Sebastian Barg

Publications (44) View all

• Source

  Available from: Patrick MacDonald

  Article: Multivesicular exocytosis in rat pancreatic beta cells.

  M B Hoppa, E Jones, J Karanauskaite, R Ramracheya, M Braun, S C Collins, Q Zhang, A Clark, L Eliasson, C Genoud, P E Macdonald, A G Monteith, S Barg, J Galvanovskis, P Rorsman
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  ABSTRACT: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 μmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.
  Diabetologia 12/2011; 55(4):1001-12. · 6.81 Impact Factor
• Source

  Available from: pnas.org

  Article: Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers.

  M K Knowles, S Barg, L Wan, M Midorikawa, X Chen, Wolfhard Almers
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  ABSTRACT: Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.
  Proceedings of the National Academy of Sciences 11/2010; 107(48):20810-5. · 9.68 Impact Factor
• Source

  Available from: pnas.org

  Article: Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

  S Barg, M K Knowles, X Chen, M Midorikawa, Wolfhard Almers
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  ABSTRACT: Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.
  Proceedings of the National Academy of Sciences 11/2010; 107(48):20804-9. · 9.68 Impact Factor
• Article: Compensatory endocytosis in chromaffin cells.

  S Barg, J D Machado
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  ABSTRACT: Exocytosis occurs via fusion of secretory granules with the cell membrane, whereupon the granule content is at least partially released and the granule membrane is temporarily added to the plasma membrane. Exocytosis is balanced by compensatory endocytosis to achieve net equilibrium of the cell surface area and to recycle and redistribute components of the exocytosis machinery. The underlying molecular mechanisms remain a matter of debate. In this review, we summarize and discuss recent progress in the understanding of compensatory endocytosis, with the focus on chromaffin cells as a useful model for studying mechanisms of regulated secretion.
  Acta Physiologica 03/2008; 192(2):195-201. · 3.09 Impact Factor
• Article: Characterization of a naturally occurring mutation (L107I) in the HNF1 alpha (MODY3) gene.

  C Cervin, M Orho-Melander, M Ridderstråle, M Lehto, S Barg, L Groop, C M Cilio
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  ABSTRACT: Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1 alpha. In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1 alpha gene. Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments. Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1 alpha in HeLa cells lacking HNF1 alpha. The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1 alpha suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1alpha protein showed normal nuclear targeting but impaired binding to an HNF1 alpha consensus sequence. Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism.
  Diabetologia 01/2003; 45(12):1703-8. · 6.81 Impact Factor