs illustrate the inhibition of intra-Golgi transport of aggregates. As in B, cells were incubated at 10°C for 5 min in the presence of the disaggregating drug to position the cargo within the cis-Golgi. Then, reaggregation was triggered by drug removal (15 min on ice) before shifting the temperature back to 10°C for 20 or 40 min. Cells were then fixed and prepared for immunofluorescence as in B. Confocal micrographs show the transport kinetics of GFP aggregates within Golgi stacks labeled with cis or trans markers. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of two independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (E) Immunoblot shows the inhibition of aggregate secretion. Cells were treated as in C, except that after the 15-min reaggregation at 4°C, the temperature was shifted to 20°C with or without the drug to allow for secretion. Media and cell fractions were analyzed by immunoblot and densitometry. Graphs show the secretion over time for each condition. Data represent the mean ± SD of two independent experiments. The dashed line indicates the percent of secretion at 10°C for disaggregated cargo, as illustrated on the gel in B.
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Figure 5: S2 cells poorly secrete aggregates, which are retained within the cis-Golgi. (A) Confocal micrograph showing separated cis- and trans-Golgi within single stacks through the cytoplasm of S2 cells. S2 cells were fixed and prepared for immunofluorescence against Gm130 (green-labeled cis-Golgi marker) and G245 (red labeled trans-Golgi marker). Graph shows Pearson's coefficient value for each imaged single Golgi stack. Red line represents the average value over 50 analyzed stacks. (B) Transport kinetics of disaggregated cargo at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10°C for various time points in the presence of the disaggregating drug, before being fixed and prepared for immunofluorescence against Gm130 (cis-Golgi) or G245 (trans-Golgi) labeled with a red dye, the green signal emanating from the GFP-tagged cargo. Confocal micrographs show representative Golgi stacks at each time point. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of three independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (C) Immunoblot showing the secretion block at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10 or 20°C for 0 or 40 min in the presence of the disaggregating drug. Media and cell contents were analyzed by immunoblot. (D) Confocal micrographs illustrate the inhibition of intra-Golgi transport of aggregates. As in B, cells were incubated at 10°C for 5 min in the presence of the disaggregating drug to position the cargo within the cis-Golgi. Then, reaggregation was triggered by drug removal (15 min on ice) before shifting the temperature back to 10°C for 20 or 40 min. Cells were then fixed and prepared for immunofluorescence as in B. Confocal micrographs show the transport kinetics of GFP aggregates within Golgi stacks labeled with cis or trans markers. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of two independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (E) Immunoblot shows the inhibition of aggregate secretion. Cells were treated as in C, except that after the 15-min reaggregation at 4°C, the temperature was shifted to 20°C with or without the drug to allow for secretion. Media and cell fractions were analyzed by immunoblot and densitometry. Graphs show the secretion over time for each condition. Data represent the mean ± SD of two independent experiments. The dashed line indicates the percent of secretion at 10°C for disaggregated cargo, as illustrated on the gel in B.

