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Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis
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 Medical Cell Biology.
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 Medical Cell Biology.
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, 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.

Place, publisher, year, edition, pages
2015. Vol. 10, no 6, e0127801
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-258769DOI: 10.1371/journal.pone.0127801ISI: 000356835000012PubMedID: 26091288OAI: oai:DiVA.org:uu-258769DiVA: diva2:842500
Funder
Swedish Research CouncilSwedish Diabetes Association
Available from: 2015-07-20 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Molecular mechanisms of biphasic insulin secretion
Open this publication in new window or tab >>Molecular mechanisms of biphasic insulin secretion
2015 (English)Doctoral 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1118
Keyword
Exocytosis, Insulin secretion, Membrane trafficking, Microscopy, Diabetes
National Category
Medical and Health Sciences
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-259071 (URN)978-91-554-9281-6 (ISBN)
Public defence
2015-09-11, B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2015-08-21 Created: 2015-07-26 Last updated: 2015-10-01

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Gandasi, Nikhil R.Helou, MariaYin, PengSaras, JanBarg, Sebastian

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