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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 protein depletion on Delta anterograde protein transport to the plasma membrane. Delta S2 cells were incubated for 96 h with ds EGFP, ds dp115, ds dGM130, and ds dsed5, or treated with brefeldin A (BFA) (see Materials and methods). (A) Representation of the three categories of plasma membrane labeling intensity, after a 1-h induction of Delta followed by a 90-min chase. One confocal section is presented. (B) Example of exclusive intracellular Delta labeling. (C) Percentage of cells exhibiting the three different intensities of plasma membrane or exclusive intracellular labeling after Delta induction for 1 h followed by 90 min of chase. Results obtained with mock-depleted cells (+ds EGFP), cells depleted of dp115 (+ds dp115), dGM130 (+ds dGM130), or dSed5p (+ds dsed5), or cells treated with brefeldin A are expressed as the percentage of total number of cells examined. The error bars represent the SD. (D) Initial rate of transport in dp115-depleted (empty symbols) and mock-depleted (filled symbols) cells induced for 25 min followed by 45, 60, and 90 min of chase. The intensity of Delta labeling at the plasma membrane was estimated as described in the Materials and methods and plotted against the chase time. The boxed results represent the total intensity obtained after a 1-h induction and 90-min chase. Immunofluorescence picture of a mock-depleted cell (E) with a control dSec23p pattern (green) and Delta at the plasma membrane (red), and of a dp115-depleted cell (F) with a fragmented dSec23p pattern (green) and the same amount of Delta at the plasma membrane (red). One confocal section is presented. Bar, 5 μm.
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fig9: Effect of protein depletion on Delta anterograde protein transport to the plasma membrane. Delta S2 cells were incubated for 96 h with ds EGFP, ds dp115, ds dGM130, and ds dsed5, or treated with brefeldin A (BFA) (see Materials and methods). (A) Representation of the three categories of plasma membrane labeling intensity, after a 1-h induction of Delta followed by a 90-min chase. One confocal section is presented. (B) Example of exclusive intracellular Delta labeling. (C) Percentage of cells exhibiting the three different intensities of plasma membrane or exclusive intracellular labeling after Delta induction for 1 h followed by 90 min of chase. Results obtained with mock-depleted cells (+ds EGFP), cells depleted of dp115 (+ds dp115), dGM130 (+ds dGM130), or dSed5p (+ds dsed5), or cells treated with brefeldin A are expressed as the percentage of total number of cells examined. The error bars represent the SD. (D) Initial rate of transport in dp115-depleted (empty symbols) and mock-depleted (filled symbols) cells induced for 25 min followed by 45, 60, and 90 min of chase. The intensity of Delta labeling at the plasma membrane was estimated as described in the Materials and methods and plotted against the chase time. The boxed results represent the total intensity obtained after a 1-h induction and 90-min chase. Immunofluorescence picture of a mock-depleted cell (E) with a control dSec23p pattern (green) and Delta at the plasma membrane (red), and of a dp115-depleted cell (F) with a fragmented dSec23p pattern (green) and the same amount of Delta at the plasma membrane (red). One confocal section is presented. Bar, 5 μm.

Mentions: We first induced Delta expression for 1 h followed by a 90-min chase as a way of measuring steady-state transport. In mock-treated and mock-depleted cells, a large majority of the staining was found at the plasma membrane (Fig. 9 A). Only 3.3 ± 2.5% of the cells exhibited exclusively intracellular (Fig. 9 B), with no plasma membrane, labeling.


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

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

Effect of protein depletion on Delta anterograde protein transport to the plasma membrane. Delta S2 cells were incubated for 96 h with ds EGFP, ds dp115, ds dGM130, and ds dsed5, or treated with brefeldin A (BFA) (see Materials and methods). (A) Representation of the three categories of plasma membrane labeling intensity, after a 1-h induction of Delta followed by a 90-min chase. One confocal section is presented. (B) Example of exclusive intracellular Delta labeling. (C) Percentage of cells exhibiting the three different intensities of plasma membrane or exclusive intracellular labeling after Delta induction for 1 h followed by 90 min of chase. Results obtained with mock-depleted cells (+ds EGFP), cells depleted of dp115 (+ds dp115), dGM130 (+ds dGM130), or dSed5p (+ds dsed5), or cells treated with brefeldin A are expressed as the percentage of total number of cells examined. The error bars represent the SD. (D) Initial rate of transport in dp115-depleted (empty symbols) and mock-depleted (filled symbols) cells induced for 25 min followed by 45, 60, and 90 min of chase. The intensity of Delta labeling at the plasma membrane was estimated as described in the Materials and methods and plotted against the chase time. The boxed results represent the total intensity obtained after a 1-h induction and 90-min chase. Immunofluorescence picture of a mock-depleted cell (E) with a control dSec23p pattern (green) and Delta at the plasma membrane (red), and of a dp115-depleted cell (F) with a fragmented dSec23p pattern (green) and the same amount of Delta at the plasma membrane (red). One confocal section is presented. Bar, 5 μm.
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fig9: Effect of protein depletion on Delta anterograde protein transport to the plasma membrane. Delta S2 cells were incubated for 96 h with ds EGFP, ds dp115, ds dGM130, and ds dsed5, or treated with brefeldin A (BFA) (see Materials and methods). (A) Representation of the three categories of plasma membrane labeling intensity, after a 1-h induction of Delta followed by a 90-min chase. One confocal section is presented. (B) Example of exclusive intracellular Delta labeling. (C) Percentage of cells exhibiting the three different intensities of plasma membrane or exclusive intracellular labeling after Delta induction for 1 h followed by 90 min of chase. Results obtained with mock-depleted cells (+ds EGFP), cells depleted of dp115 (+ds dp115), dGM130 (+ds dGM130), or dSed5p (+ds dsed5), or cells treated with brefeldin A are expressed as the percentage of total number of cells examined. The error bars represent the SD. (D) Initial rate of transport in dp115-depleted (empty symbols) and mock-depleted (filled symbols) cells induced for 25 min followed by 45, 60, and 90 min of chase. The intensity of Delta labeling at the plasma membrane was estimated as described in the Materials and methods and plotted against the chase time. The boxed results represent the total intensity obtained after a 1-h induction and 90-min chase. Immunofluorescence picture of a mock-depleted cell (E) with a control dSec23p pattern (green) and Delta at the plasma membrane (red), and of a dp115-depleted cell (F) with a fragmented dSec23p pattern (green) and the same amount of Delta at the plasma membrane (red). One confocal section is presented. Bar, 5 μm.
Mentions: We first induced Delta expression for 1 h followed by a 90-min chase as a way of measuring steady-state transport. In mock-treated and mock-depleted cells, a large majority of the staining was found at the plasma membrane (Fig. 9 A). Only 3.3 ± 2.5% of the cells exhibited exclusively intracellular (Fig. 9 B), with no plasma membrane, labeling.

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