RESUMEN
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.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinaptotagminas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestructura , Retículo Endoplásmico/química , Retículo Endoplásmico/ultraestructura , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Sinaptotagminas/química , Sinaptotagminas/genética , Levaduras/citología , Levaduras/metabolismoRESUMEN
The controlled release of mitochondrial content into the cytosol has emerged as one of the key steps in mitochondrial signaling. In particular, the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-ß) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) infection induces the release of mtDNA into the cytosol. The release of mtDNA is mediated by the viral minor capsid protein VI (pVI), which localizes to mitochondria. The presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release, whereas the viral E1B-19K protein blocked pVI-mediated mtDNA release. Surprisingly, the pVI-mediated mtDNA release did not increase but inhibited the IFN-ß gene expression. Notably, the pVI expression caused mitochondrial leakage of the HSP60 protein. The latter prevented specific phosphorylation of the interferon regulatory factor 3 (IRF3) needed for IFN-ß gene expression. Overall, we assign a new mitochondria and IFN-ß signaling-modulating function to the HAdV-C5 minor capsid protein VI. IMPORTANCE: Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.
Asunto(s)
Adenovirus Humanos , Proteínas de la Cápside , ADN Mitocondrial , Interferón beta , Mitocondrias , Transducción de Señal , Humanos , Adenovirus Humanos/fisiología , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Mitocondrias/metabolismo , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Interferón beta/metabolismo , Interferón beta/genética , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Infecciones por Adenovirus Humanos/virología , Infecciones por Adenovirus Humanos/metabolismo , Membranas Mitocondriales/metabolismo , Células HEK293 , Fosforilación , Citosol/metabolismo , Citosol/virologíaRESUMEN
Primary cilia are rod-like sensory organelles that protrude from the surface of most mammalian cells, including the cells of the islet, and mounting evidence supports important roles of these structures in the regulation of beta cell function and insulin secretion. The sensory abilities of the cilium arise from local receptor activation that is coupled to intrinsic signal transduction, and ciliary signals can propagate into the cell and influence cell function. Here, we review recent advances and studies that provide insights into intra-islet cues that trigger primary cilia signalling; how second messenger signals are generated and propagated within cilia; and how ciliary signalling affects beta cell function. We also discuss the potential involvement of primary cilia and ciliary signalling in the development and progression of type 2 diabetes, identify gaps in our current understanding of islet cell cilia function and provide suggestions on how to further our understanding of this intriguing structure.
Asunto(s)
Cilios , Diabetes Mellitus Tipo 2 , Animales , Humanos , Cilios/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal/fisiología , Mamíferos/metabolismoRESUMEN
Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic ß-cells, both lipid and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 (also known as C2CD2L) dynamically localizes to ER-PM contact sites and provides phosphatidylinositol, a precursor of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], to the PM. ß-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion, but the underlying mechanism is not known. We now show that TMEM24 only weakly interacts with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production.
Asunto(s)
Calcio , Proteínas de la Membrana , Membrana Celular , Retículo Endoplásmico/genética , Homeostasis , Proteínas de la Membrana/genéticaRESUMEN
Cancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration, which is typically reliant on bleb formation. Here we demonstrate that thrombin induces dynamic blebbing in the MDA-MB-231 breast cancer cell line and confirm that protease-activated receptor 1 (PAR1) activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we found that gentle contact compression, exerted using a custom built 'cell press' to mechanically stimulate cells, reduced thrombin-induced blebbing. Thrombin-induced blebbing was similarly attenuated using the small molecule Yoda1, an agonist of the mechanosensitive Ca2+ channel Piezo1, and this attenuation was impaired in Piezo1-depleted cells. Additionally, Piezo1 activation suppressed thrombin-induced phosphorylation of ezrin, radixin and moesin (ERM) proteins, which are implicated in the blebbing process. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.
Asunto(s)
Neoplasias de la Mama , Canales Iónicos , Movimiento Celular , Femenino , Humanos , Canales Iónicos/metabolismo , Fosforilación , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
The cytoplasmic Ca2+ concentration ([Ca2+]cyt) regulates a vast number of cellular functions, including insulin secretion from beta cells. The major physiological insulin secretagogue, glucose, triggers [Ca2+]cyt oscillations in beta cells. Synchronization of the oscillations among the beta cells within an islet underlies the generation of pulsatile insulin secretion. This review describes the mechanisms generating [Ca2+]cyt oscillations, the interactions between [Ca2+]cyt and cell metabolism, as well as the contribution of various organelles to the shaping of [Ca2+]cyt signals and insulin secretion. It also discusses how Ca2+ signals are coordinated and spread throughout the islets and data indicating that altered Ca2+ signaling is associated with beta cell dysfunction and development of type 2 diabetes.
