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AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila.

Burgess J, Jauregui M, Tan J, Rollins J, Lallet S, Leventis PA, Boulianne GL, Chang HC, Le Borgne R, Krämer H, Brill JA - Mol. Biol. Cell (2011)

Bottom Line: Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules.Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation.These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Ontario, Canada.

ABSTRACT
Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

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Glue granule biogenesis is developmentally regulated. (A–C′) Confocal micrographs of whole third-instar larval (L3) salivary glands expressing Sgs3-DsRed (red) and stained for AP-1γ (green), showing developmental timing of Sgs3-DsRed expression from stage 0 (no granules) through stage 1 (initiation of granule production) to stage 2 (fully mature granules or glands). AP-1γ is expressed in all cells of the salivary gland throughout development, whereas Sgs3-DsRed is first detected in distal (d) mid-L3 salivary gland cells (B, B′) and is expressed in more proximal (p) cells as development proceeds (C, C′). (D–F) Confocal micrographs of individual salivary gland cells showing developmental expression of Sgs3-DsRed. Sgs3-DsRed is not expressed in stage 0 (D). In stage 1, granules surround the nucleus (n) and appear uniformly small (E). In stage 2, granules are larger and occupy most of the cytoplasmic space (F). (G–I) Transmission electron micrographs (TEM) of L3 salivary glands staged using the Sgs3-DsRed marker. No granules were detected in stage 0 (G). Glue granule (Gr) maturation observed by TEM (H, I) parallels that seen by Sgs3-DsRed, validating this marker for following glue granule biogenesis (E, F). (J) Granules increase in size over time, from an average length of 1.0 μm ± 0.3 (n = 91) at stage 1 (red bar) to a maximum length of 3.5 μm ± 1.0 (n = 54) at stage 2 (green bar). (K, L) TEM of stage 1 salivary gland cells. Rough ER, transitional ER (tER), Golgi, and TGN (defined morphologically as in the work of Thomopoulos et al., 1992; Kondylis and Rabouille, 2009) are present near small glue granules (Gr) (K). Coated vesicles (CV) were also observed near glue granules (Gr) (L).
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Figure 1: Glue granule biogenesis is developmentally regulated. (A–C′) Confocal micrographs of whole third-instar larval (L3) salivary glands expressing Sgs3-DsRed (red) and stained for AP-1γ (green), showing developmental timing of Sgs3-DsRed expression from stage 0 (no granules) through stage 1 (initiation of granule production) to stage 2 (fully mature granules or glands). AP-1γ is expressed in all cells of the salivary gland throughout development, whereas Sgs3-DsRed is first detected in distal (d) mid-L3 salivary gland cells (B, B′) and is expressed in more proximal (p) cells as development proceeds (C, C′). (D–F) Confocal micrographs of individual salivary gland cells showing developmental expression of Sgs3-DsRed. Sgs3-DsRed is not expressed in stage 0 (D). In stage 1, granules surround the nucleus (n) and appear uniformly small (E). In stage 2, granules are larger and occupy most of the cytoplasmic space (F). (G–I) Transmission electron micrographs (TEM) of L3 salivary glands staged using the Sgs3-DsRed marker. No granules were detected in stage 0 (G). Glue granule (Gr) maturation observed by TEM (H, I) parallels that seen by Sgs3-DsRed, validating this marker for following glue granule biogenesis (E, F). (J) Granules increase in size over time, from an average length of 1.0 μm ± 0.3 (n = 91) at stage 1 (red bar) to a maximum length of 3.5 μm ± 1.0 (n = 54) at stage 2 (green bar). (K, L) TEM of stage 1 salivary gland cells. Rough ER, transitional ER (tER), Golgi, and TGN (defined morphologically as in the work of Thomopoulos et al., 1992; Kondylis and Rabouille, 2009) are present near small glue granules (Gr) (K). Coated vesicles (CV) were also observed near glue granules (Gr) (L).

Mentions: To develop a system in which regulated secretion could be genetically manipulated, we characterized the process of glue granule formation and maturation in third-instar larval salivary glands using a fluorescently tagged glue protein (Sgs3-DsRed) expressed under control of its own promoter (Biyasheva et al., 2001; Costantino et al., 2008). Glue expression was first visible in the distal-most cells of the salivary gland and proceeded proximally over time (Figure 1, A–C′), with the salivary gland increasing in size as glue production progressed. We defined the stages as 0 (no expression), 1 (small granules, expression in distal cells), and 2 (fully mature granules, expression in distal and proximal cells). Unlike glue protein, gamma-adaptin (AP-1γ), a large subunit of the AP-1 complex, was expressed in all cells of the salivary gland throughout third-instar larval development (Figure 1, A′–C′).


AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila.

