Olof Idevall-Hagren

PI(4,5)P2-Dependent and Ca2+-Regulated ER-PM Interactions Mediated by the Extended Synaptotagmins

Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.

Optogenetic control of phosphoinositide metabolism

Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptaseOCRL), which acts on PI(4,5)P2 and PI(3,4,5)P3, was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458–488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptaseOCRL to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P2 and PI(3,4,5)P3 biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptaseOCRL recruitment and PI(4,5)P2 dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P2 biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P3 synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial–temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.

Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion

Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](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.

cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated β-cells is driven by oscillations of the Ca2+ and cAMP concentrations in the subplasma membrane space ([Ca2+]pm and [cAMP]pm). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP]pm, [Ca2+]pm, and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca2+]pm, [cAMP]pm, and PIP3. Inhibitors of protein kinase A (PKA) markedly diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The reduced PIP3 response could be attributed to accelerated depolarization causing early rise of [Ca2+]pm that preceded the elevation of [cAMP]pm. However, the amplitude of the PIP3 response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP3 oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP3 oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca2+]pm and amplifying [cAMP]pm signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

Triggered Ca2+ influx is required for extended synaptotagmin 1‐induced ER‐plasma membrane tethering

The extended synaptotagmins (E‐Syts) are ER proteins that act as Ca2+‐regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca2+ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi‐intact cells to buffers of variable Ca2+ concentrations, we found that binding of E‐Syt1 to the PI(4,5)P2‐rich PM critically requires its C2C and C2E domains and that the EC50 of such binding is in the low micromolar Ca2+ range. Accordingly, E‐Syt1 accumulation at ER‐PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca2+ via its influx from the extracellular medium, such as store‐operated Ca2+ entry in fibroblasts and membrane depolarization in β‐cells. We also show that in spite of their very different physiological functions, membrane tethering by E‐Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca2+.

PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading.

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane-substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5-10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells. We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.

Detection and manipulation of phosphoinositides

Phosphoinositides (PIs) are minor components of cell membranes, but play key roles in cell function. Recent refinements in techniques for their detection, together with imaging methods to study their distribution and changes, have greatly facilitated the study of these lipids. Such methods have been complemented by the parallel development of techniques for the acute manipulation of their levels, which in turn allow bypassing the long-term adaptive changes implicit in genetic perturbations. Collectively, these advancements have helped elucidate the role of PIs in physiology and the impact of the dysfunction of their metabolism in disease. Combining methods for detection and manipulation enables the identification of specific roles played by each of the PIs and may eventually lead to the complete deconstruction of the PI signaling network. Here, we review current techniques used for the study and manipulation of cellular PIs and also discuss advantages and disadvantages associated with the various methods. This article is part of a Special Issue entitled Phosphoinositides.

P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion

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 β cells and determined whether DAG signaling is involved in secretagogue‐induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β 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 P2Y1, 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 distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane‐associated processes.—Wuttke, A., Idevall‐Hagren, O., Tengholm, A. P2Y1 receptor‐dependent diacylglycerol signaling microdomains in β cells promote insulin secretion. FASEB J. 27, 1610–1620 (2013). www.fasebj.org

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-Abl

Aims/hypothesisIt 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.MethodsHuman 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.ResultsImatinib 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/interpretationImatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic β Cells

By oscillating between the plasma membrane and the cytosol, Epac2 promotes pulses of insulin secretion. Sugar-Induced Oscillations Glucose stimulation of pancreatic β cells triggers increases in the concentrations of adenosine 3′,5′-monophosphate (cAMP) and Ca2+, which activate various signaling pathways that culminate in pulses of insulin release. The guanine nucleotide exchange factor Epac2 promotes insulin secretion through various mechanisms, including activation of the small guanosine triphosphatase Rap1. Idevall-Hagren et al. performed imaging and biochemical analyses in a pancreatic β cell line and β cells in isolated islets and found that glucose stimulation resulted in translocation of Epac2 from the cytosol to the plasma membrane. This translocation occurred in a cyclical, oscillatory manner that corresponded to and required oscillations in the concentrations of cAMP, which directly activates Epac2, and in Ca2+, which indirectly activates Ras, a protein that recruits Epac2 to the plasma membrane. Activation of Rap1 at the plasma membrane also occurred in an oscillatory manner. Thus, the pulsatile release of insulin from pancreatic β cells involves oscillating signals that include oscillations in the plasma membrane localization of Epac2. The cAMP (adenosine 3′,5′-monophosphate)–activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting β cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single β cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic β cells.