BBA - Molecular Cell Research (v.1864, #6)

Obituary Roger Y. Tsien (1952–2016) by By Tullio Pozzan (839-840).

Preface by Jacques Haiech; Claus W. Heizmann; Joachim Krebs (841-842).

Calcium remodeling in colorectal cancer by Carlos Villalobos; Diego Sobradillo; Miriam Hernández-Morales; Lucía Núñez (843-849).
Colorectal cancer (CRC) is the third most frequent form of cancer and the fourth leading cause of cancer-related death in the world. Basic and clinical data indicate that aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colon cancer but mechanisms remain unknown. Aspirin metabolite salicylate and other NSAIDs may inhibit tumor cell growth acting on store-operated Ca2 + entry (SOCE), suggesting an important role for this pathway in CRC. Consistently, SOCE is emerging as a novel player in different forms of cancer, including CRC. SOCE and store-operated currents (SOCs) are dramatically enhanced in CRC while Ca2 + stores are partially empty in CRC cells. These features may contribute to CRC hallmarks including enhanced cell proliferation, migration, invasion and survival. At the molecular level, enhanced SOCE and depleted stores are mediated by overexpression of Orai1, Stromal interaction protein 1 (STIM1) and Transient receptor protein channel 1 (TRPC1) and downregulation of STIM2. In normal colonic cells, SOCE is mediated by Ca2 +-release activated Ca2 + channels made of STIM1, STIM2 and Orai1. In CRC cells, SOCE is mediated by different store-operated currents (SOCs) driven by STIM1, Orai1 and TRPC1. Loss of STIM2 contributes to depletion of Ca2 + stores and enhanced resistance to cell death in CRC cells. Thus, SOCE is a novel key player in CRC and inhibition by salicylate and other NSAIDs may contribute to explain chemoprevention activity.Colorectal cancer (CRC) is the third most frequent form of cancer worldwide. Recent evidence suggests that intracellular Ca2 + remodeling may contribute to cancer hallmarks. In addition, aspirin and other NSAIDs might prevent CRC acting on remodeled Ca2 + entry pathways. In this review, we will briefly describe 1) the players involved in intracellular Ca2 + homeostasis with a particular emphasis on the mechanisms involved in SOCE activation and inactivation, 2) the evidence that aspirin metabolite salicylate and other NSAIDs inhibits tumor cell growth acting on SOCE, 3) evidences on the remodeling of intracellular Ca2 + in cancer with a particular emphasis in SOCE, 4) the remodeling of SOCE and Ca2 + store content in CRC and, finally, 5) the molecular basis of Ca2 + remodeling in CRC. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Display Omitted
Keywords: Colorectal cancer; Store-operated Ca2 + entry; Orai1; TRPC1; STIM1; STIM2; Aspirin; Non-steroidal anti-inflammatory drugs;

Ca2+ is a ubiquitous intracellular messenger that regulates numerous physiological activities in humans, animals, plants, and bacteria. Cytosolic Ca2 + is kept at a low level, but subcellular organelles such as the endoplasmic reticulum (ER) and Golgi apparatus maintain high-concentration Ca2+ stores. Under resting conditions, store Ca2+ homeostasis is dynamically regulated to equilibrate between active Ca2+ uptake and passive Ca2+ leak processes. The evolutionarily conserved Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) proteins mediate Ca2+ homeostasis and cell death. This review focuses on recent advances in functional and structural analysis of TMBIM proteins in regulation of the two related functions. The roles of TMBIM proteins in pathogen infection and cancer are also discussed with prospects for treatment. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Ca2+ homeostasis; Ca2+ signaling; Cell death; Apoptosis; Bax inhibitor-1; TMBIM; Membrane proteins; Cancer; Cellular stress; Ca2+ channel structure;

Endoplasmic reticulum-mitochondria Ca2 + crosstalk in the control of the tumor cell fate by Sonia Missiroli; Alberto Danese; Tommaso Iannitti; Simone Patergnani; Mariasole Perrone; Maurizio Previati; Carlotta Giorgi; Paolo Pinton (858-864).
Mitochondria-associated membranes are juxtaposed between the endoplasmic reticulum and mitochondria and have been identified as a critical hub in the regulation of apoptosis and tumor growth. One key function of mitochondria-associated membranes is to provide asylum to a number of proteins with tumor suppressor and oncogenic properties. In this review, we discuss how Ca2 + flux manipulation represents the primary mechanism underlying the action of several oncogenes and tumor-suppressor genes and how these networks might be manipulated to provide novel therapies for cancer. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases. Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes (MAM), in order to exchange metabolites and calcium (Ca2+) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca2+ signaling in both healthy and pathological contexts. Then we focus on recent data highlighting MAM as important hubs for hormone and nutrient signaling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss how chronic ER-mitochondria miscommunication could participate to hepatic metabolic diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Display Omitted
Keywords: Organelle communication; Mitochondria-associated membranes (MAM); Calcium signaling; Liver; Insulin resistance; Type 2 diabetes mellitus; NAFLD;

Thyroid hormones influence brain development through regulation of gene expression. This is especially true for Ca2 +-dependent regulation since a major pathway is controlled by the Ca2 +/calmodulin-dependent protein kinase IV (CaMKIV) which in turn is induced by the thyroid hormone T3. In addition, CaMKIV is involved in regulation of alternative splicing of a number of protein isoforms, among them PMCA1a, the neuronal specific isoform of the plasma membrane calcium pump. On the other hand, hypothyroidism or CaMKIV deficiency can have a severe influence on brain development. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Thyroid hormone; CaMKIV; PMCA1a; Alternative splicing; Neuronal development;

