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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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Microscopic inspection of pancreatic islets. (A) Immunofluorescent detection of insulin (green) and glucagon (red) in frozen sections (20 μm) of pancreas. Bar, 20 μm. (B) Morphometric analyses of islet size distribution and β cell mass. Total numbers of islets analyzed were 1,533 from three Grn+/Y mice (open bars) and 1,510 from three Grn−/Y mice (closed bars). (C) The numbers of total and docked insulin granules per single β cell section, the β cell size, and the granule density (granule number/cytosol area) in ultrathin sections (90 nm). 21 β cells were analyzed from each of three 12- to 20-wk-old male Grn+/Y (open bars) and Grn−/Y mice (closed bars). *, P < 0.0001. (D) Electron micrographs of pancreatic tissue section. The plasma membrane of a single β cell is framed with a black line, and docked granules whose contours touch the plasma membrane are marked by red arrowheads. Bar, 1 μm. Results are provided as mean ± SEM.
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fig3: Microscopic inspection of pancreatic islets. (A) Immunofluorescent detection of insulin (green) and glucagon (red) in frozen sections (20 μm) of pancreas. Bar, 20 μm. (B) Morphometric analyses of islet size distribution and β cell mass. Total numbers of islets analyzed were 1,533 from three Grn+/Y mice (open bars) and 1,510 from three Grn−/Y mice (closed bars). (C) The numbers of total and docked insulin granules per single β cell section, the β cell size, and the granule density (granule number/cytosol area) in ultrathin sections (90 nm). 21 β cells were analyzed from each of three 12- to 20-wk-old male Grn+/Y (open bars) and Grn−/Y mice (closed bars). *, P < 0.0001. (D) Electron micrographs of pancreatic tissue section. The plasma membrane of a single β cell is framed with a black line, and docked granules whose contours touch the plasma membrane are marked by red arrowheads. Bar, 1 μm. Results are provided as mean ± SEM.

Mentions: We performed morphological analyses of the pancreatic islets. Insulin and glucagon double immunofluorescent labeling showed a typical mantle structure between the α and β cells in the mutant islets (Fig. 3 A). Morphometric analysis on paraffin-embedded pancreatic tissue sections that had been immunostained with antiinsulin antibody revealed that the number and size distribution of the islets and the β cell mass in the sized islets were not different between the mutant and control tissues (Fig. 3 B). However, the ultrastructure of the mutant β cells showed a deficit in insulin-granule docking at the plasma membrane (Fig. 3 D). The number of docked granules was significantly reduced in the granuphilin- β cells (40 vs. 13 in control and mutant β cells, respectively; P < 0.0001), whereas the granule number per cell section, the β cell size, and the average granule density were unchanged (304 vs. 319 granule number, 82.1 vs. 86.1 μm2 cell size, 4.5 vs. 4.6 granules per μm2 cytosol area of control and mutant β cells, respectively; Fig. 3 C).


Granuphilin molecularly docks insulin granules to the fusion machinery.

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

Microscopic inspection of pancreatic islets. (A) Immunofluorescent detection of insulin (green) and glucagon (red) in frozen sections (20 μm) of pancreas. Bar, 20 μm. (B) Morphometric analyses of islet size distribution and β cell mass. Total numbers of islets analyzed were 1,533 from three Grn+/Y mice (open bars) and 1,510 from three Grn−/Y mice (closed bars). (C) The numbers of total and docked insulin granules per single β cell section, the β cell size, and the granule density (granule number/cytosol area) in ultrathin sections (90 nm). 21 β cells were analyzed from each of three 12- to 20-wk-old male Grn+/Y (open bars) and Grn−/Y mice (closed bars). *, P < 0.0001. (D) Electron micrographs of pancreatic tissue section. The plasma membrane of a single β cell is framed with a black line, and docked granules whose contours touch the plasma membrane are marked by red arrowheads. Bar, 1 μm. Results are provided as mean ± SEM.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171228&req=5

fig3: Microscopic inspection of pancreatic islets. (A) Immunofluorescent detection of insulin (green) and glucagon (red) in frozen sections (20 μm) of pancreas. Bar, 20 μm. (B) Morphometric analyses of islet size distribution and β cell mass. Total numbers of islets analyzed were 1,533 from three Grn+/Y mice (open bars) and 1,510 from three Grn−/Y mice (closed bars). (C) The numbers of total and docked insulin granules per single β cell section, the β cell size, and the granule density (granule number/cytosol area) in ultrathin sections (90 nm). 21 β cells were analyzed from each of three 12- to 20-wk-old male Grn+/Y (open bars) and Grn−/Y mice (closed bars). *, P < 0.0001. (D) Electron micrographs of pancreatic tissue section. The plasma membrane of a single β cell is framed with a black line, and docked granules whose contours touch the plasma membrane are marked by red arrowheads. Bar, 1 μm. Results are provided as mean ± SEM.
Mentions: We performed morphological analyses of the pancreatic islets. Insulin and glucagon double immunofluorescent labeling showed a typical mantle structure between the α and β cells in the mutant islets (Fig. 3 A). Morphometric analysis on paraffin-embedded pancreatic tissue sections that had been immunostained with antiinsulin antibody revealed that the number and size distribution of the islets and the β cell mass in the sized islets were not different between the mutant and control tissues (Fig. 3 B). However, the ultrastructure of the mutant β cells showed a deficit in insulin-granule docking at the plasma membrane (Fig. 3 D). The number of docked granules was significantly reduced in the granuphilin- β cells (40 vs. 13 in control and mutant β cells, respectively; P < 0.0001), whereas the granule number per cell section, the β cell size, and the average granule density were unchanged (304 vs. 319 granule number, 82.1 vs. 86.1 μm2 cell size, 4.5 vs. 4.6 granules per μm2 cytosol area of control and mutant β cells, respectively; Fig. 3 C).

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