Asunto(s)
Señalización del Calcio/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , HumanosRESUMEN
Insulin is produced by pancreatic ß-cells, and once released to the blood, the hormone stimulates glucose uptake and suppresses glucose production. Defects in both the availability and action of insulin lead to elevated plasma glucose levels and are major hallmarks of type-2 diabetes. Insulin is stored in secretory granules that form at the trans-Golgi network. The granules undergo extensive modifications en route to their release sites at the plasma membrane, including changes in both protein and lipid composition of the granule membrane and lumen. In parallel, the insulin molecules also undergo extensive modifications that render the hormone biologically active. In this review, we summarize current understanding of insulin secretory granule biogenesis, maturation, transport, docking, priming and eventual fusion with the plasma membrane. We discuss how different pools of granules form and how these pools contribute to insulin secretion under different conditions. We also highlight the role of the ß-cell in the development of type-2 diabetes and discuss how dysregulation of one or several steps in the insulin granule life cycle may contribute to disease development or progression.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretoras/metabolismo , Animales , Transporte Biológico , Glucemia/metabolismo , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2/sangre , HumanosRESUMEN
AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Homeostasis/genética , Células Secretoras de Insulina/metabolismo , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Biomarcadores/sangre , Glucemia/genética , Femenino , Hormona Folículo Estimulante/sangre , Genotipo , Humanos , Insulina/sangre , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Hipófisis/metabolismo , Caracteres Sexuales , Aumento de Peso , Pez Cebra/sangre , Pez Cebra/genética , Proteínas de Pez Cebra/sangre , Proteínas de Pez Cebra/genéticaRESUMEN
Mitochondria play an essential role in regulating insulin secretion from beta cells by providing the ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine whether the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in rodent beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria twofold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely to be important for the generation of Ca2+ microdomains required for efficient insulin granule release.
Asunto(s)
Calcio/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina , Mitocondrias/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Línea Celular , Exocitosis/fisiología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Ratones , Optogenética , RatasRESUMEN
Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are dynamic structures with important roles in the regulation of calcium (Ca2+) and lipid homeostasis. The extended synaptotagmins (E-Syts) are ER-localized lipid transport proteins that interact with PM phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. E-Syts bidirectionally transfer glycerolipids, including diacylglycerol (DAG), between the 2 juxtaposed membranes, but the biologic significance of this transport is still unclear. Using insulin-secreting cells and live-cell imaging, we now show that Ca2+-triggered exocytosis of insulin granules is followed, in sequence, by PM DAG formation and E-Syt1 recruitment. E-Syt1 counteracted the depolarization-induced DAG formation through a mechanism that required both voltage-dependent Ca2+ influx and Ca2+ release from the ER. E-Syt1 knockdown resulted in prolonged accumulation of DAG in the PM, resulting in increased glucose-stimulated insulin secretion. We conclude that Ca2+-triggered exocytosis is temporally coupled to Ca2+-triggered E-Syt1 PM recruitment and removal of DAG to negatively regulate the same process.-Xie, B., Nguyen, P. M., Idevall-Hagren, O. Feedback regulation of insulin secretion by extended synaptotagmin-1.
Asunto(s)
Insulina/metabolismo , Sinaptotagminas/metabolismo , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Exocitosis/genética , Exocitosis/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , OptogenéticaRESUMEN
The extended synaptotagmins (E-Syts) are ER proteins that act as Ca(2+)-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca(2+) 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 Ca(2+) 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 Ca(2+) range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca(2+) via its influx from the extracellular medium, such as store-operated Ca(2+) entry in fibroblasts and membrane depolarization in ß-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 Ca(2+).
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Sinaptotagmina I/metabolismo , Animales , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Retículo Endoplásmico/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Potenciales de la Membrana/fisiología , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estructura Terciaria de Proteína , Sinaptotagmina I/genéticaRESUMEN
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.
Asunto(s)
Marcación de Gen/métodos , Fosfatidilinositoles/aislamiento & purificación , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Anticuerpos/farmacología , Membrana Celular/química , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Microscopía , Optogenética , Fosfatidilinositoles/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
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-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), 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-ptase(OCRL) 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)P(2) and PI(3,4,5)P(3) 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-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) 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)P(3) 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.
