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A novel role for dp115 in the organization of tER sites in Drosophila.

Kondylis V, Rabouille C - J. Cell Biol. (2003)

Bottom Line: However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays.The effects were specific for dp115.Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, UK.

ABSTRACT
Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.

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Quantitative analysis of the morphological effects after protein depletion. (A) Percentage of Golgi stacks in profiles of S2 cells depleted of dp115 and dGM130. S2 cells were incubated for 24 to 120 h with the different dsRNAs, processed for EM, and scored for the presence of at least one Golgi stack per cell profile. The results obtained are presented as a percentage of the total number of cells examined for each condition (∼300). (B) Stereological analysis of the Golgi area after protein depletion. Representative EM pictures of mock-treated cells and cells depleted of dGM130 and dp115 at 72 and 96 h were used to estimate the percentage of Golgi membrane in total cisternae (black bars) and stacked cisternae (white bars). The error bars represent the SD. (C) Estimation of the surface density of the Golgi area (SDgo) and the total cisternae (stacked and single) (SDcis) in mock- and dp115-depleted cells for 96 h. Results are expressed in μm−1 and ± represents SD.
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fig4: Quantitative analysis of the morphological effects after protein depletion. (A) Percentage of Golgi stacks in profiles of S2 cells depleted of dp115 and dGM130. S2 cells were incubated for 24 to 120 h with the different dsRNAs, processed for EM, and scored for the presence of at least one Golgi stack per cell profile. The results obtained are presented as a percentage of the total number of cells examined for each condition (∼300). (B) Stereological analysis of the Golgi area after protein depletion. Representative EM pictures of mock-treated cells and cells depleted of dGM130 and dp115 at 72 and 96 h were used to estimate the percentage of Golgi membrane in total cisternae (black bars) and stacked cisternae (white bars). The error bars represent the SD. (C) Estimation of the surface density of the Golgi area (SDgo) and the total cisternae (stacked and single) (SDcis) in mock- and dp115-depleted cells for 96 h. Results are expressed in μm−1 and ± represents SD.

Mentions: In cells depleted of dGM130, the percentage of cells exhibiting Golgi stacks (Fig. 4 A), the number of cisternae per stack, and the mean diameter of the stacks were comparable to the figures obtained for mock-treated cells (diameter of 0.347 ± 0.078 μm and 3.5 ± 1.6 cisternae per stack).


A novel role for dp115 in the organization of tER sites in Drosophila.

Kondylis V, Rabouille C - J. Cell Biol. (2003)

Quantitative analysis of the morphological effects after protein depletion. (A) Percentage of Golgi stacks in profiles of S2 cells depleted of dp115 and dGM130. S2 cells were incubated for 24 to 120 h with the different dsRNAs, processed for EM, and scored for the presence of at least one Golgi stack per cell profile. The results obtained are presented as a percentage of the total number of cells examined for each condition (∼300). (B) Stereological analysis of the Golgi area after protein depletion. Representative EM pictures of mock-treated cells and cells depleted of dGM130 and dp115 at 72 and 96 h were used to estimate the percentage of Golgi membrane in total cisternae (black bars) and stacked cisternae (white bars). The error bars represent the SD. (C) Estimation of the surface density of the Golgi area (SDgo) and the total cisternae (stacked and single) (SDcis) in mock- and dp115-depleted cells for 96 h. Results are expressed in μm−1 and ± represents SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172793&req=5

fig4: Quantitative analysis of the morphological effects after protein depletion. (A) Percentage of Golgi stacks in profiles of S2 cells depleted of dp115 and dGM130. S2 cells were incubated for 24 to 120 h with the different dsRNAs, processed for EM, and scored for the presence of at least one Golgi stack per cell profile. The results obtained are presented as a percentage of the total number of cells examined for each condition (∼300). (B) Stereological analysis of the Golgi area after protein depletion. Representative EM pictures of mock-treated cells and cells depleted of dGM130 and dp115 at 72 and 96 h were used to estimate the percentage of Golgi membrane in total cisternae (black bars) and stacked cisternae (white bars). The error bars represent the SD. (C) Estimation of the surface density of the Golgi area (SDgo) and the total cisternae (stacked and single) (SDcis) in mock- and dp115-depleted cells for 96 h. Results are expressed in μm−1 and ± represents SD.
Mentions: In cells depleted of dGM130, the percentage of cells exhibiting Golgi stacks (Fig. 4 A), the number of cisternae per stack, and the mean diameter of the stacks were comparable to the figures obtained for mock-treated cells (diameter of 0.347 ± 0.078 μm and 3.5 ± 1.6 cisternae per stack).

Bottom Line: However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays.The effects were specific for dp115.Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, UK.

ABSTRACT
Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.

Show MeSH
Related in: MedlinePlus