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Granuphilin molecularly docks insulin granules to the fusion machinery.

Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T - J. Cell Biol. (2005)

Bottom Line: The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis.Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells.The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan.

ABSTRACT
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin- mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin- beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

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Rescue of granuphilin- β cell phenotypes by ADV-mediated granuphilin expression. (A) ADV-mediated LacZ expression in isolated islets. The islets were infected with ADV-encoding LacZ at different MOI from 20 to 500 at 37°C for 2 h and then cultured in a fresh medium for 48 h. LacZ expression was visualized by X-gal substrate enzyme activity (dark-colored cells, arrowheads). Bar, 100 μm. (B) Expression levels of granuphilin-a in islets uninfected (Uninf.) or infected for 48 h with an ADV-encoding LacZ, wild-type (WT), or L43A mutant (L43A) granuphilin-a (MOI = 500). Protein expression levels were analyzed by immunoblotting (IB) with antigranuphilin αGrp-N and anti–α-tubulin antibodies. (C–E) Electron micrographs of β cell sections from ADV-infected Grn−/Y islets (MOI = 500). Dashed lines indicate borders 200 nm distant from the plasma membrane. Bar, 1 μm. (F and G) Morphometric analyses of insulin granules in electron micrographs of ADV-infected Grn−/Y β cell sections. Relative density of granules located near the plasma membrane (F) and average granule number per cytosol area (μm2; G) were calculated as in Fig. 5 (C and D) from 12 randomly selected β cells of each group that had been infected with ADV-encoding LacZ, WT, or L43A. *, P < 0.05; **, P < 0.01; ***, P < 0.005. (H) Immunoblot analysis of ADV-infected islets. Total protein was extracted at 48 h after ADV infection and reacted with the antibodies indicated (left). Protein expression levels were quantified from six preparations (right). *, P = 0.027. Results are provided as mean ± SEM.
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fig8: Rescue of granuphilin- β cell phenotypes by ADV-mediated granuphilin expression. (A) ADV-mediated LacZ expression in isolated islets. The islets were infected with ADV-encoding LacZ at different MOI from 20 to 500 at 37°C for 2 h and then cultured in a fresh medium for 48 h. LacZ expression was visualized by X-gal substrate enzyme activity (dark-colored cells, arrowheads). Bar, 100 μm. (B) Expression levels of granuphilin-a in islets uninfected (Uninf.) or infected for 48 h with an ADV-encoding LacZ, wild-type (WT), or L43A mutant (L43A) granuphilin-a (MOI = 500). Protein expression levels were analyzed by immunoblotting (IB) with antigranuphilin αGrp-N and anti–α-tubulin antibodies. (C–E) Electron micrographs of β cell sections from ADV-infected Grn−/Y islets (MOI = 500). Dashed lines indicate borders 200 nm distant from the plasma membrane. Bar, 1 μm. (F and G) Morphometric analyses of insulin granules in electron micrographs of ADV-infected Grn−/Y β cell sections. Relative density of granules located near the plasma membrane (F) and average granule number per cytosol area (μm2; G) were calculated as in Fig. 5 (C and D) from 12 randomly selected β cells of each group that had been infected with ADV-encoding LacZ, WT, or L43A. *, P < 0.05; **, P < 0.01; ***, P < 0.005. (H) Immunoblot analysis of ADV-infected islets. Total protein was extracted at 48 h after ADV infection and reacted with the antibodies indicated (left). Protein expression levels were quantified from six preparations (right). *, P = 0.027. Results are provided as mean ± SEM.

