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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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Granule docking in isolated islets. (A and B) Electron micrographs of β cell sections. Islets were isolated from 24-wk-old male Grn+/Y and Grn−/Y mice and incubated at 37°C with 2.8 mM LG buffer for 1 h (A) and then with 25 mM HG buffer for 30 min (B). Dashed lines indicate a border 200 nm distant from the plasma membrane. Bars, 1 μm. (C and D) Morphometric analyses of insulin granules in LG-treated Grn+/Y (white bars) and Grn−/Y islets (light gray bars), and HG-treated Grn+/Y (dark gray bars) and Grn−/Y islets (black bars). For each group, 10 randomly selected β cells from four different animals were analyzed. (C) Relative density of insulin granules located near the plasma membrane. The granules were categorized into six bins according to their distance from the granule center to the plasma membrane (nm). The data were represented as a percentage of the granule density in each bin (100% corresponds to the average granule density in cytoplasm). *, P < 0.05; **, P < 0.005; ***, P < 0.0003. (D) Average granule number per cytosol area (μm2). *, P = 0.012. Results are provided as mean ± SEM.
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fig5: Granule docking in isolated islets. (A and B) Electron micrographs of β cell sections. Islets were isolated from 24-wk-old male Grn+/Y and Grn−/Y mice and incubated at 37°C with 2.8 mM LG buffer for 1 h (A) and then with 25 mM HG buffer for 30 min (B). Dashed lines indicate a border 200 nm distant from the plasma membrane. Bars, 1 μm. (C and D) Morphometric analyses of insulin granules in LG-treated Grn+/Y (white bars) and Grn−/Y islets (light gray bars), and HG-treated Grn+/Y (dark gray bars) and Grn−/Y islets (black bars). For each group, 10 randomly selected β cells from four different animals were analyzed. (C) Relative density of insulin granules located near the plasma membrane. The granules were categorized into six bins according to their distance from the granule center to the plasma membrane (nm). The data were represented as a percentage of the granule density in each bin (100% corresponds to the average granule density in cytoplasm). *, P < 0.05; **, P < 0.005; ***, P < 0.0003. (D) Average granule number per cytosol area (μm2). *, P = 0.012. Results are provided as mean ± SEM.

Mentions: Torii et al. (2004) showed that the overexpression of granuphilin in MIN6 cells induces a redistribution of insulin granules to the peripheral plasma membrane area. Thus, in the present study we performed a detailed electron microscopic analysis in isolated islets. The islets were incubated with either a low-glucose (LG) or a high-glucose (HG) buffer and fixed in the fixative (Fig. 5, A and B). The insulin granules were categorized into six bins according to their distance from the granule center to the plasma membrane, and the relative density of granules in each bin was calculated. The density of granules with centers that resided within 100 nm of the plasma membrane (first bin) was much lower than the average granule density in the cytoplasm (i.e., 100%; Fig. 5 C), which is not surprising considering that the diameter of insulin granules is ∼350 nm (Kasai et al., 2005; also see the end of this paragraph). By contrast, the density of the granules with centers that resided at 100–200 nm (second bin) was markedly greater than the average for the wild-type cells, which indicates an accumulation of granules docked to the plasma membrane, as reported previously (Kasai et al., 2005). The densities of the granules in both fractions were drastically reduced in the granuphilin- β cells irrespective of the preincubated glucose concentrations (for granules within 0–100 nm, 10.2 ± 3.2% and 7.1 ± 2.0% in LG- and HG-treated control cells, respectively, vs. 1.6 ± 1.1% and 1.9 ± 1.2% in LG- and HG-treated mutant cells, respectively; for granules within 100–200 nm, 182.5 ± 23.2% and 219.4 ± 21.9% in LG- and HG-treated control cells, respectively, vs. 60.0 ± 10.9% and 63.8 ± 9.4% in LG- and HG-treated mutant cells, respectively). By contrast, the relative densities of the granules within 300–400 nm (fourth bin) and at >500 nm (sixth bin) were significantly increased (for granules within 300–400 nm, 87.2 ± 8.0% and 81.9 ± 3.7% in LG- and HG-treated control cells, respectively, vs. 144.0 ± 8.3% and 123.1 ± 11.0% in LG- and HG-treated mutant cells, respectively; for granules at >500 nm, 91.7 ± 4.0% in HG-treated control cells vs. 104.3 ± 1.2% in HG-treated mutant cells). Although the mean granule diameter was slightly larger in the mutant β cells (324.9 and 339.4 nm in LG- and HG-treated control cells, respectively, vs. 345.7 and 377.6 nm in LG- and HG-treated mutant cells, respectively; P < 0.001), these 6–11% changes should not affect the granule locations. The mean granule density was significantly decreased in the HG-treated mutant β cells (3.81 ± 0.31 in control cells vs. 2.80 ± 0.19 in mutant cells; Fig. 5 D), which could be related to the enhanced insulin secretion. These results strongly indicate that granuphilin is essential for the docking of insulin granules to the plasma membrane.