Mentions: Drosophila S2 cells naturally harbor Golgi stacks that are dispersed through the cytoplasm (Kondylis and Rabouille, 2003). As for microtubule-induced ministacks, cis- and trans-Golgi cisternae can be resolved at the light level (Figure 5A and graph). We first generated a plasmid compatible with the expression of the drug-controlled GFP aggregate within S2 cells. S2 cells normally grow at 25°C, and this prevented the direct application of the16°C reaggregation temperature block that we established with HeLa cells, which normally grow at 37°C. We empirically tested several lower temperatures to determine which one would be most appropriate to slow down trafficking of the disaggregated human growth hormone (hGH) cargo, such that it would be positioned within the cis-Golgi before its reaggregation was triggered. We found that, at 10°C, the disaggregated cargo reached the cis-Golgi after 5 min and then progressively reached the trans-Golgi within the next 20 min (Figure 5B and graph). Importantly, after 40 min at 10°C, the vast majority of the disaggregated cargo remained at the trans-Golgi (Figure 5B), and only a very small portion could be detected in the media (<5%, Figure 5C). We concluded that the 10°C incubation slowed down the trafficking within S2 cells and triggered retention of the cargo within the trans-Golgi, thereby mimicking the well-known 20°C temperature block often used in mammalian cells. Now knowing precisely the kinetics of the sequential ER→cis-Golgi→trans-Golgi transport at 10°C, we decided to incubate the S2 cells at 10°C for 5 min in the presence of the disaggregating drug to position the cargo within the cis-Golgi before triggering (or not) its reaggregation for 10 min on ice, which did not alter the microtubule network (Figure S4). The cells were then either incubated for 20–40 min at 10°C before being processed for confocal microscopy or were incubated at 20°C to analyze and compare the rate of secretion of each reaggregated and disaggregated cargo. As judged by confocal microscopy, the reaggregated cargo remained associated longer within the cis-Golgi (Figure 5D and graph; up to 40 min instead of 5–10 min for the disaggregated cargo). A portion of the aggregates reached the trans-Golgi, but again was more than two times slower than the disaggregated cargo. Analysis of the bulk secretion when transport was resumed at 20°C revealed that the secretion of the aggregates was inhibited by a factor of two when compared with the disaggregated cargo (Figure 5E and graph). Note that almost 100% of the disaggregated hGH was released from the S2 cells, whereas only 50% was released from the HeLa cells. We attribute this difference to the transfection efficiency and the protein overexpression level, which is considerably higher in HeLa cells than in S2 cells, resulting in a large portion of the ER aggregates remaining insensitive to the disaggregating drug in HeLa cells. This, however, has no impact on our interpretation of the results.


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)

S2 cells poorly secrete aggregates, which are retained within the cis-Golgi. (A) Confocal micrograph showing separated cis- and trans-Golgi within single stacks through the cytoplasm of S2 cells. S2 cells were fixed and prepared for immunofluorescence against Gm130 (green-labeled cis-Golgi marker) and G245 (red labeled trans-Golgi marker). Graph shows Pearson's coefficient value for each imaged single Golgi stack. Red line represents the average value over 50 analyzed stacks. (B) Transport kinetics of disaggregated cargo at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10°C for various time points in the presence of the disaggregating drug, before being fixed and prepared for immunofluorescence against Gm130 (cis-Golgi) or G245 (trans-Golgi) labeled with a red dye, the green signal emanating from the GFP-tagged cargo. Confocal micrographs show representative Golgi stacks at each time point. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of three independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (C) Immunoblot showing the secretion block at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10 or 20°C for 0 or 40 min in the presence of the disaggregating drug. Media and cell contents were analyzed by immunoblot. (D) Confocal micrographs illustrate the inhibition of intra-Golgi transport of aggregates. As in B, cells were incubated at 10°C for 5 min in the presence of the disaggregating drug to position the cargo within the cis-Golgi. Then, reaggregation was triggered by drug removal (15 min on ice) before shifting the temperature back to 10°C for 20 or 40 min. Cells were then fixed and prepared for immunofluorescence as in B. Confocal micrographs show the transport kinetics of GFP aggregates within Golgi stacks labeled with cis or trans markers. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of two independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (E) Immunoblot shows the inhibition of aggregate secretion. Cells were treated as in C, except that after the 15-min reaggregation at 4°C, the temperature was shifted to 20°C with or without the drug to allow for secretion. Media and cell fractions were analyzed by immunoblot and densitometry. Graphs show the secretion over time for each condition. Data represent the mean ± SD of two independent experiments. The dashed line indicates the percent of secretion at 10°C for disaggregated cargo, as illustrated on the gel in B.
