Calcium Signaling in Cell Compartments - the Importance of Sphingosine Kinase 1 and ORP5/8

För att upprätthålla liv använder cellerna i en organism intrikata signaleringsräckor för regleringen av fysiologiska processer inom enskilda celler och mellan olika celler och vävnaderna som utgör en organism. Reglering av detta slag kallas cellsignalering. En av de mest centrala molekylerna i cellsignaleringen är kalciumjonen, Ca2+. Många centrala biologiska processer, såsom muskelkontraktion, befruktning, cellproliferation, cellmigration, cellulär energiproduktion och celldöd, moduleras av Ca2+-signalering. Dysfunktionell Ca2+-signalering kan bidra till utvecklingen av olika sjukdomstillstånd såsom cancer och neurologiska sjukdomar. I de publikationer som utgör basen för denna avhandling studerade vi hur tre proteiner av intresse påverkar regleringen av Ca2+-hantering i specifika cellulära domäner, främst mitokondrier och caveoler. Mitokondrier är organeller som är centrala för cellulär energiproduktion och bidrar även till andra viktiga processer som Ca2+-signalering och celldöd. Caveoler är små inbuktningar av cellmembranet och deltar i regleringen av viktiga cellulära funktioner. Caveoler fungerar till exempel som orkestratorer av cellsignalering. För att studera Ca2+-signalering i cellmembranets domän nära caveoler skapade och karakteriserade vi en ny Ca2+-indikator kopplad till det caveolära proteinet caveolin-1. I dessa undersökningar fann vi att överuttryck av ett lipidkinasprotein, sfingosinkinas 1 (SK1), resulterar i ökad agonist-inducerad frigivning av Ca2+ från det endoplasmatiska retiklet (cellens huvudsakliga Ca2+-förråd), samtidigt som det ökar Ca2+-koncentrationen i mitokondriernas matrix och vid caveolerna. De SK1-inducerade Ca2+-effekterna kan också vara bidragande faktorer i regleringen av cellandning och migration, som båda vi fann vara förstärkta av SK1-överuttryck. Dessa fynd bekräftar de tidigare indikationerna på att SK1 är involverad i processer relaterade till cancerfysiologi, möjligen genom Ca2+- medierade processer. Därtill fann vi att överuttryck av de oxysterolbindande proteinerna 5 respektive 8 (ORP5/8) ökar Ca2+-koncentrationen i mitokondriernas matrix och i de caveolära regionerna i cellmembranet. Vi fann också att överuttryck av både ORP5 och ORP8 ökade cellproliferation. ORP5/8 är lokaliserade vid, och reglerar, membrankontakterna, och signaleringen mellan det endoplasmatiska retiklet, mitokondrierna och cellmembranet. Våra resultat som visar ORP5/8-medierade effekter i Ca2+-signalering i mitokondriernas matrix och vid de caveolära cellmembrandomänerna belyser således den fysiologiska rollen som ORP5/8-proteinerna spelar som reglerare av signaleringen vid membrankontakterna i dessa intracellulära domäner. Sammantaget visar dessa resultat att SK1- och ORP5/8-proteiner är involverade i regleringen av intracellulära Ca2+-signaler vid specifika cellulära domäner.To maintain life, the cells within an organism employ intricate signaling pathways to regulate physiological processes within single cells and between the various cells and tissues that constitute an organism. Regulation of this kind is termed cell signaling. One of the key molecules in cell signaling is the calcium ion, Ca2+. Many central biological processes, such as muscle contraction, fertilization, cell proliferation, cell migration, cellular energy production and cell death, are modulated by Ca2+ signaling. Dysfunctional Ca2+ signaling may contribute to the development of various disease conditions such as cancer and neurological disease. In the original publications of this thesis, we studied how three proteins of interest affect the regulation of Ca2+ handling in specific cellular compartments, mainly the mitochondria and the caveolae. The mitochondria are organelles that are key in cellular energy production and contribute also to other important processes such as Ca2+ signaling and cell death. The caveolae are small invaginations of the cell membrane with important functions, for instance as orchestrators of cell signaling events. To study the Ca2+ signaling events at the caveolar domain of the cell membrane, we created and characterized a novel Ca2+ indicator linked to the key caveolar protein, caveolin-1. We found that overexpression of a lipid kinase protein, sphingosine kinase 1 (SK1), results in increased agonist-induced release of Ca2+ from the endoplasmic reticulum, while also increasing the Ca2+ concentration in the mitochondrial matrix and at the caveolae. The SK1-induced Ca2+ effects may also be contributing factors in the regulation of cell respiration and migration, both of which we found to be augmented by SK1 overexpression. These findings corroborate the previous indications of SK1 being involved in processes related to cancer physiology, possibly through Ca2+ mediated processes. In addition, we found that overexpression of the oxysterol-binding proteins 5 and 8 (ORP5/8), respectively, increases the Ca2+ concentration in the mitochondrial matrix and at the caveolar regions of the cell membrane. We found that overexpression of ORP5 and ORP8 also increased cell proliferation. ORP5/8 are known to reside at and regulate the membrane contacts and signaling between the endoplasmic reticulum (the main Ca2+ store of the cell) and the mitochondria and the cell membrane. Our results showing ORP5/8-mediated Ca2+ signaling in the mitochondrial matrix and at caveolar cell membrane domains thus further elucidate the physiological role of the ORP5/8 proteins as regulators of signaling events at the membrane contact sites of these intracellular compartments. Taken together, our results show that SK1 and ORP5/8 proteins are involved in the regulation intracellular Ca2+ signaling at specific cellular compartments

