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Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena.

Briguglio JS, Kumar S, Turkewitz AP - J. Cell Biol. (2013)

Bottom Line: We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis.In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation.Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.

ABSTRACT
Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

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Δsor4 cells are defective in regulated exocytosis and in sorting of a second Grt family protein. (A) A qualitative assay for mucocyst discharge. Individual T. thermophila cells, fixed and photographed after treatment with the secretagogue Alcian blue. The wild-type cell (left) is surrounded by a translucent capsule made up of the released contents of exocytosed mucocysts. In contrast, Δsor4 cells (right) never form visible capsules after stimulation. Images are differential interference contrast micrographs. The same wild-type control is shown again in Fig. S4 A. Bars, 5 µm. (B) A semiquantitative assay for mucocyst discharge. Identical numbers of wild-type and Δsor4 cells were stimulated with dibucaine, and immediately centrifuged. The wild-type culture produces a two-layer pellet, in which a thick layer of flocculent (below the broken line) resulting from mucocyst discharge sits atop of the packed cells (below the dotted line). Stimulated Δsor4 cultures, in contrast, produce no flocculent layer. Stimulated Δsor2 cultures generate an intermediate amount of the mucocyst-derived flocculent. (C) Δsor4 cells show defective sorting to mucocysts of a second Grt family protein, Igr1p. Igr1p-GFP, expressed from an inducible promoter, accumulates in docked mucocysts in wild-type cells (left), but is absent from the periphery of Δsor4, instead found in small highly mobile cytoplasmic puncta. Images are of GFP autofluorescence in live, immobilized cells. Bars, 5 µm.
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fig4: Δsor4 cells are defective in regulated exocytosis and in sorting of a second Grt family protein. (A) A qualitative assay for mucocyst discharge. Individual T. thermophila cells, fixed and photographed after treatment with the secretagogue Alcian blue. The wild-type cell (left) is surrounded by a translucent capsule made up of the released contents of exocytosed mucocysts. In contrast, Δsor4 cells (right) never form visible capsules after stimulation. Images are differential interference contrast micrographs. The same wild-type control is shown again in Fig. S4 A. Bars, 5 µm. (B) A semiquantitative assay for mucocyst discharge. Identical numbers of wild-type and Δsor4 cells were stimulated with dibucaine, and immediately centrifuged. The wild-type culture produces a two-layer pellet, in which a thick layer of flocculent (below the broken line) resulting from mucocyst discharge sits atop of the packed cells (below the dotted line). Stimulated Δsor4 cultures, in contrast, produce no flocculent layer. Stimulated Δsor2 cultures generate an intermediate amount of the mucocyst-derived flocculent. (C) Δsor4 cells show defective sorting to mucocysts of a second Grt family protein, Igr1p. Igr1p-GFP, expressed from an inducible promoter, accumulates in docked mucocysts in wild-type cells (left), but is absent from the periphery of Δsor4, instead found in small highly mobile cytoplasmic puncta. Images are of GFP autofluorescence in live, immobilized cells. Bars, 5 µm.

Mentions: Missorting of Grt1p would not by itself produce a dramatic mucocyst defect, as cells in which GRT1 was deleted together with the closely related GRT2 showed only a mild secretion phenotype (Rahaman et al., 2009). In contrast, Δsor4 cells showed a complete absence of mucocyst discharge on stimulation, as assessed by two different methods. In wild-type T. thermophila, the entire set of docked mucocysts undergo exocytosis after cells are exposed to either the polycation Alcian blue or dibucaine. Alcian blue binds to acidic mucocyst proteins as they exit, entrapping each wild-type cell in a blue-stained capsule (Tiedtke, 1976), but Δsor4 cells showed no trace of capsule formation (Fig. 4 A). The mucocyst contents released from dibucaine-treated wild-type cells form large pelletable aggregates (Satir, 1977), but these were entirely absent in dibucaine-treated Δsor4 cultures, and greatly reduced in Δsor2 cultures (Fig. 4 B). Both the capsule-formation defect and the absence of flocculent are comparable to mutants in mucocyst formation or exocytosis that have previously been characterized in this organism (Orias et al., 1983; Melia et al., 1998; Cowan et al., 2005). The disparity between the strong Δsor4 and mild Δgrt1 phenotypes indicated that Grt1p was unlikely to be the only ligand that depends on Sor4p for sorting to mucocysts. Because Grt1p belongs to a 13-member family of mucocyst content proteins, the other members were obvious candidates for Sor4p ligands.


Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena.