Asunto(s)
Endocitosis/fisiología , Endocitosis/efectos de la radiación , Luz , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Actinas/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión/fisiología , Células COS , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Chlorocebus aethiops , Criptocromos/genética , Criptocromos/metabolismo , Humanos , Canal de Potasio KCNQ2/fisiología , Canal de Potasio KCNQ3/fisiología , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Células PC12 , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación/fisiología , Fosforilación/efectos de la radiación , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Transducción de Señal/efectos de la radiaciónRESUMEN
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 ß cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse ß cells, and human ß 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.
Asunto(s)
Diglicéridos/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteína Quinasa C/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Transducción de Señal , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Exocitosis/efectos de los fármacos , Glucosa/metabolismo , Humanos , Secreción de Insulina , Ratones , Transducción de Señal/efectos de los fármacosRESUMEN
Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca2+-dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation.
Asunto(s)
Colesterol , Retículo Endoplásmico , Secreción de Insulina , Insulina , Antígenos de Histocompatibilidad Menor , Receptores de Esteroides , Vesículas Secretoras , Retículo Endoplásmico/metabolismo , Vesículas Secretoras/metabolismo , Animales , Colesterol/metabolismo , Insulina/metabolismo , Receptores de Esteroides/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ratones , Humanos , Calcio/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Glucosa/metabolismoRESUMEN
Introduction: High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions. Methods: Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied. Results: We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents. Discussion: It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.
Asunto(s)
Adenosina , Células Secretoras de Insulina , Proteínas Proto-Oncogénicas c-akt , Humanos , Adenosina/farmacología , Apoptosis , Glucosa/farmacología , Glucosa/metabolismo , Insulina/metabolismo , Palmitatos , Fosfatidilinositol 3-Quinasas , Células Secretoras de Insulina/citologíaRESUMEN
Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting ß-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarization-triggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in ß-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.
Asunto(s)
Calcio , Células Secretoras de Insulina , Calcio/metabolismo , Células Secretoras de Insulina/metabolismo , Transducción de Señal , Retículo Endoplásmico/metabolismo , Colorantes/metabolismo , Colorantes/farmacología , Señalización del Calcio , Adenosina Trifosfato/metabolismoRESUMEN
The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting ß-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet ß-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.
Asunto(s)
Calcio , Cilios , Islotes Pancreáticos , Receptores de GABA-B , Ácido gamma-Aminobutírico , Animales , Ratones , Calcio/metabolismo , Células Cultivadas , Cilios/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Receptores de GABA-B/metabolismo , CitosolRESUMEN
A wide range of fluorescent sensors with different properties have been developed for imaging of cAMP signals in living cells and tissues. Most cAMP reporters have been designed to undergo changes in fluorescence resonance energy transfer but there are alternative techniques with advantages for certain applications. Here, we describe protocols for cAMP recordings in the sub-plasma membrane space based on detection of translocation of engineered, fluorescent protein-tagged protein kinase A subunits between the plasma membrane and the cytoplasm. Changes in reporter localization can be detected with either confocal or total internal reflection fluorescence microscopy but signal changes are more robust and image analyses less complicated with the latter technique. We show how translocation reporters can be used to study sub-plasma membrane cAMP signals, including oscillations, in insulin-secreting ß-cells stimulated with glucose and G-protein-coupled receptor agonists. We also demonstrate how translocation reporters can be combined with other sensors for simultaneous recordings of the cytosolic Ca2+ concentration, protein kinase A activity or plasma-membrane binding of the cAMP effector protein Epac2. Fluorescent translocation reporters thus provide a versatile complement to the growing cAMP imaging toolkit for elucidating sub-plasma membrane cAMP signals in various types of cells.
Asunto(s)
AMP Cíclico , Células Secretoras de Insulina , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Células Secretoras de Insulina/metabolismoRESUMEN
Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca(2+) and cAMP concentrations in the subplasma membrane space ([Ca(2+)](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca(2+)](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca(2+)](pm), [cAMP](pm), and PIP(3). Inhibitors of protein kinase A (PKA) markedly diminished the PIP(3) response when applied before glucose stimulation, but did not affect already manifested PIP(3) oscillations. The reduced PIP(3) response could be attributed to accelerated depolarization causing early rise of [Ca(2+)](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP(3) 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 PIP(3) 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 PIP(3) oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca(2+)](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.