Burgess J, Jauregui M, Tan J, Rollins J, Lallet S, Leventis PA, Boulianne GL, Chang HC, Le Borgne R, Krämer H, Brill JA - Mol. Biol. Cell (2011)

Glue granule biogenesis is developmentally regulated. (A–C′) Confocal micrographs of whole third-instar larval (L3) salivary glands expressing Sgs3-DsRed (red) and stained for AP-1γ (green), showing developmental timing of Sgs3-DsRed expression from stage 0 (no granules) through stage 1 (initiation of granule production) to stage 2 (fully mature granules or glands). AP-1γ is expressed in all cells of the salivary gland throughout development, whereas Sgs3-DsRed is first detected in distal (d) mid-L3 salivary gland cells (B, B′) and is expressed in more proximal (p) cells as development proceeds (C, C′). (D–F) Confocal micrographs of individual salivary gland cells showing developmental expression of Sgs3-DsRed. Sgs3-DsRed is not expressed in stage 0 (D). In stage 1, granules surround the nucleus (n) and appear uniformly small (E). In stage 2, granules are larger and occupy most of the cytoplasmic space (F). (G–I) Transmission electron micrographs (TEM) of L3 salivary glands staged using the Sgs3-DsRed marker. No granules were detected in stage 0 (G). Glue granule (Gr) maturation observed by TEM (H, I) parallels that seen by Sgs3-DsRed, validating this marker for following glue granule biogenesis (E, F). (J) Granules increase in size over time, from an average length of 1.0 μm ± 0.3 (n = 91) at stage 1 (red bar) to a maximum length of 3.5 μm ± 1.0 (n = 54) at stage 2 (green bar). (K, L) TEM of stage 1 salivary gland cells. Rough ER, transitional ER (tER), Golgi, and TGN (defined morphologically as in the work of Thomopoulos et al., 1992; Kondylis and Rabouille, 2009) are present near small glue granules (Gr) (K). Coated vesicles (CV) were also observed near glue granules (Gr) (L).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113773&req=5

Figure 1: Glue granule biogenesis is developmentally regulated. (A–C′) Confocal micrographs of whole third-instar larval (L3) salivary glands expressing Sgs3-DsRed (red) and stained for AP-1γ (green), showing developmental timing of Sgs3-DsRed expression from stage 0 (no granules) through stage 1 (initiation of granule production) to stage 2 (fully mature granules or glands). AP-1γ is expressed in all cells of the salivary gland throughout development, whereas Sgs3-DsRed is first detected in distal (d) mid-L3 salivary gland cells (B, B′) and is expressed in more proximal (p) cells as development proceeds (C, C′). (D–F) Confocal micrographs of individual salivary gland cells showing developmental expression of Sgs3-DsRed. Sgs3-DsRed is not expressed in stage 0 (D). In stage 1, granules surround the nucleus (n) and appear uniformly small (E). In stage 2, granules are larger and occupy most of the cytoplasmic space (F). (G–I) Transmission electron micrographs (TEM) of L3 salivary glands staged using the Sgs3-DsRed marker. No granules were detected in stage 0 (G). Glue granule (Gr) maturation observed by TEM (H, I) parallels that seen by Sgs3-DsRed, validating this marker for following glue granule biogenesis (E, F). (J) Granules increase in size over time, from an average length of 1.0 μm ± 0.3 (n = 91) at stage 1 (red bar) to a maximum length of 3.5 μm ± 1.0 (n = 54) at stage 2 (green bar). (K, L) TEM of stage 1 salivary gland cells. Rough ER, transitional ER (tER), Golgi, and TGN (defined morphologically as in the work of Thomopoulos et al., 1992; Kondylis and Rabouille, 2009) are present near small glue granules (Gr) (K). Coated vesicles (CV) were also observed near glue granules (Gr) (L).
Mentions: To develop a system in which regulated secretion could be genetically manipulated, we characterized the process of glue granule formation and maturation in third-instar larval salivary glands using a fluorescently tagged glue protein (Sgs3-DsRed) expressed under control of its own promoter (Biyasheva et al., 2001; Costantino et al., 2008). Glue expression was first visible in the distal-most cells of the salivary gland and proceeded proximally over time (Figure 1, A–C′), with the salivary gland increasing in size as glue production progressed. We defined the stages as 0 (no expression), 1 (small granules, expression in distal cells), and 2 (fully mature granules, expression in distal and proximal cells). Unlike glue protein, gamma-adaptin (AP-1γ), a large subunit of the AP-1 complex, was expressed in all cells of the salivary gland throughout third-instar larval development (Figure 1, A′–C′).

Bottom Line: Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules.Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation.These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Ontario, Canada.

ABSTRACT
Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

Show MeSH
Related in: MedlinePlus