Briguglio JS, Kumar S, Turkewitz AP - J. Cell Biol. (2013)

Δsor4 cells are defective in regulated exocytosis and in sorting of a second Grt family protein. (A) A qualitative assay for mucocyst discharge. Individual T. thermophila cells, fixed and photographed after treatment with the secretagogue Alcian blue. The wild-type cell (left) is surrounded by a translucent capsule made up of the released contents of exocytosed mucocysts. In contrast, Δsor4 cells (right) never form visible capsules after stimulation. Images are differential interference contrast micrographs. The same wild-type control is shown again in Fig. S4 A. Bars, 5 µm. (B) A semiquantitative assay for mucocyst discharge. Identical numbers of wild-type and Δsor4 cells were stimulated with dibucaine, and immediately centrifuged. The wild-type culture produces a two-layer pellet, in which a thick layer of flocculent (below the broken line) resulting from mucocyst discharge sits atop of the packed cells (below the dotted line). Stimulated Δsor4 cultures, in contrast, produce no flocculent layer. Stimulated Δsor2 cultures generate an intermediate amount of the mucocyst-derived flocculent. (C) Δsor4 cells show defective sorting to mucocysts of a second Grt family protein, Igr1p. Igr1p-GFP, expressed from an inducible promoter, accumulates in docked mucocysts in wild-type cells (left), but is absent from the periphery of Δsor4, instead found in small highly mobile cytoplasmic puncta. Images are of GFP autofluorescence in live, immobilized cells. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824020&req=5

fig4: Δsor4 cells are defective in regulated exocytosis and in sorting of a second Grt family protein. (A) A qualitative assay for mucocyst discharge. Individual T. thermophila cells, fixed and photographed after treatment with the secretagogue Alcian blue. The wild-type cell (left) is surrounded by a translucent capsule made up of the released contents of exocytosed mucocysts. In contrast, Δsor4 cells (right) never form visible capsules after stimulation. Images are differential interference contrast micrographs. The same wild-type control is shown again in Fig. S4 A. Bars, 5 µm. (B) A semiquantitative assay for mucocyst discharge. Identical numbers of wild-type and Δsor4 cells were stimulated with dibucaine, and immediately centrifuged. The wild-type culture produces a two-layer pellet, in which a thick layer of flocculent (below the broken line) resulting from mucocyst discharge sits atop of the packed cells (below the dotted line). Stimulated Δsor4 cultures, in contrast, produce no flocculent layer. Stimulated Δsor2 cultures generate an intermediate amount of the mucocyst-derived flocculent. (C) Δsor4 cells show defective sorting to mucocysts of a second Grt family protein, Igr1p. Igr1p-GFP, expressed from an inducible promoter, accumulates in docked mucocysts in wild-type cells (left), but is absent from the periphery of Δsor4, instead found in small highly mobile cytoplasmic puncta. Images are of GFP autofluorescence in live, immobilized cells. Bars, 5 µm.
Mentions: Missorting of Grt1p would not by itself produce a dramatic mucocyst defect, as cells in which GRT1 was deleted together with the closely related GRT2 showed only a mild secretion phenotype (Rahaman et al., 2009). In contrast, Δsor4 cells showed a complete absence of mucocyst discharge on stimulation, as assessed by two different methods. In wild-type T. thermophila, the entire set of docked mucocysts undergo exocytosis after cells are exposed to either the polycation Alcian blue or dibucaine. Alcian blue binds to acidic mucocyst proteins as they exit, entrapping each wild-type cell in a blue-stained capsule (Tiedtke, 1976), but Δsor4 cells showed no trace of capsule formation (Fig. 4 A). The mucocyst contents released from dibucaine-treated wild-type cells form large pelletable aggregates (Satir, 1977), but these were entirely absent in dibucaine-treated Δsor4 cultures, and greatly reduced in Δsor2 cultures (Fig. 4 B). Both the capsule-formation defect and the absence of flocculent are comparable to mutants in mucocyst formation or exocytosis that have previously been characterized in this organism (Orias et al., 1983; Melia et al., 1998; Cowan et al., 2005). The disparity between the strong Δsor4 and mild Δgrt1 phenotypes indicated that Grt1p was unlikely to be the only ligand that depends on Sor4p for sorting to mucocysts. Because Grt1p belongs to a 13-member family of mucocyst content proteins, the other members were obvious candidates for Sor4p ligands.

Bottom Line: We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis.In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation.Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.

ABSTRACT
Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

Show MeSH
Related in: MedlinePlus