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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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Related in: MedlinePlus

Clathrin heavy chain and the clathrin adaptor AP-1 colocalize at the trans-Golgi network. Confocal micrographs of stage 0 salivary gland cells. (A–A″) RFP-Chc (green) localizes adjacent to, but does not overlap with, the cis-Golgi marker Lva (red). (B–B″) Endogenous AP-1γ (green) localizes adjacent to Lva (red). (C–C″) Projection of a series of spinning-disk confocal images of salivary gland cells stained for AP-1γ (green), Lva (red), and DNA (stained with DAPI; blue) reveals numerous Golgi bodies scattered throughout the cytoplasm (C). A three-dimensional rotation of a single Golgi body shows AP-1γ (green) adjacent to the cup-shaped Lva-positive cis-Golgi (red) (C’–C″). Images were generated from Z stacks of 28 (C) or 5 (C’–C″) optical sections acquired at a distance of 0.3 μm (see Materials and Methods). (D–D″) AP-1γ (green) and RFP-Chc (red) colocalize adjacent to Lva (blue). Colocalization of AP-1γ and RFP-Chc appears yellow in the merged image. (E–E″) Spinning-disk confocal images reveal that VFP-AP-47 (green) does not colocalize with mCherry-AP3δ (red). Boxed region is shown at 2× higher magnification in the insets.
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Figure 2: Clathrin heavy chain and the clathrin adaptor AP-1 colocalize at the trans-Golgi network. Confocal micrographs of stage 0 salivary gland cells. (A–A″) RFP-Chc (green) localizes adjacent to, but does not overlap with, the cis-Golgi marker Lva (red). (B–B″) Endogenous AP-1γ (green) localizes adjacent to Lva (red). (C–C″) Projection of a series of spinning-disk confocal images of salivary gland cells stained for AP-1γ (green), Lva (red), and DNA (stained with DAPI; blue) reveals numerous Golgi bodies scattered throughout the cytoplasm (C). A three-dimensional rotation of a single Golgi body shows AP-1γ (green) adjacent to the cup-shaped Lva-positive cis-Golgi (red) (C’–C″). Images were generated from Z stacks of 28 (C) or 5 (C’–C″) optical sections acquired at a distance of 0.3 μm (see Materials and Methods). (D–D″) AP-1γ (green) and RFP-Chc (red) colocalize adjacent to Lva (blue). Colocalization of AP-1γ and RFP-Chc appears yellow in the merged image. (E–E″) Spinning-disk confocal images reveal that VFP-AP-47 (green) does not colocalize with mCherry-AP3δ (red). Boxed region is shown at 2× higher magnification in the insets.

Mentions: To identify coats that might function in granule biogenesis, we examined the subcellular distribution of clathrin heavy chain, as well as subunits of the clathrin adaptor protein complexes AP-1 and AP-3, which reside on intracellular organelles (note that Drosophila lacks AP-4; Boehm and Bonifacino, 2001). We first examined clathrin, AP-1, and AP-3 in salivary gland cells at stage 0, just prior to glue production. At this stage, Golgi bodies are easily visualized using antibodies directed against the golgin Lava lamp (Lva), which localizes to the cis-Golgi (Sisson et al., 2000) (Figure 2, A–D″). Note that the cis-Golgi has a cup-shaped appearance. A monomeric red fluorescent protein fusion to clathrin heavy chain (RFP-Chc) predominantly localized to large puncta adjacent to the concave face of the cis-Golgi (Figure 2, A–A″), consistent with a previous report showing localization of endogenous Chc to intracellular puncta in these cells (Wingen et al., 2009). Endogenous AP-1γ showed a similar distribution (Figure 2, B–B″). A projection constructed from serial confocal sections revealed numerous Golgi units scattered throughout the cytoplasm (Figure 2C). There was a one-to-one correspondence between AP-1γ– and Lva-positive structures, with the cis-Golgi cups surrounding AP-1γ in a manner consistent with AP-1 localizing to the TGN (Figure 2, C–C’″). Indeed AP-1γ and RFP-Chc colocalized with the trans-Golgi protein EpsinR (also called Liquid facets-Related or LqfR; Lee et al., 2009) (Supplemental Figure S1A). In contrast, AP-1 showed only minimal overlap with the recycling endosome regulator Rab11 (Buszczak et al., 2007; Lighthouse et al., 2008) (Supplemental Figure S1B). AP-1γ and RFP-Chc colocalized at the TGN (Figure 2, D–D′″), although AP-1γ distribution appeared slightly more diffuse in salivary gland cells expressing RFP-Chc than in nonexpressing cells (compare Figure 2, B and D). Localization of AP-1 to the TGN is adaptor-protein specific, because a functional monomeric cherry fluorescent protein (mCherry) fusion to AP-3δ (called Garnet in Drosophila) showed no overlap with a Venus fluorescent protein (VFP) fusion to AP-1μ (called AP-47 in Drosophila) (Figure 2, E–E″), but rather colocalized with the late endosome marker Rab7 (unpublished data). Given the high degree of colocalization of clathrin and AP-1, we wondered whether AP-1 might be required to recruit clathrin to the TGN.