Oxysterol-binding protein related-proteins (ORPs) 5 and 8 regulate calcium signaling at specific cell compartments

Oxysterol-binding protein related-protein 5 and 8 (ORP5/8) localize to the membrane contact sites (MCS) of the endoplasmic reticulum (ER) and the mitochondria, as well as to the ER-plasma membrane (PM) MCS. The MCS are emerging as important regulators of cell signaling events, including calcium (Ca2+) signaling. ORP5/8 have been shown to interact with phosphatidylinositol-4,5-bisphosphate (PIP2) in the PM, and to modulate mitochondrial respiration and morphology. PIP2 is the direct precursor of inositol trisphosphate (IP3), a key second messenger responsible for Ca2+-release from the intracellular Ca2+ stores. Further, mitochondrial respiration is linked to Ca2+ transfer from the ER to the mitochondria. Hence, we asked whether ORP5/8 would affect Ca2+ signaling in these cell compartments, and employed genetically engineered aequorin Ca2+ probes to investigate the effect of ORP5/8 in the regulation of mitochondrial and caveolar Ca2+. Our results show that ORP5/8 overexpression leads to increased mitochondrial matrix Ca2+ as well as to increased Ca2+ concentration at the caveolar subdomains of the PM during histamine stimulation, while having no effect on the cytoplasmic Ca2+. Also, we found that ORP5/8 overexpression increases cell proliferation. Our results show that ORP5/8 regulate Ca2+ signaling at specific MCS foci. These local ORP5/8-mediated Ca2+ signaling events are likely to play roles in processes such as mitochondrial respiration and cell proliferation.Peer reviewe

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells

Sphingosine kinase 1 (SKI) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (SIP). SW binds to G-protein-coupled receptors (S1PR(1-5)) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SKI augments agonist-induced Ca2+ release from the ER resulting in increased mitochondria] matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.Peer reviewe

Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes

Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.Peer reviewe

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Severe neonatal MEGDHEL syndrome with a homozygous truncating mutation in SERAC1

In the diagnostic work-up of a newborn infant with a metabolic crisis, lethal multiorgan failure on day six of life, and increased excretion of 3-methylglutaconic acid, we found using whole genome sequencing a homozygous SERAC1 mutation indicating MEGDHEL syndrome (3-methylglutaconic aciduria with deafness-dystonia, hepatopathy, encephalopathy, and Leigh-like syndrome). The SERAC1 protein is located at the contact site between mitochondria and the endoplasmic reticulum (ER) and is crucial for cholesterol trafficking. Our aim was to investigate the effect of the homozygous truncating mutation on mitochondrial structure and function. In the patient fibroblasts, no SERAC1 protein was detected, the mitochondrial network was severely fragmented, and the cristae morphology was altered. Filipin staining showed uneven localization of unesterified cholesterol. The calcium buffer function between cytoplasm and mitochondria was deficient. In liver mitochondria, complexes I, III, and IV were clearly decreased. In transfected COS-1 cells the mutant protein with the a 45-amino acid C-terminal truncation was distributed throughout the cell, whereas wild-type SERAC1 partially colocalized with the mitochondrial marker MT-001. The structural and functional mitochondrial abnormalities, caused by the loss of SERAC1, suggest that the crucial disease mechanism is disrupted interplay between the ER and mitochondria leading to decreased influx of calcium to mitochondria and secondary respiratory chain deficiency.Peer reviewe

Complexation of c6-ceramide with cholesteryl phosphocholine - a potent solvent-free ceramide delivery formulation for cells in culture.

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca(2+) influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca(2+) homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides

A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²⁺ microdomain in the caveolar compartment

Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1-Aequorin fusion protein (Cav1-Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca2+]cav). In HeLa cells, Cav1-Aeq reported different [Ca2+] as compared to the plasma membrane [Ca2+] in general (reported by SNAP25-Aeq) or as compared to the cytosolic [Ca2+] (reported by cyt-Aeq). The Ca2+ signals detected by Cav1-Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca2+]cav during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+ as well as during receptor-operated Ca2+ entry (ROCE). The SK1-induced increase in [Ca2+]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca2+]cav during ROCE. S1P treatment stimulated the [Ca2+]cav upon ROCE. The Ca2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium

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C6-Cer/CholPC and C6-dihydroCer reduce mitochondrial calcium uptake.

<p>HeLa cells were preincubated for 180 min with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO (panels A and B), and changes in intracellular Ca²⁺ levels were measured using mtAEQ. DMSO was added to control cells. Panel A shows kinetics of changes in mitochondrial Ca²⁺ after challenge with histamine. In panel B, the change in Ca²⁺ response was quantitated. The cells were challenged with 100 µM histamine as indicated by the arrow. Panel C shows kinetics of the Ca²⁺ response after180 min exposure of cells to C6-dihydroCer. Traces (panel A and C) are averages of 3 measurements, each representing the average luminescence from a cell population of 150 000–200 000 cells. In panel B, the bar shows the average change in [Ca²⁺]_mito during histamine-induced Ca²⁺ release (± SEM, n = 3). The data were analyzed using one-way Anova and Bonferroni’s multiple comparison test (***p<0.001 compared with DMSO; ¤¤¤p<0.001 compared with C6-Cer).</p