Mentions: We performed rescue experiments to examine whether defects in granuphilin-deficient β cells are reversibly and specifically restored by the expression of exogenous granuphilin. We first examined the optimum infective condition using ADV-encoding β-galactosidase (LacZ) to probe the infection efficiency in isolated islets (Fig. 8 A). Although LacZ expression was weak and inhomogeneous in the islet cells at a multiplicity of infection (MOI) <100, the higher titers at MOI 250–500 achieved overall expression and thus were used for the rescue experiments. Infection of the mutant islets with ADV-encoding wild-type granuphilin-a induced a protein level equivalent to the endogenous level in uninfected wild-type islets (Fig. 8 B and Fig. S1). Moreover, it restored morphologically docked granules in the mutant β cells, in contrast to the expression of control LacZ (Fig. 8, C, D, and F). The relative density of granules near the plasma membrane in granuphilin-a–expressed mutant cells was comparable with that observed in noninfected wild-type β cells (Fig. 8 F vs. Fig. 5 C). The granule density, however, was reduced in infected β cells compared with uninfected β cells (Fig. 8 G vs. Fig. 5 D), a result that occurred irrespective of the expressed protein and could be ascribed to the background effect of the virus infection. Because of this effect, we did not examine the effect on insulin secretion, although we suspect that granuphilin expression decreases the evoked insulin secretion as was reported in MIN6 cells (Torii et al., 2002). Importantly, the restoration of morphologically docked granules was not seen by the expression of the granuphilin-a mutant L43A, which is specifically defective in binding to syntaxin-1a (Torii et al., 2002; Fig. 8, E and F). The differential effect should be specific to the protein expressed because it was observed in cells close to the outer margin of the islets, where infection occurs most efficiently (unpublished data), and because we infected the islets under conditions in which almost all cells express exogenous proteins (Fig. 8 A). Consistently, we previously demonstrated that the overexpression of the L43A mutant in MIN6 cells fails to promote the targeting of insulin granules onto the plasma membrane or to inhibit high K+-induced insulin secretion efficiently, in contrast to the case of the wild type (Torii et al., 2002, 2004). Besides the effect on granule docking, the expression of wild-type granuphilin-a, but not of the L43 mutant, significantly increased the protein level of syntaxin-1a (Fig. 8 H). By contrast, the expression of granuphilin-a did not alter the protein level of Munc18-1. These findings suggest that the reductions in docked granules and syntaxin-1a expression are direct consequences of granuphilin deficiency, whereas the decreased level of Munc18-1 is a secondary phenomenon resulting from the chronic deficiency in granuphilin and/or syntaxin-1a.


Granuphilin molecularly docks insulin granules to the fusion machinery.

Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T - J. Cell Biol. (2005)

