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  • 1. Barig, Susann
    et al.
    Alisch, Romy
    Nieland, Susanne
    Wuttke, Anne
    Graeser, Yvonne
    Huddar, Mahesh
    Schnitzlein, Klaus
    Stahmann, Klaus-Peter
    Monoseptic growth of fungal lipase producers under minimized sterile conditions: Cultivation of Phialemonium curvatum in 350 L scale2011In: Engineering in Life Sciences, ISSN 1618-0240, E-ISSN 1618-2863, Vol. 11, no 4, p. 387-394Article in journal (Refereed)
  • 2. Bergenholtz, Sa Schoug
    et al.
    Wessman, Per
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Håkansson, Sebastian
    A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying2012In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 64, no 3, p. 152-159Article in journal (Refereed)
    Abstract [en]

    Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.

  • 3.
    Dyachok, Oleg
    et al.
    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.
    Sågetorp, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Arrieumerlou, Cecile
    Infection Biology, Biozentrum, University of Basel, Switzerland.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gylfe, Erik
    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.
    Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion2008In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 8, no 1, p. 26-37Article in journal (Refereed)
    Abstract [en]

    Cyclic AMP (cAMP) and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting β-cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the detailed kinetics of cAMP signalling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the sub-membrane space of single MIN6-cells and primary mouse β-cells. These oscillations were preceded and enhanced by elevations of [Ca2+]i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca2+]i rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP, and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

  • 4.
    Mokhtari, Dariush
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Al-Amin, Abdullah
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Turpaev, Kyrill
    Li, Tingting
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Idevall-Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Li, Jia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fred, Rikard G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ravassard, Philippe
    Scharfmann, R
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl2013In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, no 6, p. 1327-1338Article in journal (Refereed)
    Abstract [en]

    AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

  • 5.
    Tengholm, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Autocrine signals mediate plasma membrane translocation of protein kinase C in insulin-secreting cells2013In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, p. S207-S207Article in journal (Other academic)
  • 6.
    Thore, Sophia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Wuttke, Anne
    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.
    Rapid turnover of phosphatidylinositol-4,5-bisphosphate in insulin-secreting cells mediated by Ca2+ and the ATP-to-ADP ratio2007In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 56, no 3, p. 818-826Article in journal (Refereed)
    Abstract [en]

    Phosphatidylinositol-4,5-bisphosphate (PIP2) is important for a variety of cellular processes as a precursor for second messengers and by regulating ion channels, the cytoskeleton, and vesicle traffic in many types of cells, including insulin-secreting β-cells. Here, we applied evanescent wave microscopy and the PIP2-binding pleckstrin homology domain from phospholipase C (PLC)-δ fused to the green fluorescent protein to characterize the regulation of plasma membrane PIP2 in individual insulin-secreting MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mmol/l evoked antisynchronous oscillations of [PIP2] and cytoplasmic Ca2+concentration, consistent with PLC being periodically activated by the voltage-dependent Ca2+ influx. The effect of adenine nucleotides on [PIP2] was studied in cells permeabilized with α-toxin. ATP dose- dependently stimulated PIP2 synthesis with half-maximal effect at 300 μmol/l. Omission of the nucleotide resulted in rapid loss of PIP2 with t1/2 < 40 s. ADP also stimulated PIP2 formation, but this effect reflected local ATP formation and was prevented by the adenylate kinase inhibitor diadenosine-pentaphosphate. The ATP-induced PIP2 synthesis was counteracted by the ADP analog adenosine-5′-O-2-thiodiphosphate. We conclude that plasma membrane PIP2 is dynamically regulated by intracellular Ca2+ and the ATP-to-ADP ratio in insulin-secreting cells. The rapid turnover allows maintenance of PIP2 levels while generating second messengers of critical importance for insulin secretion.

  • 7.
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lipid Signalling Dynamics at the beta-cell Plasma Membrane2015In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 116, no 4, p. 281-290Article, review/survey (Refereed)
    Abstract [en]

    Pancreatic -cells are clustered in islets of Langerhans and secrete insulin in response to increased concentrations of circulating glucose. Insulin in turn acts on liver, muscle and fat tissue to store energy and normalize the blood glucose level. Inappropriate insulin release may lead to impaired glucose tolerance and diabetes. In addition to glucose, other nutrients, neural stimuli and hormonal stimuli control insulin secretion. Many of these signals are perceived at the plasma membrane, which is also the site where insulin granules undergo exocytosis. Therefore, it is not surprising that membrane lipids play an important role in the regulation of insulin secretion. -cells release insulin in a pulsatile fashion. Signalling lipids integrate the nutrient and neurohormonal inputs to fine-tune, shape and co-ordinate the pulsatility. An important group of signalling lipids are phosphoinositides and their downstream messengers. This MiniReview will discuss new insights into lipid signalling dynamics in -cells obtained from live-cell imaging experiments with fluorescent translocation biosensors. The plasma membrane concentration of several phosphoinositides and of their downstream messengers changes rapidly upon nutrient or neurohormonal stimulation. Glucose induces the most complex spatio-temporal patterns, typically involving oscillations of messenger concentrations, which sometimes are locally restricted. The tightly controlled levels of lipid messengers can mediate specific binding of downstream effectors to the plasma membrane, contributing to the appropriate regulation of insulin secretion.

  • 8.
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lipid Signalling Dynamics in Insulin-secreting β-cells2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.