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Figure 5: S2 cells poorly secrete aggregates, which are retained within the cis-Golgi. (A) Confocal micrograph showing separated cis- and trans-Golgi within single stacks through the cytoplasm of S2 cells. S2 cells were fixed and prepared for immunofluorescence against Gm130 (green-labeled cis-Golgi marker) and G245 (red labeled trans-Golgi marker). Graph shows Pearson's coefficient value for each imaged single Golgi stack. Red line represents the average value over 50 analyzed stacks. (B) Transport kinetics of disaggregated cargo at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10°C for various time points in the presence of the disaggregating drug, before being fixed and prepared for immunofluorescence against Gm130 (cis-Golgi) or G245 (trans-Golgi) labeled with a red dye, the green signal emanating from the GFP-tagged cargo. Confocal micrographs show representative Golgi stacks at each time point. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of three independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (C) Immunoblot showing the secretion block at 10°C. The S2 cells expressing GFP-FM4-hGH were incubated at 10 or 20°C for 0 or 40 min in the presence of the disaggregating drug. Media and cell contents were analyzed by immunoblot. (D) Confocal micrographs illustrate the inhibition of intra-Golgi transport of aggregates. As in B, cells were incubated at 10°C for 5 min in the presence of the disaggregating drug to position the cargo within the cis-Golgi. Then, reaggregation was triggered by drug removal (15 min on ice) before shifting the temperature back to 10°C for 20 or 40 min. Cells were then fixed and prepared for immunofluorescence as in B. Confocal micrographs show the transport kinetics of GFP aggregates within Golgi stacks labeled with cis or trans markers. Graph shows the Pearson's coefficient over time for each combination. Data represent the mean ± SD of two independent experiments. For each time point of each experiment, at least 30 stacks were analyzed. *, p values < 0.01. (E) Immunoblot shows the inhibition of aggregate secretion. Cells were treated as in C, except that after the 15-min reaggregation at 4°C, the temperature was shifted to 20°C with or without the drug to allow for secretion. Media and cell fractions were analyzed by immunoblot and densitometry. Graphs show the secretion over time for each condition. Data represent the mean ± SD of two independent experiments. The dashed line indicates the percent of secretion at 10°C for disaggregated cargo, as illustrated on the gel in B.
Mentions: Drosophila S2 cells naturally harbor Golgi stacks that are dispersed through the cytoplasm (Kondylis and Rabouille, 2003). As for microtubule-induced ministacks, cis- and trans-Golgi cisternae can be resolved at the light level (Figure 5A and graph). We first generated a plasmid compatible with the expression of the drug-controlled GFP aggregate within S2 cells. S2 cells normally grow at 25°C, and this prevented the direct application of the16°C reaggregation temperature block that we established with HeLa cells, which normally grow at 37°C. We empirically tested several lower temperatures to determine which one would be most appropriate to slow down trafficking of the disaggregated human growth hormone (hGH) cargo, such that it would be positioned within the cis-Golgi before its reaggregation was triggered. We found that, at 10°C, the disaggregated cargo reached the cis-Golgi after 5 min and then progressively reached the trans-Golgi within the next 20 min (Figure 5B and graph). Importantly, after 40 min at 10°C, the vast majority of the disaggregated cargo remained at the trans-Golgi (Figure 5B), and only a very small portion could be detected in the media (<5%, Figure 5C). We concluded that the 10°C incubation slowed down the trafficking within S2 cells and triggered retention of the cargo within the trans-Golgi, thereby mimicking the well-known 20°C temperature block often used in mammalian cells. Now knowing precisely the kinetics of the sequential ER→cis-Golgi→trans-Golgi transport at 10°C, we decided to incubate the S2 cells at 10°C for 5 min in the presence of the disaggregating drug to position the cargo within the cis-Golgi before triggering (or not) its reaggregation for 10 min on ice, which did not alter the microtubule network (Figure S4). The cells were then either incubated for 20–40 min at 10°C before being processed for confocal microscopy or were incubated at 20°C to analyze and compare the rate of secretion of each reaggregated and disaggregated cargo. As judged by confocal microscopy, the reaggregated cargo remained associated longer within the cis-Golgi (Figure 5D and graph; up to 40 min instead of 5–10 min for the disaggregated cargo). A portion of the aggregates reached the trans-Golgi, but again was more than two times slower than the disaggregated cargo. Analysis of the bulk secretion when transport was resumed at 20°C revealed that the secretion of the aggregates was inhibited by a factor of two when compared with the disaggregated cargo (Figure 5E and graph). Note that almost 100% of the disaggregated hGH was released from the S2 cells, whereas only 50% was released from the HeLa cells. We attribute this difference to the transfection efficiency and the protein overexpression level, which is considerably higher in HeLa cells than in S2 cells, resulting in a large portion of the ER aggregates remaining insensitive to the disaggregating drug in HeLa cells. This, however, has no impact on our interpretation of the results.

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