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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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AP-1 is essential for glue granule biogenesis. Confocal fluorescence micrographs of late–third-instar (stage 2) larval salivary glands. (A–B’’) AP-1μ (AP-47SHE-11) mutant clones (cells marked by the absence of GFP (green) and outlined in yellow) exhibit a complete block in production of Sgs3-DsRed–containing glue granules (red) (A’ and A’’) or strikingly small glue granules (B’ and B’’). Note that cells with two copies of wild-type AP-47 (marked by two copies of GFP) have larger granules than heterozygous cells (marked by one copy of GFP). (C–I) Confocal fluorescence micrographs of stage 2 larval salivary gland cells expressing Sgs3-DsRed. (C) Control salivary gland cells expressing the AB1-GAL4 driver alone have granules of normal size (C, boxed region; shown at 2× higher magnification in inset). (D) Salivary gland cells expressing both the AB1-GAL4 and a UAS-AP-1γ RNAi transgene completely lack glue granules (outlined cell) or have strikingly small glue granules (D, boxed region; shown at 2× higher magnification in inset). See also Supplemental Figure S2. (E–I) Spinning-disk confocal micrographs of salivary gland cells expressing Sgs3-DsRed. (E) Control wild-type cells showing normal-sized glue granules. (F) Larvae bearing the heteroallelic genotype AP-47SHE-11/EP1112 exhibit intermediate-sized granules. (G) Depletion of clathrin heavy chain by RNAi in cells expressing AB1-GAL4 and a UAS-Chc RNAi transgene causes a complete block in glue production in most cells, whereas a minority of cells produced small amounts of glue. (H and I) Strong loss-of-function mutations in AP-3μ (carmine1 [cm1]) (H) or AP-3δ (garnet50e [g50e]) (I) have no effect on glue granule biogenesis.
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Figure 5: AP-1 is essential for glue granule biogenesis. Confocal fluorescence micrographs of late–third-instar (stage 2) larval salivary glands. (A–B’’) AP-1μ (AP-47SHE-11) mutant clones (cells marked by the absence of GFP (green) and outlined in yellow) exhibit a complete block in production of Sgs3-DsRed–containing glue granules (red) (A’ and A’’) or strikingly small glue granules (B’ and B’’). Note that cells with two copies of wild-type AP-47 (marked by two copies of GFP) have larger granules than heterozygous cells (marked by one copy of GFP). (C–I) Confocal fluorescence micrographs of stage 2 larval salivary gland cells expressing Sgs3-DsRed. (C) Control salivary gland cells expressing the AB1-GAL4 driver alone have granules of normal size (C, boxed region; shown at 2× higher magnification in inset). (D) Salivary gland cells expressing both the AB1-GAL4 and a UAS-AP-1γ RNAi transgene completely lack glue granules (outlined cell) or have strikingly small glue granules (D, boxed region; shown at 2× higher magnification in inset). See also Supplemental Figure S2. (E–I) Spinning-disk confocal micrographs of salivary gland cells expressing Sgs3-DsRed. (E) Control wild-type cells showing normal-sized glue granules. (F) Larvae bearing the heteroallelic genotype AP-47SHE-11/EP1112 exhibit intermediate-sized granules. (G) Depletion of clathrin heavy chain by RNAi in cells expressing AB1-GAL4 and a UAS-Chc RNAi transgene causes a complete block in glue production in most cells, whereas a minority of cells produced small amounts of glue. (H and I) Strong loss-of-function mutations in AP-3μ (carmine1 [cm1]) (H) or AP-3δ (garnet50e [g50e]) (I) have no effect on glue granule biogenesis.

Mentions: To determine whether AP-1 is required for glue granule formation, we examined AP-47SHE-11 homozygous mutant cells in late–third-instar larvae, when glue granules are fully mature (stage 2). AP-47SHE-11 mutant cells either lacked detectible Sgs3-DsRed–containing glue granules (8 of 13 cells) (Figure 5, A–A″) or accumulated small granules in the cytoplasm (5 of 13 cells) (Figure 5, B–B’’). This difference is likely due to variations in perdurance of AP-1μ protein in mutant cells. In addition, AP-47SHE-11 mutant cells also appeared smaller, suggesting that additional secretory pathways involved in cell growth might be affected. Remarkably, AP-1μ showed dosage dependence, in that cells with only one wild-type copy of AP-47 (marked by one copy of GFP) had intermediate-sized glue granules, whereas cells with two functional copies of AP-47 (marked by two copies of GFP) had granules of normal size (Figure 5, A–A’’). In support of the idea that AP-1 is limiting for granule biogenesis, third-instar larvae heterozygous for AP-47SHE-11 and the hypomorphic allele AP-47EP1112 were viable and exhibited glue granules of intermediate size (compare Figure 5, E and F).