Rescue of granuphilin- β cell phenotypes by ADV-mediated granuphilin expression. (A) ADV-mediated LacZ expression in isolated islets. The islets were infected with ADV-encoding LacZ at different MOI from 20 to 500 at 37°C for 2 h and then cultured in a fresh medium for 48 h. LacZ expression was visualized by X-gal substrate enzyme activity (dark-colored cells, arrowheads). Bar, 100 μm. (B) Expression levels of granuphilin-a in islets uninfected (Uninf.) or infected for 48 h with an ADV-encoding LacZ, wild-type (WT), or L43A mutant (L43A) granuphilin-a (MOI = 500). Protein expression levels were analyzed by immunoblotting (IB) with antigranuphilin αGrp-N and anti–α-tubulin antibodies. (C–E) Electron micrographs of β cell sections from ADV-infected Grn−/Y islets (MOI = 500). Dashed lines indicate borders 200 nm distant from the plasma membrane. Bar, 1 μm. (F and G) Morphometric analyses of insulin granules in electron micrographs of ADV-infected Grn−/Y β cell sections. Relative density of granules located near the plasma membrane (F) and average granule number per cytosol area (μm2; G) were calculated as in Fig. 5 (C and D) from 12 randomly selected β cells of each group that had been infected with ADV-encoding LacZ, WT, or L43A. *, P < 0.05; **, P < 0.01; ***, P < 0.005. (H) Immunoblot analysis of ADV-infected islets. Total protein was extracted at 48 h after ADV infection and reacted with the antibodies indicated (left). Protein expression levels were quantified from six preparations (right). *, P = 0.027. Results are provided as mean ± SEM.
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fig8: Rescue of granuphilin- β cell phenotypes by ADV-mediated granuphilin expression. (A) ADV-mediated LacZ expression in isolated islets. The islets were infected with ADV-encoding LacZ at different MOI from 20 to 500 at 37°C for 2 h and then cultured in a fresh medium for 48 h. LacZ expression was visualized by X-gal substrate enzyme activity (dark-colored cells, arrowheads). Bar, 100 μm. (B) Expression levels of granuphilin-a in islets uninfected (Uninf.) or infected for 48 h with an ADV-encoding LacZ, wild-type (WT), or L43A mutant (L43A) granuphilin-a (MOI = 500). Protein expression levels were analyzed by immunoblotting (IB) with antigranuphilin αGrp-N and anti–α-tubulin antibodies. (C–E) Electron micrographs of β cell sections from ADV-infected Grn−/Y islets (MOI = 500). Dashed lines indicate borders 200 nm distant from the plasma membrane. Bar, 1 μm. (F and G) Morphometric analyses of insulin granules in electron micrographs of ADV-infected Grn−/Y β cell sections. Relative density of granules located near the plasma membrane (F) and average granule number per cytosol area (μm2; G) were calculated as in Fig. 5 (C and D) from 12 randomly selected β cells of each group that had been infected with ADV-encoding LacZ, WT, or L43A. *, P < 0.05; **, P < 0.01; ***, P < 0.005. (H) Immunoblot analysis of ADV-infected islets. Total protein was extracted at 48 h after ADV infection and reacted with the antibodies indicated (left). Protein expression levels were quantified from six preparations (right). *, P = 0.027. Results are provided as mean ± SEM.
Mentions: We performed rescue experiments to examine whether defects in granuphilin-deficient β cells are reversibly and specifically restored by the expression of exogenous granuphilin. We first examined the optimum infective condition using ADV-encoding β-galactosidase (LacZ) to probe the infection efficiency in isolated islets (Fig. 8 A). Although LacZ expression was weak and inhomogeneous in the islet cells at a multiplicity of infection (MOI) <100, the higher titers at MOI 250–500 achieved overall expression and thus were used for the rescue experiments. Infection of the mutant islets with ADV-encoding wild-type granuphilin-a induced a protein level equivalent to the endogenous level in uninfected wild-type islets (Fig. 8 B and Fig. S1). Moreover, it restored morphologically docked granules in the mutant β cells, in contrast to the expression of control LacZ (Fig. 8, C, D, and F). The relative density of granules near the plasma membrane in granuphilin-a–expressed mutant cells was comparable with that observed in noninfected wild-type β cells (Fig. 8 F vs. Fig. 5 C). The granule density, however, was reduced in infected β cells compared with uninfected β cells (Fig. 8 G vs. Fig. 5 D), a result that occurred irrespective of the expressed protein and could be ascribed to the background effect of the virus infection. Because of this effect, we did not examine the effect on insulin secretion, although we suspect that granuphilin expression decreases the evoked insulin secretion as was reported in MIN6 cells (Torii et al., 2002). Importantly, the restoration of morphologically docked granules was not seen by the expression of the granuphilin-a mutant L43A, which is specifically defective in binding to syntaxin-1a (Torii et al., 2002; Fig. 8, E and F). The differential effect should be specific to the protein expressed because it was observed in cells close to the outer margin of the islets, where infection occurs most efficiently (unpublished data), and because we infected the islets under conditions in which almost all cells express exogenous proteins (Fig. 8 A). Consistently, we previously demonstrated that the overexpression of the L43A mutant in MIN6 cells fails to promote the targeting of insulin granules onto the plasma membrane or to inhibit high K+-induced insulin secretion efficiently, in contrast to the case of the wild type (Torii et al., 2002, 2004). Besides the effect on granule docking, the expression of wild-type granuphilin-a, but not of the L43 mutant, significantly increased the protein level of syntaxin-1a (Fig. 8 H). By contrast, the expression of granuphilin-a did not alter the protein level of Munc18-1. These findings suggest that the reductions in docked granules and syntaxin-1a expression are direct consequences of granuphilin deficiency, whereas the decreased level of Munc18-1 is a secondary phenomenon resulting from the chronic deficiency in granuphilin and/or syntaxin-1a.

Bottom Line: The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis.Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells.The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan.

ABSTRACT
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin- mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin- beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

Show MeSH
Related in: MedlinePlus