Limits...
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.

Show MeSH

Related in: MedlinePlus

grasp55/65 silencing scatters the Golgi ribbon and inhibits the secretion of aggregates and collagen I. (A) Confocal micrographs illustrating the Golgi scattering mediated by grasp 55/65 silencing. HeLa cells were treated with either scrambled or grasp55/65 siRNA for 3 d. Cells were fixed and prepared for immunofluorescence against COPI (red channel) and Grasp55 (green channel). Using ImageJ, we delineated the Golgi shape within the red channel (mask). For each Golgi, we measured the Golgi area (within the red channel) to evaluate the Golgi scattering and the G55 intensity (within the green channel) to evaluate the efficiency of silencing. Red boxes show higher magnification of indicated Golgi. Graphs show Golgi scattering in red and G55 intensity in green for each condition. Values represent the mean ± SD of two independent experiments. For each condition, we analyzed at least 50 ministacks. (B) Immunoblots showing the inhibition of hGH secretion in grasp55/65-silenced cells. Cells expressing ssGFP-FM4-GFP were treated with grasp55/65 or scrambled siRNA for 3 d. Secretion of disaggregated and reaggregated cargo was then measured as described in Figure 1A (minus the nocodazole treatment) after a 2-h chase at 37°C. siRNA silencing efficiency was confirmed by immunoblot analysis against G55 and G65 within the cell fraction. Graph represents the percent of secretion for each condition. Data represent the mean ± SD of three independent experiments. (C) Immunoblots showing the inhibition of collagen I secretion in grasp55/65 silenced cells. Saos2 cells were treated with siRNA against G55/G65 for 3 d. siRNA silencing efficiency within Saos-2 cells was confirmed by immunoblot analysis (upper panel). MMP-2 and collagen I secretion were then assessed as described in Figure 3A (minus the nocodazole treatment). Graphs represent the normalized rate of secretion over time, for each condition. MMP-2 and collagen I secretion at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230591&req=5

Figure 4: grasp55/65 silencing scatters the Golgi ribbon and inhibits the secretion of aggregates and collagen I. (A) Confocal micrographs illustrating the Golgi scattering mediated by grasp 55/65 silencing. HeLa cells were treated with either scrambled or grasp55/65 siRNA for 3 d. Cells were fixed and prepared for immunofluorescence against COPI (red channel) and Grasp55 (green channel). Using ImageJ, we delineated the Golgi shape within the red channel (mask). For each Golgi, we measured the Golgi area (within the red channel) to evaluate the Golgi scattering and the G55 intensity (within the green channel) to evaluate the efficiency of silencing. Red boxes show higher magnification of indicated Golgi. Graphs show Golgi scattering in red and G55 intensity in green for each condition. Values represent the mean ± SD of two independent experiments. For each condition, we analyzed at least 50 ministacks. (B) Immunoblots showing the inhibition of hGH secretion in grasp55/65-silenced cells. Cells expressing ssGFP-FM4-GFP were treated with grasp55/65 or scrambled siRNA for 3 d. Secretion of disaggregated and reaggregated cargo was then measured as described in Figure 1A (minus the nocodazole treatment) after a 2-h chase at 37°C. siRNA silencing efficiency was confirmed by immunoblot analysis against G55 and G65 within the cell fraction. Graph represents the percent of secretion for each condition. Data represent the mean ± SD of three independent experiments. (C) Immunoblots showing the inhibition of collagen I secretion in grasp55/65 silenced cells. Saos2 cells were treated with siRNA against G55/G65 for 3 d. siRNA silencing efficiency within Saos-2 cells was confirmed by immunoblot analysis (upper panel). MMP-2 and collagen I secretion were then assessed as described in Figure 3A (minus the nocodazole treatment). Graphs represent the normalized rate of secretion over time, for each condition. MMP-2 and collagen I secretion at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments.

Mentions: One interpretation of the results reported above would be to consider that the phenotype is mainly due to the alteration of the microtubule network and is only an indirect consequence of the ribbon breakdown. To test this hypothesis, we decided to knock down grasp55/65, which is known to disrupt the ribbon structure of the Golgi without affecting the microtubule organization (Puthenveedu et al., 2006; Feinstein and Linstedt, 2008; Lee et al., 2014). As judged by immunoblotting and immunofluorescence analysis (Figure 4, A–C), at least 75% of the grasp55/65 proteins were efficiently silenced within cells treated with specific siRNA. As previously reported (Feinstein and Linstedt, 2008), we confirmed that these cells harbored a mildly scattered Golgi (Figure 4A and graph), without showing any alteration of the microtubules (Figure S3).