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)

AP-1 is essential for glue granule biogenesis. Confocal fluorescence micrographs of late–third-instar (stage 2) larval salivary glands. (A–B’’) AP-1μ (AP-47SHE-11) mutant clones (cells marked by the absence of GFP (green) and outlined in yellow) exhibit a complete block in production of Sgs3-DsRed–containing glue granules (red) (A’ and A’’) or strikingly small glue granules (B’ and B’’). Note that cells with two copies of wild-type AP-47 (marked by two copies of GFP) have larger granules than heterozygous cells (marked by one copy of GFP). (C–I) Confocal fluorescence micrographs of stage 2 larval salivary gland cells expressing Sgs3-DsRed. (C) Control salivary gland cells expressing the AB1-GAL4 driver alone have granules of normal size (C, boxed region; shown at 2× higher magnification in inset). (D) Salivary gland cells expressing both the AB1-GAL4 and a UAS-AP-1γ RNAi transgene completely lack glue granules (outlined cell) or have strikingly small glue granules (D, boxed region; shown at 2× higher magnification in inset). See also Supplemental Figure S2. (E–I) Spinning-disk confocal micrographs of salivary gland cells expressing Sgs3-DsRed. (E) Control wild-type cells showing normal-sized glue granules. (F) Larvae bearing the heteroallelic genotype AP-47SHE-11/EP1112 exhibit intermediate-sized granules. (G) Depletion of clathrin heavy chain by RNAi in cells expressing AB1-GAL4 and a UAS-Chc RNAi transgene causes a complete block in glue production in most cells, whereas a minority of cells produced small amounts of glue. (H and I) Strong loss-of-function mutations in AP-3μ (carmine1 [cm1]) (H) or AP-3δ (garnet50e [g50e]) (I) have no effect on glue granule biogenesis.
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Related In: Results  -  Collection

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Figure 5: AP-1 is essential for glue granule biogenesis. Confocal fluorescence micrographs of late–third-instar (stage 2) larval salivary glands. (A–B’’) AP-1μ (AP-47SHE-11) mutant clones (cells marked by the absence of GFP (green) and outlined in yellow) exhibit a complete block in production of Sgs3-DsRed–containing glue granules (red) (A’ and A’’) or strikingly small glue granules (B’ and B’’). Note that cells with two copies of wild-type AP-47 (marked by two copies of GFP) have larger granules than heterozygous cells (marked by one copy of GFP). (C–I) Confocal fluorescence micrographs of stage 2 larval salivary gland cells expressing Sgs3-DsRed. (C) Control salivary gland cells expressing the AB1-GAL4 driver alone have granules of normal size (C, boxed region; shown at 2× higher magnification in inset). (D) Salivary gland cells expressing both the AB1-GAL4 and a UAS-AP-1γ RNAi transgene completely lack glue granules (outlined cell) or have strikingly small glue granules (D, boxed region; shown at 2× higher magnification in inset). See also Supplemental Figure S2. (E–I) Spinning-disk confocal micrographs of salivary gland cells expressing Sgs3-DsRed. (E) Control wild-type cells showing normal-sized glue granules. (F) Larvae bearing the heteroallelic genotype AP-47SHE-11/EP1112 exhibit intermediate-sized granules. (G) Depletion of clathrin heavy chain by RNAi in cells expressing AB1-GAL4 and a UAS-Chc RNAi transgene causes a complete block in glue production in most cells, whereas a minority of cells produced small amounts of glue. (H and I) Strong loss-of-function mutations in AP-3μ (carmine1 [cm1]) (H) or AP-3δ (garnet50e [g50e]) (I) have no effect on glue granule biogenesis.
Mentions: To determine whether AP-1 is required for glue granule formation, we examined AP-47SHE-11 homozygous mutant cells in late–third-instar larvae, when glue granules are fully mature (stage 2). AP-47SHE-11 mutant cells either lacked detectible Sgs3-DsRed–containing glue granules (8 of 13 cells) (Figure 5, A–A″) or accumulated small granules in the cytoplasm (5 of 13 cells) (Figure 5, B–B’’). This difference is likely due to variations in perdurance of AP-1μ protein in mutant cells. In addition, AP-47SHE-11 mutant cells also appeared smaller, suggesting that additional secretory pathways involved in cell growth might be affected. Remarkably, AP-1μ showed dosage dependence, in that cells with only one wild-type copy of AP-47 (marked by one copy of GFP) had intermediate-sized glue granules, whereas cells with two functional copies of AP-47 (marked by two copies of GFP) had granules of normal size (Figure 5, A–A’’). In support of the idea that AP-1 is limiting for granule biogenesis, third-instar larvae heterozygous for AP-47SHE-11 and the hypomorphic allele AP-47EP1112 were viable and exhibited glue granules of intermediate size (compare Figure 5, E and F).

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