Granuphilin molecularly docks insulin granules to the fusion machinery.

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

Granule docking in isolated islets. (A and B) Electron micrographs of β cell sections. Islets were isolated from 24-wk-old male Grn+/Y and Grn−/Y mice and incubated at 37°C with 2.8 mM LG buffer for 1 h (A) and then with 25 mM HG buffer for 30 min (B). Dashed lines indicate a border 200 nm distant from the plasma membrane. Bars, 1 μm. (C and D) Morphometric analyses of insulin granules in LG-treated Grn+/Y (white bars) and Grn−/Y islets (light gray bars), and HG-treated Grn+/Y (dark gray bars) and Grn−/Y islets (black bars). For each group, 10 randomly selected β cells from four different animals were analyzed. (C) Relative density of insulin granules located near the plasma membrane. The granules were categorized into six bins according to their distance from the granule center to the plasma membrane (nm). The data were represented as a percentage of the granule density in each bin (100% corresponds to the average granule density in cytoplasm). *, P < 0.05; **, P < 0.005; ***, P < 0.0003. (D) Average granule number per cytosol area (μm2). *, P = 0.012. Results are provided as mean ± SEM.
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fig5: Granule docking in isolated islets. (A and B) Electron micrographs of β cell sections. Islets were isolated from 24-wk-old male Grn+/Y and Grn−/Y mice and incubated at 37°C with 2.8 mM LG buffer for 1 h (A) and then with 25 mM HG buffer for 30 min (B). Dashed lines indicate a border 200 nm distant from the plasma membrane. Bars, 1 μm. (C and D) Morphometric analyses of insulin granules in LG-treated Grn+/Y (white bars) and Grn−/Y islets (light gray bars), and HG-treated Grn+/Y (dark gray bars) and Grn−/Y islets (black bars). For each group, 10 randomly selected β cells from four different animals were analyzed. (C) Relative density of insulin granules located near the plasma membrane. The granules were categorized into six bins according to their distance from the granule center to the plasma membrane (nm). The data were represented as a percentage of the granule density in each bin (100% corresponds to the average granule density in cytoplasm). *, P < 0.05; **, P < 0.005; ***, P < 0.0003. (D) Average granule number per cytosol area (μm2). *, P = 0.012. Results are provided as mean ± SEM.
Mentions: Torii et al. (2004) showed that the overexpression of granuphilin in MIN6 cells induces a redistribution of insulin granules to the peripheral plasma membrane area. Thus, in the present study we performed a detailed electron microscopic analysis in isolated islets. The islets were incubated with either a low-glucose (LG) or a high-glucose (HG) buffer and fixed in the fixative (Fig. 5, A and B). The insulin granules were categorized into six bins according to their distance from the granule center to the plasma membrane, and the relative density of granules in each bin was calculated. The density of granules with centers that resided within 100 nm of the plasma membrane (first bin) was much lower than the average granule density in the cytoplasm (i.e., 100%; Fig. 5 C), which is not surprising considering that the diameter of insulin granules is ∼350 nm (Kasai et al., 2005; also see the end of this paragraph). By contrast, the density of the granules with centers that resided at 100–200 nm (second bin) was markedly greater than the average for the wild-type cells, which indicates an accumulation of granules docked to the plasma membrane, as reported previously (Kasai et al., 2005). The densities of the granules in both fractions were drastically reduced in the granuphilin- β cells irrespective of the preincubated glucose concentrations (for granules within 0–100 nm, 10.2 ± 3.2% and 7.1 ± 2.0% in LG- and HG-treated control cells, respectively, vs. 1.6 ± 1.1% and 1.9 ± 1.2% in LG- and HG-treated mutant cells, respectively; for granules within 100–200 nm, 182.5 ± 23.2% and 219.4 ± 21.9% in LG- and HG-treated control cells, respectively, vs. 60.0 ± 10.9% and 63.8 ± 9.4% in LG- and HG-treated mutant cells, respectively). By contrast, the relative densities of the granules within 300–400 nm (fourth bin) and at >500 nm (sixth bin) were significantly increased (for granules within 300–400 nm, 87.2 ± 8.0% and 81.9 ± 3.7% in LG- and HG-treated control cells, respectively, vs. 144.0 ± 8.3% and 123.1 ± 11.0% in LG- and HG-treated mutant cells, respectively; for granules at >500 nm, 91.7 ± 4.0% in HG-treated control cells vs. 104.3 ± 1.2% in HG-treated mutant cells). Although the mean granule diameter was slightly larger in the mutant β cells (324.9 and 339.4 nm in LG- and HG-treated control cells, respectively, vs. 345.7 and 377.6 nm in LG- and HG-treated mutant cells, respectively; P < 0.001), these 6–11% changes should not affect the granule locations. The mean granule density was significantly decreased in the HG-treated mutant β cells (3.81 ± 0.31 in control cells vs. 2.80 ± 0.19 in mutant cells; Fig. 5 D), which could be related to the enhanced insulin secretion. These results strongly indicate that granuphilin is essential for the docking of insulin granules to the plasma membrane.

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