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)

Clathrin heavy chain and the clathrin adaptor AP-1 colocalize at the trans-Golgi network. Confocal micrographs of stage 0 salivary gland cells. (A–A″) RFP-Chc (green) localizes adjacent to, but does not overlap with, the cis-Golgi marker Lva (red). (B–B″) Endogenous AP-1γ (green) localizes adjacent to Lva (red). (C–C″) Projection of a series of spinning-disk confocal images of salivary gland cells stained for AP-1γ (green), Lva (red), and DNA (stained with DAPI; blue) reveals numerous Golgi bodies scattered throughout the cytoplasm (C). A three-dimensional rotation of a single Golgi body shows AP-1γ (green) adjacent to the cup-shaped Lva-positive cis-Golgi (red) (C’–C″). Images were generated from Z stacks of 28 (C) or 5 (C’–C″) optical sections acquired at a distance of 0.3 μm (see Materials and Methods). (D–D″) AP-1γ (green) and RFP-Chc (red) colocalize adjacent to Lva (blue). Colocalization of AP-1γ and RFP-Chc appears yellow in the merged image. (E–E″) Spinning-disk confocal images reveal that VFP-AP-47 (green) does not colocalize with mCherry-AP3δ (red). Boxed region is shown at 2× higher magnification in the insets.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
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Figure 2: Clathrin heavy chain and the clathrin adaptor AP-1 colocalize at the trans-Golgi network. Confocal micrographs of stage 0 salivary gland cells. (A–A″) RFP-Chc (green) localizes adjacent to, but does not overlap with, the cis-Golgi marker Lva (red). (B–B″) Endogenous AP-1γ (green) localizes adjacent to Lva (red). (C–C″) Projection of a series of spinning-disk confocal images of salivary gland cells stained for AP-1γ (green), Lva (red), and DNA (stained with DAPI; blue) reveals numerous Golgi bodies scattered throughout the cytoplasm (C). A three-dimensional rotation of a single Golgi body shows AP-1γ (green) adjacent to the cup-shaped Lva-positive cis-Golgi (red) (C’–C″). Images were generated from Z stacks of 28 (C) or 5 (C’–C″) optical sections acquired at a distance of 0.3 μm (see Materials and Methods). (D–D″) AP-1γ (green) and RFP-Chc (red) colocalize adjacent to Lva (blue). Colocalization of AP-1γ and RFP-Chc appears yellow in the merged image. (E–E″) Spinning-disk confocal images reveal that VFP-AP-47 (green) does not colocalize with mCherry-AP3δ (red). Boxed region is shown at 2× higher magnification in the insets.
Mentions: To identify coats that might function in granule biogenesis, we examined the subcellular distribution of clathrin heavy chain, as well as subunits of the clathrin adaptor protein complexes AP-1 and AP-3, which reside on intracellular organelles (note that Drosophila lacks AP-4; Boehm and Bonifacino, 2001). We first examined clathrin, AP-1, and AP-3 in salivary gland cells at stage 0, just prior to glue production. At this stage, Golgi bodies are easily visualized using antibodies directed against the golgin Lava lamp (Lva), which localizes to the cis-Golgi (Sisson et al., 2000) (Figure 2, A–D″). Note that the cis-Golgi has a cup-shaped appearance. A monomeric red fluorescent protein fusion to clathrin heavy chain (RFP-Chc) predominantly localized to large puncta adjacent to the concave face of the cis-Golgi (Figure 2, A–A″), consistent with a previous report showing localization of endogenous Chc to intracellular puncta in these cells (Wingen et al., 2009). Endogenous AP-1γ showed a similar distribution (Figure 2, B–B″). A projection constructed from serial confocal sections revealed numerous Golgi units scattered throughout the cytoplasm (Figure 2C). There was a one-to-one correspondence between AP-1γ– and Lva-positive structures, with the cis-Golgi cups surrounding AP-1γ in a manner consistent with AP-1 localizing to the TGN (Figure 2, C–C’″). Indeed AP-1γ and RFP-Chc colocalized with the trans-Golgi protein EpsinR (also called Liquid facets-Related or LqfR; Lee et al., 2009) (Supplemental Figure S1A). In contrast, AP-1 showed only minimal overlap with the recycling endosome regulator Rab11 (Buszczak et al., 2007; Lighthouse et al., 2008) (Supplemental Figure S1B). AP-1γ and RFP-Chc colocalized at the TGN (Figure 2, D–D′″), although AP-1γ distribution appeared slightly more diffuse in salivary gland cells expressing RFP-Chc than in nonexpressing cells (compare Figure 2, B and D). Localization of AP-1 to the TGN is adaptor-protein specific, because a functional monomeric cherry fluorescent protein (mCherry) fusion to AP-3δ (called Garnet in Drosophila) showed no overlap with a Venus fluorescent protein (VFP) fusion to AP-1μ (called AP-47 in Drosophila) (Figure 2, E–E″), but rather colocalized with the late endosome marker Rab7 (unpublished data). Given the high degree of colocalization of clathrin and AP-1, we wondered whether AP-1 might be required to recruit clathrin to the TGN.

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