Limits...
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.

Show MeSH

Related in: MedlinePlus

AP-1 is required to recruit clathrin to the trans-Golgi network. (A–C’’’) Confocal micrographs of stage 0 salivary glands showing mutant clones (cells) marked by absence of GFP (green) and outlined in yellow. (A–A’’) AP-1γ (red) localization is lost in an AP-1μ (AP-47SHE-11) mutant cell. (B–B’’) Rab5-positive early endosomes (red) are unaffected in AP-47SHE-11 mutant cells. (C–C’’’) RFP-Chc (red) becomes largely cytoplasmic in an AP-47SHE-11 mutant cell, whereas the distribution of the cis-Golgi marker Lva (blue) is unaltered. Note that Lva shows a gradient of signal intensity due to incomplete antibody penetration of the tissue. (D–D’’’) Control salivary gland cells expressing the AB1-GAL4 driver alone show colocalization of AP-1γ (green) and RFP-Chc (red) adjacent to Lva (blue). (E–E’’) Salivary gland cells expressing both the AB1-GAL4 driver and a UAS-AP-1γ RNAi transgene are depleted of AP-1γ (green) and show cytosolic distribution of RFP-Chc (red), whereas Lva (blue) is largely unaffected. See also Supplemental Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113773&req=5

Figure 3: AP-1 is required to recruit clathrin to the trans-Golgi network. (A–C’’’) Confocal micrographs of stage 0 salivary glands showing mutant clones (cells) marked by absence of GFP (green) and outlined in yellow. (A–A’’) AP-1γ (red) localization is lost in an AP-1μ (AP-47SHE-11) mutant cell. (B–B’’) Rab5-positive early endosomes (red) are unaffected in AP-47SHE-11 mutant cells. (C–C’’’) RFP-Chc (red) becomes largely cytoplasmic in an AP-47SHE-11 mutant cell, whereas the distribution of the cis-Golgi marker Lva (blue) is unaltered. Note that Lva shows a gradient of signal intensity due to incomplete antibody penetration of the tissue. (D–D’’’) Control salivary gland cells expressing the AB1-GAL4 driver alone show colocalization of AP-1γ (green) and RFP-Chc (red) adjacent to Lva (blue). (E–E’’) Salivary gland cells expressing both the AB1-GAL4 driver and a UAS-AP-1γ RNAi transgene are depleted of AP-1γ (green) and show cytosolic distribution of RFP-Chc (red), whereas Lva (blue) is largely unaffected. See also Supplemental Figure S2.

Mentions: To test whether AP-1 recruits clathrin to the TGN, we made use of a μ1-adaptin allele, AP-47SHE-11 (see Materials and Methods). To bypass late embryonic lethality caused by this allele, we generated mosaic clones in the salivary gland using FLP-FRT–based recombination (see Materials and Methods). Briefly, the wild-type chromosome carries a copy of green fluorescent protein (GFP) such that homozygous mutant cells are marked by the absence of GFP expression and heterozygous and wild-type cells are marked by one or two copies of GFP, respectively. AP-47SHE-11 clones were generated during embryogenesis and analyzed in third-instar larval salivary glands at stage 0, just prior to glue production. To determine whether other AP-1 subunits can localize to the TGN in the absence of AP-47, we examined the distribution of AP-1γ and found that its punctate localization was entirely lost in AP-47SHE-11 mutant cells (Figure 3, A–A″). Hence AP-47 is required for efficient recruitment or stability of AP-1γ, similar to what was previously observed in μ1-adaptin–deficient mouse embryonic fibroblasts (Meyer et al., 2000). Not all trafficking markers were affected by the loss of AP-47, as the early endosome marker Rab5 was unperturbed (Figure 3, B–B″).


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 required to recruit clathrin to the trans-Golgi network. (A–C’’’) Confocal micrographs of stage 0 salivary glands showing mutant clones (cells) marked by absence of GFP (green) and outlined in yellow. (A–A’’) AP-1γ (red) localization is lost in an AP-1μ (AP-47SHE-11) mutant cell. (B–B’’) Rab5-positive early endosomes (red) are unaffected in AP-47SHE-11 mutant cells. (C–C’’’) RFP-Chc (red) becomes largely cytoplasmic in an AP-47SHE-11 mutant cell, whereas the distribution of the cis-Golgi marker Lva (blue) is unaltered. Note that Lva shows a gradient of signal intensity due to incomplete antibody penetration of the tissue. (D–D’’’) Control salivary gland cells expressing the AB1-GAL4 driver alone show colocalization of AP-1γ (green) and RFP-Chc (red) adjacent to Lva (blue). (E–E’’) Salivary gland cells expressing both the AB1-GAL4 driver and a UAS-AP-1γ RNAi transgene are depleted of AP-1γ (green) and show cytosolic distribution of RFP-Chc (red), whereas Lva (blue) is largely unaffected. See also Supplemental Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: AP-1 is required to recruit clathrin to the trans-Golgi network. (A–C’’’) Confocal micrographs of stage 0 salivary glands showing mutant clones (cells) marked by absence of GFP (green) and outlined in yellow. (A–A’’) AP-1γ (red) localization is lost in an AP-1μ (AP-47SHE-11) mutant cell. (B–B’’) Rab5-positive early endosomes (red) are unaffected in AP-47SHE-11 mutant cells. (C–C’’’) RFP-Chc (red) becomes largely cytoplasmic in an AP-47SHE-11 mutant cell, whereas the distribution of the cis-Golgi marker Lva (blue) is unaltered. Note that Lva shows a gradient of signal intensity due to incomplete antibody penetration of the tissue. (D–D’’’) Control salivary gland cells expressing the AB1-GAL4 driver alone show colocalization of AP-1γ (green) and RFP-Chc (red) adjacent to Lva (blue). (E–E’’) Salivary gland cells expressing both the AB1-GAL4 driver and a UAS-AP-1γ RNAi transgene are depleted of AP-1γ (green) and show cytosolic distribution of RFP-Chc (red), whereas Lva (blue) is largely unaffected. See also Supplemental Figure S2.
Mentions: To test whether AP-1 recruits clathrin to the TGN, we made use of a μ1-adaptin allele, AP-47SHE-11 (see Materials and Methods). To bypass late embryonic lethality caused by this allele, we generated mosaic clones in the salivary gland using FLP-FRT–based recombination (see Materials and Methods). Briefly, the wild-type chromosome carries a copy of green fluorescent protein (GFP) such that homozygous mutant cells are marked by the absence of GFP expression and heterozygous and wild-type cells are marked by one or two copies of GFP, respectively. AP-47SHE-11 clones were generated during embryogenesis and analyzed in third-instar larval salivary glands at stage 0, just prior to glue production. To determine whether other AP-1 subunits can localize to the TGN in the absence of AP-47, we examined the distribution of AP-1γ and found that its punctate localization was entirely lost in AP-47SHE-11 mutant cells (Figure 3, A–A″). Hence AP-47 is required for efficient recruitment or stability of AP-1γ, similar to what was previously observed in μ1-adaptin–deficient mouse embryonic fibroblasts (Meyer et al., 2000). Not all trafficking markers were affected by the loss of AP-47, as the early endosome marker Rab5 was unperturbed (Figure 3, B–B″).

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