| 1. |
- Gandasi, Nikhil R, et al.
(author)
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Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
- 2018
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In: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 27:2, s. 470-478
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Journal article (peer-reviewed)abstract
- Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.
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| 2. |
- Yin, Peng, 1982-
(author)
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Architecture and function of the insulin granule secretion machinery
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Insulin is released into the blood stream to normalize elevated blood glucose, for example after a meal. The hormone is synthesized by β-cells in the endocrine pancreas, and stored in small vesicles, known as secretory granules, until required. When glucose is elevated, these granules undergo regulated exocytosis and thereby secrete the hormone. The primary trigger for this is a glucose-dependent elevation in cytosolic Ca2+, which enters the cell through voltage-gated Ca2+ channels. Glucose-stimulated insulin secretion follows a biphasic timecourse, with a rapid 1st phase that lasts for a few minutes, followed by a slowly developing sustained 2nd phase. Compromised 1st phase secretion is an early sign of developing type-2 diabetes. Biphasic secretion is thought reflect the vastly different probabilities of individual insulin granules, but direct evidence for this is still lacking. In this thesis, high resolution TIRF microscopy was used to identify rate limiting steps for insulin granule exocytosis in health and in type-2 diabetes, and to understand these steps at the molecular level. It is shown that granule docking is critical for sustained insulin secretion. In β-cells from type-2 diabetic donors, docking is compromised and no longer responsive to glucose. Expression analysis in a large donor cohort suggests that this is due to decreased expression of proteins involved in the docking step. One of these proteins, the SNARE protein syntaxin-1, is well-known to cluster at the site of docked granules, which initiates the formation of functional release sites. Analysis using a series of syntaxin-1 mutations indicates that this clustering depends on specific features in its N-terminal Habc domain and involves binding of the S/M protein munc-18. The data suggest that the closed conformation of syntaxin-1 mediates the interaction between granule and plasma membrane. Finally, it is shown that voltage-gated L-type Ca2+ channels are slowly recruited to the sites of docked granules, which depends on interaction with the granule priming factor Munc13. This arrangement leads to localized the Ca2+ influx near a subset of the docked granules, which dramatically increases their release probability. Importantly, the interaction between Ca2+ channels and granules fails in type-2 diabetic β cells. In summary, the thesis highlights the importance of the spatial organization of the secretory machinery for adequate insulin secretion, and suggests that defects in this process partly underlie the disturbed blood glucose regulation in type-2 diabetes.
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| 3. |
- Yin, Peng, 1982-, et al.
(author)
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Syntaxin clusters at secretory granules in a munc18-bound conformation
- 2018
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 29:22, s. 2700-2708
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Journal article (peer-reviewed)abstract
- Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1-bound conformation, providing a rationale for the requirement for both proteins for granule docking.
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| 4. |
- Yin, Peng, 1982-, et al.
(author)
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Syntaxin clusters at secretory granules in its closed munc18-bound conformation
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Other publication (other academic/artistic)abstract
- Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, the protein is recruited specifically into small clusters at the granule docking site. Here we determined structural features that allow interaction of stx-1 with the docking site. We find in live insulin-secreting cells that stx-1 and stx-3 (but not stx-4 or stx-11) accumulate at docked granules, and that stx-1 (but not stx-4) rescues docking in cells expressing botulinum toxin-C. Using a series of stx-1 deletion mutants and stx-1/4 chimeras we provide evidence that all four helical domains (Ha, Hb, Hc, H3) and the short N-peptide contribute to the recruitment to granules. However, only the Hc domain confers specificity and must be derived from stx-1 for recruitment to occur. We show further that mutations interfering with binding of stx-1’s Habc-domain or N-peptide to munc18-1 prevent recruitment and docking. We conclude that stx-1 (and -3) are recruited to the docking site in the closed, munc18-1 bound conformation, which provides a rationale for the requirement of both proteins during granule docking.
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