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)

grasp55/65 silencing scatters the Golgi ribbon and inhibits the secretion of aggregates and collagen I. (A) Confocal micrographs illustrating the Golgi scattering mediated by grasp 55/65 silencing. HeLa cells were treated with either scrambled or grasp55/65 siRNA for 3 d. Cells were fixed and prepared for immunofluorescence against COPI (red channel) and Grasp55 (green channel). Using ImageJ, we delineated the Golgi shape within the red channel (mask). For each Golgi, we measured the Golgi area (within the red channel) to evaluate the Golgi scattering and the G55 intensity (within the green channel) to evaluate the efficiency of silencing. Red boxes show higher magnification of indicated Golgi. Graphs show Golgi scattering in red and G55 intensity in green for each condition. Values represent the mean ± SD of two independent experiments. For each condition, we analyzed at least 50 ministacks. (B) Immunoblots showing the inhibition of hGH secretion in grasp55/65-silenced cells. Cells expressing ssGFP-FM4-GFP were treated with grasp55/65 or scrambled siRNA for 3 d. Secretion of disaggregated and reaggregated cargo was then measured as described in Figure 1A (minus the nocodazole treatment) after a 2-h chase at 37°C. siRNA silencing efficiency was confirmed by immunoblot analysis against G55 and G65 within the cell fraction. Graph represents the percent of secretion for each condition. Data represent the mean ± SD of three independent experiments. (C) Immunoblots showing the inhibition of collagen I secretion in grasp55/65 silenced cells. Saos2 cells were treated with siRNA against G55/G65 for 3 d. siRNA silencing efficiency within Saos-2 cells was confirmed by immunoblot analysis (upper panel). MMP-2 and collagen I secretion were then assessed as described in Figure 3A (minus the nocodazole treatment). Graphs represent the normalized rate of secretion over time, for each condition. MMP-2 and collagen I secretion at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230591&req=5

Figure 4: grasp55/65 silencing scatters the Golgi ribbon and inhibits the secretion of aggregates and collagen I. (A) Confocal micrographs illustrating the Golgi scattering mediated by grasp 55/65 silencing. HeLa cells were treated with either scrambled or grasp55/65 siRNA for 3 d. Cells were fixed and prepared for immunofluorescence against COPI (red channel) and Grasp55 (green channel). Using ImageJ, we delineated the Golgi shape within the red channel (mask). For each Golgi, we measured the Golgi area (within the red channel) to evaluate the Golgi scattering and the G55 intensity (within the green channel) to evaluate the efficiency of silencing. Red boxes show higher magnification of indicated Golgi. Graphs show Golgi scattering in red and G55 intensity in green for each condition. Values represent the mean ± SD of two independent experiments. For each condition, we analyzed at least 50 ministacks. (B) Immunoblots showing the inhibition of hGH secretion in grasp55/65-silenced cells. Cells expressing ssGFP-FM4-GFP were treated with grasp55/65 or scrambled siRNA for 3 d. Secretion of disaggregated and reaggregated cargo was then measured as described in Figure 1A (minus the nocodazole treatment) after a 2-h chase at 37°C. siRNA silencing efficiency was confirmed by immunoblot analysis against G55 and G65 within the cell fraction. Graph represents the percent of secretion for each condition. Data represent the mean ± SD of three independent experiments. (C) Immunoblots showing the inhibition of collagen I secretion in grasp55/65 silenced cells. Saos2 cells were treated with siRNA against G55/G65 for 3 d. siRNA silencing efficiency within Saos-2 cells was confirmed by immunoblot analysis (upper panel). MMP-2 and collagen I secretion were then assessed as described in Figure 3A (minus the nocodazole treatment). Graphs represent the normalized rate of secretion over time, for each condition. MMP-2 and collagen I secretion at 2 h were set to 100%. Data represent the mean ± SD of three independent experiments.
Mentions: One interpretation of the results reported above would be to consider that the phenotype is mainly due to the alteration of the microtubule network and is only an indirect consequence of the ribbon breakdown. To test this hypothesis, we decided to knock down grasp55/65, which is known to disrupt the ribbon structure of the Golgi without affecting the microtubule organization (Puthenveedu et al., 2006; Feinstein and Linstedt, 2008; Lee et al., 2014). As judged by immunoblotting and immunofluorescence analysis (Figure 4, A–C), at least 75% of the grasp55/65 proteins were efficiently silenced within cells treated with specific siRNA. As previously reported (Feinstein and Linstedt, 2008), we confirmed that these cells harbored a mildly scattered Golgi (Figure 4A and graph), without showing any alteration of the microtubules (Figure S3).

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