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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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MMP-2 flows through nocodazole-induced stacks, whereas collagen I is retained within the cis-Golgi. (A) Immunoblots show the inhibition of collagen I secretion under nocodazole treatment. Saos-2 cells were incubated overnight in ascorbate-depleted media. During the last 2 h, cells were treated or not with nocodazole, which was maintained during the experiment. Chase was performed at 37°C in media containing ascorbate and CHX. TCA-precipitated media was analyzed by SDS–PAGE/immunoblotting, followed by densitometry measurement. (B) Graph represents the normalized percent of secretion over time for each condition. MMP-2 and collagen I secretions at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments. (C) Two-color STED micrographs show that collagen I is retained at the cis face of Golgi ministacks. Saos2 cells were treated as described above, except that the chase was performed at 20°C for 1 h. Cells were fixed and prepared for immunofluorescence against collagen I and the cis-Golgi marker gpp130 or the trans-Golgi marker p230. Primary antibodies were detected with secondary antibodies coupled with STED-compatible dyes. Graph represents the Pearson's coefficient for each marker combination for each condition. Values represent the mean ± SD. For each condition, we analyzed between 67 and 83 ministacks. *, p values < 0.01.
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Figure 3: MMP-2 flows through nocodazole-induced stacks, whereas collagen I is retained within the cis-Golgi. (A) Immunoblots show the inhibition of collagen I secretion under nocodazole treatment. Saos-2 cells were incubated overnight in ascorbate-depleted media. During the last 2 h, cells were treated or not with nocodazole, which was maintained during the experiment. Chase was performed at 37°C in media containing ascorbate and CHX. TCA-precipitated media was analyzed by SDS–PAGE/immunoblotting, followed by densitometry measurement. (B) Graph represents the normalized percent of secretion over time for each condition. MMP-2 and collagen I secretions at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments. (C) Two-color STED micrographs show that collagen I is retained at the cis face of Golgi ministacks. Saos2 cells were treated as described above, except that the chase was performed at 20°C for 1 h. Cells were fixed and prepared for immunofluorescence against collagen I and the cis-Golgi marker gpp130 or the trans-Golgi marker p230. Primary antibodies were detected with secondary antibodies coupled with STED-compatible dyes. Graph represents the Pearson's coefficient for each marker combination for each condition. Values represent the mean ± SD. For each condition, we analyzed between 67 and 83 ministacks. *, p values < 0.01.

Mentions: The cargo used above was artificially aggregated and may not perfectly reflect the behavior of physiologically large cargoes. Collagen I, an abundant component of the extracellular matrix, is an example of a physiological large cargo (Ricard-Blum, 2011). We repeated our experiments using Saos-2 cells that secrete endogenous collagen I (Saito et al., 2009; Lavieu et al., 2013). We took advantage of the ascorbate-dependent folding of collagen to control the wave of collagen secretion (Harwood et al., 1976; Mironov et al., 2001). Cells were cultured overnight in ascorbate-depleted media to block collagen within the ER and then, during the last 2 h, cells were pretreated with nocodazole before ascorbate was added back to allow for collagen folding and subsequent exit from the ER. We then assessed the secretion of endogenous collagen I (large cargo) and endogenous MMP2 (small cargo) simultaneously within the same samples. This allows for the direct comparison of the secretion rates of the two endogenous cargoes using a bulk biochemical secretion assay that leaves no room for biased interpretation. The rate of secretion of collagen I was strongly diminished (70% reduction) in the presence of nocodazole, whereas the secretion of MMP-2 was not altered by more than 20% (Figure 3, A and B).


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)

MMP-2 flows through nocodazole-induced stacks, whereas collagen I is retained within the cis-Golgi. (A) Immunoblots show the inhibition of collagen I secretion under nocodazole treatment. Saos-2 cells were incubated overnight in ascorbate-depleted media. During the last 2 h, cells were treated or not with nocodazole, which was maintained during the experiment. Chase was performed at 37°C in media containing ascorbate and CHX. TCA-precipitated media was analyzed by SDS–PAGE/immunoblotting, followed by densitometry measurement. (B) Graph represents the normalized percent of secretion over time for each condition. MMP-2 and collagen I secretions at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments. (C) Two-color STED micrographs show that collagen I is retained at the cis face of Golgi ministacks. Saos2 cells were treated as described above, except that the chase was performed at 20°C for 1 h. Cells were fixed and prepared for immunofluorescence against collagen I and the cis-Golgi marker gpp130 or the trans-Golgi marker p230. Primary antibodies were detected with secondary antibodies coupled with STED-compatible dyes. Graph represents the Pearson's coefficient for each marker combination for each condition. Values represent the mean ± SD. For each condition, we analyzed between 67 and 83 ministacks. *, p values < 0.01.
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Figure 3: MMP-2 flows through nocodazole-induced stacks, whereas collagen I is retained within the cis-Golgi. (A) Immunoblots show the inhibition of collagen I secretion under nocodazole treatment. Saos-2 cells were incubated overnight in ascorbate-depleted media. During the last 2 h, cells were treated or not with nocodazole, which was maintained during the experiment. Chase was performed at 37°C in media containing ascorbate and CHX. TCA-precipitated media was analyzed by SDS–PAGE/immunoblotting, followed by densitometry measurement. (B) Graph represents the normalized percent of secretion over time for each condition. MMP-2 and collagen I secretions at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments. (C) Two-color STED micrographs show that collagen I is retained at the cis face of Golgi ministacks. Saos2 cells were treated as described above, except that the chase was performed at 20°C for 1 h. Cells were fixed and prepared for immunofluorescence against collagen I and the cis-Golgi marker gpp130 or the trans-Golgi marker p230. Primary antibodies were detected with secondary antibodies coupled with STED-compatible dyes. Graph represents the Pearson's coefficient for each marker combination for each condition. Values represent the mean ± SD. For each condition, we analyzed between 67 and 83 ministacks. *, p values < 0.01.
Mentions: The cargo used above was artificially aggregated and may not perfectly reflect the behavior of physiologically large cargoes. Collagen I, an abundant component of the extracellular matrix, is an example of a physiological large cargo (Ricard-Blum, 2011). We repeated our experiments using Saos-2 cells that secrete endogenous collagen I (Saito et al., 2009; Lavieu et al., 2013). We took advantage of the ascorbate-dependent folding of collagen to control the wave of collagen secretion (Harwood et al., 1976; Mironov et al., 2001). Cells were cultured overnight in ascorbate-depleted media to block collagen within the ER and then, during the last 2 h, cells were pretreated with nocodazole before ascorbate was added back to allow for collagen folding and subsequent exit from the ER. We then assessed the secretion of endogenous collagen I (large cargo) and endogenous MMP2 (small cargo) simultaneously within the same samples. This allows for the direct comparison of the secretion rates of the two endogenous cargoes using a bulk biochemical secretion assay that leaves no room for biased interpretation. The rate of secretion of collagen I was strongly diminished (70% reduction) in the presence of nocodazole, whereas the secretion of MMP-2 was not altered by more than 20% (Figure 3, A and B).

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