    List of papers
    1. Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells
    Open this publication in new window or tab >>Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells
    2010 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 123, no 9, p. 1492-1502Article in journal (Refereed) Published
    Abstract [en]

    Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P-2 concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5)P-2. We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P-2 dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P-2 induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca2+ entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca2+ independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca2+- and PKC-dependent changes in the plasma-membrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P-2. These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P-2 pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.

    Keywords
    Ca2+, Glucose, Insulin-secreting cells, Oscillations, PI4-kinase, PtdIns(4)P, PtdIns(4, 5)P-2
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-136347 (URN)10.1242/jcs.060525 (DOI)000276912300013 ()
    Available from: 2010-12-11 Created: 2010-12-11 Last updated: 2018-01-12Bibliographically approved
    2. P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion
    Open this publication in new window or tab >>P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion
    2013 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 27, no 4, p. 1610-1620Article in journal (Refereed) Published
    Abstract [en]

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic beta cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse beta cells, and human beta cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 +/- 0.4 s) but high-amplitude elevations (up to 109 +/- 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y(1), but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinctmicrodomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.-Wuttke, A., Idevall-Hagren, O., Tengholm, A. P2Y(1) receptor-dependent diacylglycerol signaling microdomains in beta cells promote insulin secretion. 

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-198038 (URN)10.1096/fj.12-221499 (DOI)000316940800031 ()
    Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2018-01-11Bibliographically approved
    3. Autocrine signalling underlies fast repetitive plasma membrane translocation of protein kinase C in β-cells
    Open this publication in new window or tab >>Autocrine signalling underlies fast repetitive plasma membrane translocation of protein kinase C in β-cells
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-198040 (URN)
    Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2018-01-11
    4. Continuous imaging of exocytosis in β-cells reveals negative feedback of insulin
    Open this publication in new window or tab >>Continuous imaging of exocytosis in β-cells reveals negative feedback of insulin
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-198041 (URN)
    Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2018-01-11
  • 9.
    Wuttke, Anne
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Emanuelsson, H.
    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.
    Glucose and muscarinic stimulation trigger distinct diacylglycerol signals in pancreatic beta cells2010Conference paper (Other academic)
  • 10.
    Wuttke, Anne
    et al.
    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.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Exocytotic release of ATP triggers diacylglycerol spiking in insulin-secreting cells2011In: Minutes Of The 46th General Assembly Of The European Association For The Study Of Diabetes: held in Stockholmsmässan, Stockholm, Sweden on 23 September 2010, 2011, p. S194-S195Conference paper (Other academic)
  • 11.
    Wuttke, Anne
    et al.
    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.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Imaging phosphoinositide dynamics in living cells2010In: Inositol Phosphates and Lipids: Methods and Protocols / [ed] Christopher J. Barker, Humana Press , 2010, Vol. 645, p. 219-235Chapter in book (Other academic)
    Abstract [en]

    To improve our understanding of the important roles played by inositol lipid derivatives in signalling and other cellular processes, it is crucial to measure phosphoinositide concentration changes in individual cells with high spatial and temporal resolution. A number of protein domains that interact with inositol lipids in a specific manner have been identified. Tagged with the green fluorescent protein or its colour variants, these protein modules can be used as probes to visualize various phosphoinositide species in different sub-cellular compartments. Here, we present protocols for fluorescence imaging of phosphoinositide dynamics in single living cells. Total internal reflection fluorescence microscopy is particularly powerful for time-lapse recordings of phosphoinositides in the plasma membrane. We demonstrate how this technique can be used to record phospholipase C- and PI3-kinase-induced changes in inositol lipids in insulin-secreting cells. These procedures should be applicable to studies of the spatio-temporal regulation of phosphoinositide metabolism in many types of cells.

  • 12.
    Wuttke, Anne
    et al.
    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.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion2013In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 27, no 4, p. 1610-1620Article in journal (Refereed)
    Abstract [en]

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic beta cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse beta cells, and human beta cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 +/- 0.4 s) but high-amplitude elevations (up to 109 +/- 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y(1), but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinctmicrodomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.-Wuttke, A., Idevall-Hagren, O., Tengholm, A. P2Y(1) receptor-dependent diacylglycerol signaling microdomains in beta cells promote insulin secretion. 

  • 13.
    Wuttke, Anne
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sagetorp, J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Nils
    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.
    Type III phosphatidylinositol 4-kinases mediate plasma membrane PIP2 synthesis in insulin-secreting cells2007In: 43rd easd annual meeting of the european association for the study of diabetes: Amsterdam, The Netherlands, 18 – 21 September 2007, 2007, Vol. 50, p. S216-S216Conference paper (Other academic)
  • 14.
    Wuttke, Anne
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sågetorp, Jenny
    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.
    Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells2010In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 123, no 9, p. 1492-1502Article in journal (Refereed)
    Abstract [en]

    Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P-2 concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5)P-2. We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P-2 dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P-2 induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca2+ entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca2+ independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca2+- and PKC-dependent changes in the plasma-membrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P-2. These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P-2 pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.

  • 15.
    Wuttke, Anne
    et al.
    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.
    In situ characterisation of plasma membrane PtdIns(4)P and PtdIns(4,5)P 2 turnover in individual insulin secreting cells2008Conference paper (Other academic)
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    Wuttke, Anne
    et al.
    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.
    Secretagogue-induced elevation of the plasma membrane PtdIns4P concentration in individual insulin-secreting cells2009In: 45th Annual Meeting of the European-Association-for-the-Study-of-Diabetes, Vienna, SEP 30-OCT 02, 2009, 2009, Vol. 52, p. S181-S181Conference paper (Other academic)
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