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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 dSec23p and d120kd in dp115-depleted cells. S2 cells depleted of dp115 were processed for IEM and double labeled for dSec23p (10-nm gold) and d120kd (15-nm gold), as described in the legend of Fig. 5. (A–C) dSec23p-positive clusters. (C) d120kd-positive clusters. (D–F) Mixed clusters. A small arrow in B points to an ER bud labeled for dSec23p. Large arrows in D–F point to profiles reminiscent of Golgi cisternal remnants. Arrowhead in E points to a gold particle corresponding to d120kd associated with an ER cisterna. Note that in D–F, the labeling for dSec23p and d120kd marks differential regions of the same cluster. N, nucleus. Bars, 200 nm.
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fig7: Localization of dSec23p and d120kd in dp115-depleted cells. S2 cells depleted of dp115 were processed for IEM and double labeled for dSec23p (10-nm gold) and d120kd (15-nm gold), as described in the legend of Fig. 5. (A–C) dSec23p-positive clusters. (C) d120kd-positive clusters. (D–F) Mixed clusters. A small arrow in B points to an ER bud labeled for dSec23p. Large arrows in D–F point to profiles reminiscent of Golgi cisternal remnants. Arrowhead in E points to a gold particle corresponding to d120kd associated with an ER cisterna. Note that in D–F, the labeling for dSec23p and d120kd marks differential regions of the same cluster. N, nucleus. Bars, 200 nm.

Mentions: In dp115-depleted cells observed by IEM, the dSec23p-positive small and scattered dots observed in immunofluorescence represented pleiomorphic membranes containing vesicles and tubules, reminiscent of those observed in control cells, but with a smaller size (compare Fig. 5 A with Fig. 7, A and B; see Fig. S2, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200301136/DC1). They appeared more dispersed throughout the cytoplasm than in control cells and sometimes exhibited a reduced labeling density (Fig. 7, B and E). The number of these dSec23p-positive sites per cell section was 6.7 ± 2.3, and only 20% of them were positive for d120kd. This is to be compared with 2.7 ± 1.5 dSec23p-positive sites per section of mock-depleted cells, 90% of them also positive for d120kd (Fig. 5 C), suggesting that small tER sites have been generated that lack the spatial relationship with Golgi areas.


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

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

Localization of dSec23p and d120kd in dp115-depleted cells. S2 cells depleted of dp115 were processed for IEM and double labeled for dSec23p (10-nm gold) and d120kd (15-nm gold), as described in the legend of Fig. 5. (A–C) dSec23p-positive clusters. (C) d120kd-positive clusters. (D–F) Mixed clusters. A small arrow in B points to an ER bud labeled for dSec23p. Large arrows in D–F point to profiles reminiscent of Golgi cisternal remnants. Arrowhead in E points to a gold particle corresponding to d120kd associated with an ER cisterna. Note that in D–F, the labeling for dSec23p and d120kd marks differential regions of the same cluster. N, nucleus. Bars, 200 nm.
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Related In: Results  -  Collection

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fig7: Localization of dSec23p and d120kd in dp115-depleted cells. S2 cells depleted of dp115 were processed for IEM and double labeled for dSec23p (10-nm gold) and d120kd (15-nm gold), as described in the legend of Fig. 5. (A–C) dSec23p-positive clusters. (C) d120kd-positive clusters. (D–F) Mixed clusters. A small arrow in B points to an ER bud labeled for dSec23p. Large arrows in D–F point to profiles reminiscent of Golgi cisternal remnants. Arrowhead in E points to a gold particle corresponding to d120kd associated with an ER cisterna. Note that in D–F, the labeling for dSec23p and d120kd marks differential regions of the same cluster. N, nucleus. Bars, 200 nm.
Mentions: In dp115-depleted cells observed by IEM, the dSec23p-positive small and scattered dots observed in immunofluorescence represented pleiomorphic membranes containing vesicles and tubules, reminiscent of those observed in control cells, but with a smaller size (compare Fig. 5 A with Fig. 7, A and B; see Fig. S2, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200301136/DC1). They appeared more dispersed throughout the cytoplasm than in control cells and sometimes exhibited a reduced labeling density (Fig. 7, B and E). The number of these dSec23p-positive sites per cell section was 6.7 ± 2.3, and only 20% of them were positive for d120kd. This is to be compared with 2.7 ± 1.5 dSec23p-positive sites per section of mock-depleted cells, 90% of them also positive for d120kd (Fig. 5 C), suggesting that small tER sites have been generated that lack the spatial relationship with Golgi areas.

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