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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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Localization of d120kd and dSec23p in S2 cells. Cryosections of Drosophila S2 cells, fixed with PFA/GA (A–C) or PFA alone (D and E), were labeled (A, D, and E) with a polyclonal anti-Sec23p antibody (10-nm gold) or (B) a monoclonal antibody against d120kd (10-nm gold). (C) Sections were double labeled with the d120kd antibody (15-nm gold) and the anti-Sec23p antibody (10-nm gold). (F) Delta S2 cells were induced with CuSO4 for 25 min and processed for immunofluorescence. Delta and dSec23p were labeled using C594.9B (red) and the anti-Sec23p antibody (green), respectively. The merge projections of 30 confocal sections are presented, and the overlap is yellow. The COPII coats labeled for dSec23p are indicated with an arrow. G, Golgi stacks. Bars: (A–E) 200 nm; (F) 5 μm.
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fig5: Localization of d120kd and dSec23p in S2 cells. Cryosections of Drosophila S2 cells, fixed with PFA/GA (A–C) or PFA alone (D and E), were labeled (A, D, and E) with a polyclonal anti-Sec23p antibody (10-nm gold) or (B) a monoclonal antibody against d120kd (10-nm gold). (C) Sections were double labeled with the d120kd antibody (15-nm gold) and the anti-Sec23p antibody (10-nm gold). (F) Delta S2 cells were induced with CuSO4 for 25 min and processed for immunofluorescence. Delta and dSec23p were labeled using C594.9B (red) and the anti-Sec23p antibody (green), respectively. The merge projections of 30 confocal sections are presented, and the overlap is yellow. The COPII coats labeled for dSec23p are indicated with an arrow. G, Golgi stacks. Bars: (A–E) 200 nm; (F) 5 μm.

Mentions: When used in immunoelectron microscopy (IEM), the dp115/584 antibody gave a specific signal (Fig. 1, C–F). In S2 cells, 30 ± 5% of the gold particles were on the cytoplasm. The Golgi area was labeled by 25 ± 8% of the membrane-associated gold particles. The ER cisternae were decorated by 21 ± 6%. Pleiomorphic membranes (Fig. 1, C and D, asterisk) comprising tubules and vesicles (50–70 nm in diameter, but also larger vessels) close to the Golgi stacks or in their neighborhood contained 54 ± 7% of the membrane-associated labeling. A double labeling with an antibody recognizing the Drosophila homologue of Sec23p (dSec23p; Fig. 1, E and F) suggested that these pleiomorphic membranes represented tERs (see Fig. 5). The linear density of gold particles over the membrane of these three compartments was estimated to be 0.57 gold/μm on the Golgi membrane, 0.37 on tER membrane, and 0.15 on ER cisternae. A similar pattern of distribution was observed when salivary glands of third instar larvae were labeled with the same antibody (Fig. 1 D).


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

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

Localization of d120kd and dSec23p in S2 cells. Cryosections of Drosophila S2 cells, fixed with PFA/GA (A–C) or PFA alone (D and E), were labeled (A, D, and E) with a polyclonal anti-Sec23p antibody (10-nm gold) or (B) a monoclonal antibody against d120kd (10-nm gold). (C) Sections were double labeled with the d120kd antibody (15-nm gold) and the anti-Sec23p antibody (10-nm gold). (F) Delta S2 cells were induced with CuSO4 for 25 min and processed for immunofluorescence. Delta and dSec23p were labeled using C594.9B (red) and the anti-Sec23p antibody (green), respectively. The merge projections of 30 confocal sections are presented, and the overlap is yellow. The COPII coats labeled for dSec23p are indicated with an arrow. G, Golgi stacks. Bars: (A–E) 200 nm; (F) 5 μm.
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fig5: Localization of d120kd and dSec23p in S2 cells. Cryosections of Drosophila S2 cells, fixed with PFA/GA (A–C) or PFA alone (D and E), were labeled (A, D, and E) with a polyclonal anti-Sec23p antibody (10-nm gold) or (B) a monoclonal antibody against d120kd (10-nm gold). (C) Sections were double labeled with the d120kd antibody (15-nm gold) and the anti-Sec23p antibody (10-nm gold). (F) Delta S2 cells were induced with CuSO4 for 25 min and processed for immunofluorescence. Delta and dSec23p were labeled using C594.9B (red) and the anti-Sec23p antibody (green), respectively. The merge projections of 30 confocal sections are presented, and the overlap is yellow. The COPII coats labeled for dSec23p are indicated with an arrow. G, Golgi stacks. Bars: (A–E) 200 nm; (F) 5 μm.
Mentions: When used in immunoelectron microscopy (IEM), the dp115/584 antibody gave a specific signal (Fig. 1, C–F). In S2 cells, 30 ± 5% of the gold particles were on the cytoplasm. The Golgi area was labeled by 25 ± 8% of the membrane-associated gold particles. The ER cisternae were decorated by 21 ± 6%. Pleiomorphic membranes (Fig. 1, C and D, asterisk) comprising tubules and vesicles (50–70 nm in diameter, but also larger vessels) close to the Golgi stacks or in their neighborhood contained 54 ± 7% of the membrane-associated labeling. A double labeling with an antibody recognizing the Drosophila homologue of Sec23p (dSec23p; Fig. 1, E and F) suggested that these pleiomorphic membranes represented tERs (see Fig. 5). The linear density of gold particles over the membrane of these three compartments was estimated to be 0.57 gold/μm on the Golgi membrane, 0.37 on tER membrane, and 0.15 on ER cisternae. A similar pattern of distribution was observed when salivary glands of third instar larvae were labeled with the same antibody (Fig. 1 D).

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