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  • 1.
    Alenkvist, Ida
    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.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis2017In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 66, no 10, p. 2610-2622Article in journal (Refereed)
    Abstract [en]

    Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including -cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the -cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

  • 2.
    Cortese, Giuliana
    et al.
    Univ Padua, Dept Stat Sci, Padua, Italy.
    Gandasi, Nikhil R.
    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.
    Pedersen, Morten Gram
    Univ Padua, Dept Informat Engn, Padua, Italy.
    Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic beta-cells2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0167282Article in journal (Refereed)
    Abstract [en]

    Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic beta-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level.

  • 3.
    Gandasi, Nikhil R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Molecular mechanisms of biphasic insulin secretion2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.

    List of papers
    1. Granule docking is rate limiting during sustained insulin secretion in human β-cells
    Open this publication in new window or tab >>Granule docking is rate limiting during sustained insulin secretion in human β-cells
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-259066 (URN)
    Available from: 2015-07-26 Created: 2015-07-26 Last updated: 2015-10-01Bibliographically approved
    2. Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site
    Open this publication in new window or tab >>Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site
    2014 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, article id 3914Article in journal (Refereed) Published
    Abstract [en]

    Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins' N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13's reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-226097 (URN)10.1038/ncomms4914 (DOI)000337504500001 ()24835618 (PubMedID)
    Available from: 2014-06-11 Created: 2014-06-11 Last updated: 2018-01-11Bibliographically approved
    3. Dynamic clustering of L-ˇ‐type Ca2+ -ˇ‐channels enables fast synchronized insulin secretion through localized Ca2+  nanodomains
    Open this publication in new window or tab >>Dynamic clustering of L-ˇ‐type Ca2+ -ˇ‐channels enables fast synchronized insulin secretion through localized Ca2+  nanodomains
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-259062 (URN)
    Available from: 2015-07-25 Created: 2015-07-25 Last updated: 2015-10-01Bibliographically approved
    4. Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis
    Open this publication in new window or tab >>Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis
    Show others...
    2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, article id e0127801Article in journal (Refereed) Published
    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.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-258769 (URN)10.1371/journal.pone.0127801 (DOI)000356835000012 ()26091288 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Diabetes Association
    Available from: 2015-07-20 Created: 2015-07-20 Last updated: 2019-02-01Bibliographically approved
  • 4.
    Gandasi, Nikhil R
    et al.
    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.
    Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, article id 3914Article in journal (Refereed)
    Abstract [en]

    Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins' N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13's reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors.

  • 5.
    Gandasi, Nikhil R.
    et al.
    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.
    Quantitative Imaging of the Exocytosis Machinery Assembly2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 102A-102AArticle in journal (Other academic)
  • 6.
    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.

  • 7.
    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.

  • 8.
    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.

  • 9.
    Gandasi R., Nikhil
    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.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Granule docking is rate limiting during sustained insulin secretion in human β-cellsManuscript (preprint) (Other academic)
  • 10.
    Gandasi R., Nikhil
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Riz, Michela
    Cortese, Guiliana
    Chibaliana, Margarita
    Rorsman, Patrik
    Sherman, Arthur
    Pedersen G, Morten
    Barg, Sebastian
    Dynamic clustering of L-ˇ‐type Ca2+ -ˇ‐channels enables fast synchronized insulin secretion through localized Ca2+  nanodomainsManuscript (preprint) (Other academic)
  • 11.
    Gucek, Alenka
    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.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bakke, Marit
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Doskeland, Stein O.
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Tengholm, Anders
    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.
    Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e41711Article in journal (Refereed)
    Abstract [en]

    Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2(-/-) (Rapgef4(-/-)) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

  • 12.
    Korol, Sergiy V
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Jin, Zhe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Jin, Yang
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Bhandage, Amol K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Tengholm, Anders
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Espes, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Laver, Derek
    University of Newcastle, Callaghan, Australia.
    Birnir, Bryndis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells2018In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30Article in journal (Refereed)
    Abstract [en]

    In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.

  • 13. Krus, Ulrika
    et al.
    King, Ben C
    Nagaraj, Vini
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sjölander, Jonatan
    Buda, Pawel
    Garcia-Vaz, Eliana
    Gomez, Maria F
    Ottosson-Laakso, Emilia
    Storm, Petter
    Fex, Malin
    Vikman, Petter
    Zhang, Enming
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Blom, Anna M
    Renström, Erik
    The Complement Inhibitor CD59 Regulates Insulin Secretion by Modulating Exocytotic Events2014In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 19, no 5, p. 883-890Article in journal (Refereed)
    Abstract [en]

    Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic β cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes.

