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  • 1.
    Gandasi, Nikhil R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Vesto, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Helou, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Saras, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, article id e0127801Article in journal (Refereed)
    Abstract [en]

    Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.

  • 2.
    Gandasi, Nikhil R
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ottosson Laakso, Emilia
    Lund University Diabetes Centre, Malmö, Sweden.
    Vikman, Petter
    Lund University Diabetes Centre, Malmö, Sweden.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes2018In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 2, p. 470-478Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Gandasi, Nikhil R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Riz, Michela
    Univ Padua, Dept Informat Engn, Padua, Italy..
    Chibalina, Margarita V
    Univ Oxford, Oxford Ctr Diabet Endocrinol & Metab, Oxford, England..
    Cortese, Giuliana
    Univ Padua, Dept Stat Sci, Padua, Italy..
    Lund, Per-Eric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Matveev, Victor
    New Jersey Inst Technol, Dept Math Sci, Newark, NJ 07102 USA..
    Rorsman, Patrik
    Univ Oxford, Oxford Ctr Diabet Endocrinol & Metab, Oxford, England..
    Sherman, Arthur
    NIDDK, Lab Biol Modeling, NIH, Bethesda, MD 20892 USA..
    Pedersen, Morten G
    Univ Padua, Dept Informat Engn, Padua, Italy..
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes2017In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 127, no 6, p. 2353-2364Article in journal (Refereed)
    Abstract [en]

    Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

  • 4.
    Yin, Peng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Arora, Swati
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Saras, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Syntaxin clusters at secretory granules in a munc18-bound conformation2018In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 29, no 22, p. 2700-2708Article in journal (Refereed)
    Abstract [en]

    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.

1 - 4 of 4
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