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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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Schematic model for docked granules. Morphologically docked granules, whose centers reside within 200 nm of the plasma membrane in this figure, consist of molecularly docked granules (red) and those just stochastically located close to the plasma membrane (blue). Granuphilin links insulin granules to the fusion-incompetent, closed form of syntaxin-1a, although it is currently unknown whether it interacts with free syntaxin-1a or the syntaxin-1a–Munc18-1 complex. Such molecularly docked granules need to be primed for fusion. The priming reaction somehow disassembles the docking protein complex and enables syntaxin-1a to adopt an open configuration and thus to form a complex with other SNARE proteins. In response to a secretory stimulus, primed molecularly docked granules (some of the red granules), molecularly undocked but morphologically docked granules (blue), and even undocked granules (black) can be released from a region where secretagogue-dependent signals extend (inside of the dashed line). Although there are no molecularly docked granules (red), evoked exocytosis is augmented in granuphilin-deficient cells, indicating that molecular docking is inhibitory for fusion.
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fig9: Schematic model for docked granules. Morphologically docked granules, whose centers reside within 200 nm of the plasma membrane in this figure, consist of molecularly docked granules (red) and those just stochastically located close to the plasma membrane (blue). Granuphilin links insulin granules to the fusion-incompetent, closed form of syntaxin-1a, although it is currently unknown whether it interacts with free syntaxin-1a or the syntaxin-1a–Munc18-1 complex. Such molecularly docked granules need to be primed for fusion. The priming reaction somehow disassembles the docking protein complex and enables syntaxin-1a to adopt an open configuration and thus to form a complex with other SNARE proteins. In response to a secretory stimulus, primed molecularly docked granules (some of the red granules), molecularly undocked but morphologically docked granules (blue), and even undocked granules (black) can be released from a region where secretagogue-dependent signals extend (inside of the dashed line). Although there are no molecularly docked granules (red), evoked exocytosis is augmented in granuphilin-deficient cells, indicating that molecular docking is inhibitory for fusion.

Mentions: In summary, we demonstrated increased evoked exocytosis despite a severely reduced docking of insulin granules in granuphilin-deficient pancreatic β cells. The existence of a predocked vesicle pool is a hallmark of regulated exocytosis, in which constitutive fusion of incoming vesicles is inhibited. The prevailing view has been that a readily releasable pool of vesicles resides within the docked pool that has been primed (Burgoyne and Morgan, 2003; Rorsman and Renström, 2003), although the definition of the pool is often vague, especially in the case of secretory granules, whose release has a relatively slow onset and extends over a relatively long time scale. Our model is schematically shown in Fig. 9, which demonstrates that morphologically docked granules are composed of heterogeneous populations: those just stochastically located close to the membrane and those molecularly docked to the fusion machinery. The docking machinery in regulated exocytosis not only promotes vesicles in apposition to the plasma membrane but simultaneously imposes a temporal constraint to inhibit subsequent fusion. The priming reaction, which is also unique to regulated exocytosis, may be only rate limiting for molecularly docked vesicles to release the docking constraint. Thus, a readily, if not immediately, releasable pool cannot be defined morphologically. Besides imposing the need for priming on docked vesicles, docking occupies a part of the fusion machinery and thereby may further restrict the fusion of incoming undocked vesicles. As such, docking likely contributes to the fine-tuning of secretion in response to external stimuli. Finally, the presence of enhanced glucose tolerance in granuphilin- mice suggests that the modification of granuphilin function should be explored as a novel pharmaceutical target strategy for the treatment of diabetes.


Granuphilin molecularly docks insulin granules to the fusion machinery.

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

Schematic model for docked granules. Morphologically docked granules, whose centers reside within 200 nm of the plasma membrane in this figure, consist of molecularly docked granules (red) and those just stochastically located close to the plasma membrane (blue). Granuphilin links insulin granules to the fusion-incompetent, closed form of syntaxin-1a, although it is currently unknown whether it interacts with free syntaxin-1a or the syntaxin-1a–Munc18-1 complex. Such molecularly docked granules need to be primed for fusion. The priming reaction somehow disassembles the docking protein complex and enables syntaxin-1a to adopt an open configuration and thus to form a complex with other SNARE proteins. In response to a secretory stimulus, primed molecularly docked granules (some of the red granules), molecularly undocked but morphologically docked granules (blue), and even undocked granules (black) can be released from a region where secretagogue-dependent signals extend (inside of the dashed line). Although there are no molecularly docked granules (red), evoked exocytosis is augmented in granuphilin-deficient cells, indicating that molecular docking is inhibitory for fusion.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Schematic model for docked granules. Morphologically docked granules, whose centers reside within 200 nm of the plasma membrane in this figure, consist of molecularly docked granules (red) and those just stochastically located close to the plasma membrane (blue). Granuphilin links insulin granules to the fusion-incompetent, closed form of syntaxin-1a, although it is currently unknown whether it interacts with free syntaxin-1a or the syntaxin-1a–Munc18-1 complex. Such molecularly docked granules need to be primed for fusion. The priming reaction somehow disassembles the docking protein complex and enables syntaxin-1a to adopt an open configuration and thus to form a complex with other SNARE proteins. In response to a secretory stimulus, primed molecularly docked granules (some of the red granules), molecularly undocked but morphologically docked granules (blue), and even undocked granules (black) can be released from a region where secretagogue-dependent signals extend (inside of the dashed line). Although there are no molecularly docked granules (red), evoked exocytosis is augmented in granuphilin-deficient cells, indicating that molecular docking is inhibitory for fusion.
Mentions: In summary, we demonstrated increased evoked exocytosis despite a severely reduced docking of insulin granules in granuphilin-deficient pancreatic β cells. The existence of a predocked vesicle pool is a hallmark of regulated exocytosis, in which constitutive fusion of incoming vesicles is inhibited. The prevailing view has been that a readily releasable pool of vesicles resides within the docked pool that has been primed (Burgoyne and Morgan, 2003; Rorsman and Renström, 2003), although the definition of the pool is often vague, especially in the case of secretory granules, whose release has a relatively slow onset and extends over a relatively long time scale. Our model is schematically shown in Fig. 9, which demonstrates that morphologically docked granules are composed of heterogeneous populations: those just stochastically located close to the membrane and those molecularly docked to the fusion machinery. The docking machinery in regulated exocytosis not only promotes vesicles in apposition to the plasma membrane but simultaneously imposes a temporal constraint to inhibit subsequent fusion. The priming reaction, which is also unique to regulated exocytosis, may be only rate limiting for molecularly docked vesicles to release the docking constraint. Thus, a readily, if not immediately, releasable pool cannot be defined morphologically. Besides imposing the need for priming on docked vesicles, docking occupies a part of the fusion machinery and thereby may further restrict the fusion of incoming undocked vesicles. As such, docking likely contributes to the fine-tuning of secretion in response to external stimuli. Finally, the presence of enhanced glucose tolerance in granuphilin- mice suggests that the modification of granuphilin function should be explored as a novel pharmaceutical target strategy for the treatment of diabetes.

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