  • 14. Krus, Ulrika
    et al.
    King, Benjamin C.
    Nagaraj, Vini
    Gandasi, Nikhil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sjolander, Jonatan
    Buda, Pawel
    Garcia-Vaz, Eliana
    Gomez, Maria
    Ottosson-Laakso, Emilia
    Storm, Petter
    Fex, Malin
    Vikman, Petter
    Zhang, Enming
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Blom, Anna M.
    Renstrom, Erik
    CD59 is required for pancreatic beta cell insulin exocytosis2014In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 61, no 2, p. 279-279Article in journal (Other academic)
  • 15.
    Nguyen, Phuoc My
    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.
    Idevall-Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    The lipid phosphatase INPP5F regulates insulin secretion2018In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 61, p. S197-S197Article in journal (Other academic)
  • 16.
    Nguyen, Phuoc My
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xie, Beichen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sugahara, Sari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Univ Tokyo, Grad Sch Pharmaceut Sci, Lab Hlth Chem, Tokyo, Japan.
    Xu, Yingke
    Zhejiang Univ, Dept Biomed Engn, Key Lab Biomed Engn,Minist Educ, Zhejiang Prov Key Lab Cardiocerebral Vasc Detect, Hangzhou, Zhejiang, Peoples R China.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    The PI(4)P phosphatase Sac2 controls insulin granule docking and release2019In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 218, no 11, p. 3714-3729Article in journal (Refereed)
    Abstract [en]

    Insulin granule biogenesis involves transport to, and stable docking at, the plasma membrane before priming and fusion. Defects in this pathway result in impaired insulin secretion and are a hallmark of type 2 diabetes. We now show that the phosphatidylinositol 4-phosphate phosphatase Sac2 localizes to insulin granules in a substrate-dependent manner and that loss of Sac2 results in impaired insulin secretion. Sac2 operates upstream of granule docking, since loss of Sac2 prevented granule tethering to the plasma membrane and resulted in both reduced granule density and number of exocytic events. Sac2 levels correlated positively with the number of docked granules and exocytic events in clonal beta cells and with insulin secretion in human pancreatic islets, and Sac2 expression was reduced in islets from type 2 diabetic subjects. Taken together, we identified a phosphoinositide switch on the surface on insulin granules that is required for stable granule docking at the plasma membrane and impaired in human type 2 diabetes.

  • 17.
    Omar-Hmeadi, Muhmmad
    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.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Plasma membrane PI(4,5)P-2 is critical for secretory granule exocytosis2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 18.
    Omar-Hmeadi, Muhmmad
    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.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    PtdIns(4,5)P2 is not required for secretory granule docking2018In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 19, no 6, p. 436-445Article in journal (Refereed)
    Abstract [en]

    Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P-2) by recruitment of a 5-phosphatase strongly inhibited Ca2+-dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by -toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P-2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

  • 19.
    Omar-Hmeadi, Muhmmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lund, Per-Eric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    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.
    Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetesManuscript (preprint) (Other academic)
  • 20.
    Salunkhe, V. A.
    et al.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Ofori, J.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Gandasi, Nikhil R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Salo, S. A.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden.;Roskilde Univ, Dept Sci Syst & Models, Roskilde, Denmark..
    Hansson, S.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Andersson, M. E.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Wendt, A.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Esguerra, J. L. S.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Eliasson, L.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    MiR-335 regulates exocytotic proteins and affects glucose-stimulated insulin secretion through decreased Ca2+-dependent exocytosis in beta cells2015In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no Suppl. 1, p. S128-S128Article in journal (Other academic)
  • 21.
    Salunkhe, Vishal A.
    et al.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Ofori, Jones K.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Salo, Sofia A.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Hansson, Sofia
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Andersson, Markus E.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Wendt, Anna
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Esguerra, Jonathan L. S.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Eliasson, Lena
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles2017In: Physiological Reports, E-ISSN 2051-817X, Vol. 5, no 21, article id e13493Article in journal (Refereed)
    Abstract [en]

    MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic beta-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates beta-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-beta H1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.

  • 22.
    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 - 22 of 22
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