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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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Effect of dSed5p depletion on the Golgi stack morphology and tER organization. (A) S2 cells were incubated with ds dsed5 up to 96 h. Extracts of mock- and dSed5p-depleted S2 cells were Western blotted using JSEE1 antibody. The 35-kD band corresponding to dSed5p is efficiently depleted (arrow). (B) The Golgi stack morphology in depleted cells was assessed by conventional EM. The cells exhibited no Golgi stacks, but extended areas completely vesiculated in >95% of the profiles examined. (C) The tER organization was monitored by immunofluorescence using the anti-Sec23p antibody, as described in the legend of Fig. 6, in control cells, (D) in cells depleted of dSed5p, and (E) in cells depleted of dGM130. N, nucleus. Bars: (B) 200 nm; (C–E) 5 μm.
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fig8: Effect of dSed5p depletion on the Golgi stack morphology and tER organization. (A) S2 cells were incubated with ds dsed5 up to 96 h. Extracts of mock- and dSed5p-depleted S2 cells were Western blotted using JSEE1 antibody. The 35-kD band corresponding to dSed5p is efficiently depleted (arrow). (B) The Golgi stack morphology in depleted cells was assessed by conventional EM. The cells exhibited no Golgi stacks, but extended areas completely vesiculated in >95% of the profiles examined. (C) The tER organization was monitored by immunofluorescence using the anti-Sec23p antibody, as described in the legend of Fig. 6, in control cells, (D) in cells depleted of dSed5p, and (E) in cells depleted of dGM130. N, nucleus. Bars: (B) 200 nm; (C–E) 5 μm.

Mentions: These effects were specific for dp115 depletion. dGM130 depletion did not have any effect either on the structure of the Golgi complex (Fig. 3 B) or on tER organization (Fig. 8 E). On the other hand, depletion of dSed5p, the Drosophila homologue of mammalian syntaxin 5 (Banfield et al., 1994), had a very strong effect on the Golgi stack morphology (complete and quantitative vesiculation; Fig. 8, A and B), but the organization of the tERs was kept intact (Fig. 8 D) when compared with mock-depleted cells (Fig. 8 C). The fragmentation of the Golgi stacks therefore did not cause the redistribution of the tER sites.


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

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

Effect of dSed5p depletion on the Golgi stack morphology and tER organization. (A) S2 cells were incubated with ds dsed5 up to 96 h. Extracts of mock- and dSed5p-depleted S2 cells were Western blotted using JSEE1 antibody. The 35-kD band corresponding to dSed5p is efficiently depleted (arrow). (B) The Golgi stack morphology in depleted cells was assessed by conventional EM. The cells exhibited no Golgi stacks, but extended areas completely vesiculated in >95% of the profiles examined. (C) The tER organization was monitored by immunofluorescence using the anti-Sec23p antibody, as described in the legend of Fig. 6, in control cells, (D) in cells depleted of dSed5p, and (E) in cells depleted of dGM130. N, nucleus. Bars: (B) 200 nm; (C–E) 5 μm.
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Related In: Results  -  Collection

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fig8: Effect of dSed5p depletion on the Golgi stack morphology and tER organization. (A) S2 cells were incubated with ds dsed5 up to 96 h. Extracts of mock- and dSed5p-depleted S2 cells were Western blotted using JSEE1 antibody. The 35-kD band corresponding to dSed5p is efficiently depleted (arrow). (B) The Golgi stack morphology in depleted cells was assessed by conventional EM. The cells exhibited no Golgi stacks, but extended areas completely vesiculated in >95% of the profiles examined. (C) The tER organization was monitored by immunofluorescence using the anti-Sec23p antibody, as described in the legend of Fig. 6, in control cells, (D) in cells depleted of dSed5p, and (E) in cells depleted of dGM130. N, nucleus. Bars: (B) 200 nm; (C–E) 5 μm.
Mentions: These effects were specific for dp115 depletion. dGM130 depletion did not have any effect either on the structure of the Golgi complex (Fig. 3 B) or on tER organization (Fig. 8 E). On the other hand, depletion of dSed5p, the Drosophila homologue of mammalian syntaxin 5 (Banfield et al., 1994), had a very strong effect on the Golgi stack morphology (complete and quantitative vesiculation; Fig. 8, A and B), but the organization of the tERs was kept intact (Fig. 8 D) when compared with mock-depleted cells (Fig. 8 C). The fragmentation of the Golgi stacks therefore did not cause the redistribution of the tER sites.

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