TRP channels in calcium homeostasis: from hormonal control to structure-function relationship of TRPV5 and TRPV6 by Mark K.C. van Goor; Joost G.J. Hoenderop; Jenny van der Wijst (883-893).
Maintaining plasma calcium levels within a narrow range is of vital importance for many physiological functions. Therefore, calcium transport processes in the intestine, bone and kidney are tightly regulated to fine-tune the rate of absorption, storage and excretion. The TRPV5 and TRPV6 calcium channels are viewed as the gatekeepers of epithelial calcium transport. Several calciotropic hormones control the channels at the level of transcription, membrane expression, and function. Recent technological advances have provided the first near-atomic resolution structural models of several TRPV channels, allowing insight into their architecture. While this field is still in its infancy, it has increased our understanding of molecular channel regulation and holds great promise for future structure-function studies of these ion channels. This review will summarize the mechanisms that control the systemic calcium balance, as well as extrapolate structural views to the molecular functioning of TRPV5/6 channels in epithelial calcium transport.
Keywords: Calcium; TRP channels; Structure; Hormones;

Measuring Ca2 + inside intracellular organelles with luminescent and fluorescent aequorin-based sensors by María Teresa Alonso; Jonathan Rojo-Ruiz; Paloma Navas-Navarro; Macarena Rodríguez-Prados; Javier García-Sancho (894-899).
GFP-Aequorin Protein (GAP) can be used to measure [Ca2 +] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca2 +. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca2 + measurements ex vivo and in vivo to explore animal models of health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Biosensor; Calcium; Organelles; GFP; Aequorin; GECI; Endoplasmic reticulum; Golgi apparatus;

The functions of store-operated calcium channels by James W. Putney; Natacha Steinckwich-Besançon; Takuro Numaga-Tomita; Felicity M. Davis; Pooja N. Desai; Diane M. D'Agostin; Shilan Wu; Gary S. Bird (900-906).
Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca2 + stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Store-operated calcium channels; Calcium signaling; Mouse models; Exocrine glands; Neutrophils; Keratinocytes;

Cardiac inositol 1,4,5-trisphosphate receptors by M. Iveth Garcia; Darren Boehning (907-914).
Calcium is a second messenger that regulates almost all cellular functions. In cardiomyocytes, calcium plays an integral role in many functions including muscle contraction, gene expression, and cell death. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of calcium channels that are ubiquitously expressed in all tissues. In the heart, IP3Rs have been associated with regulation of cardiomyocyte function in response to a variety of neurohormonal agonists, including those implicated in cardiac disease. Notably, IP3R activity is thought to be essential for mediating the hypertrophic response to multiple stimuli including endothelin-1 and angiotensin II. In this review, we will explore the functional implications of IP3R activity in the heart in health and disease.Display Omitted
Keywords: IP3 Receptor; Cardiac hypertrophy; Calcium channels;

Src-family tyrosine kinases and the Ca2 + signal by Estefanía Anguita; Antonio Villalobo (915-932).
In this review, we shall describe the rich crosstalk between non-receptor Src-family kinases (SFKs) and the Ca2 + transient generated in activated cells by a variety of extracellular and intracellular stimuli, resulting in diverse signaling events. The exchange of information between SFKs and Ca2 + is reciprocal, as it flows in both directions. These kinases are main actors in pathways leading to the generation of the Ca2 + signal, and reciprocally, the Ca2 + signal modulates SFKs activity and functions. We will cover how SFKs participate in the generation of the cytosolic Ca2 + rise upon activation of a series of receptors and the mechanism of clearance of this Ca2 + signal. The role of SFKs modulating Ca2 +-translocating channels participating in these events will be amply discussed. Finally, the role of the Ca2 + sensor protein calmodulin on the activity of c-Src, and potentially on other SFKs, will be outlined as well. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Calcium; Calmodulin; Cell signaling; Channels; Phospho-tyrosine phosphatases; Receptors; Src kinases;

Annexin A6 in the liver: From the endocytic compartment to cellular physiology by Carlos Enrich; Carles Rentero; Thomas Grewal (933-946).
Annexin A6 (AnxA6) belongs to the conserved annexin family — a group of Ca2 +-dependent membrane binding proteins. AnxA6 is the largest of all annexins and highly expressed in smooth muscle, hepatocytes, endothelial cells and cardiomyocytes. Upon activation, AnxA6 binds to negatively charged phospholipids in a wide range of intracellular localizations, in particular the plasma membrane, late endosomes/pre-lysosomes, but also synaptic vesicles and sarcolemma. In these cellular sites, AnxA6 is believed to contribute to the organization of membrane microdomains, such as cholesterol-rich lipid rafts and confer multiple regulatory functions, ranging from vesicle fusion, endocytosis and exocytosis to programmed cell death and muscle contraction. Growing evidence supports that Ca2 + and Ca2 +-binding proteins control endocytosis and autophagy. Their regulatory role seems to operate at the level of the signalling pathways that initiate autophagy or at later stages, when autophagosomes fuse with endolysosomal compartments. The convergence of the autophagic and endocytic vesicles to lysosomes shares several features that depend on Ca2 + originating from lysosomes/late endosomes and seems to depend on proteins that are subsequently activated by this cation. However, the involvement of Ca2 + and its effector proteins in these autophagic and endocytic stages still remains poorly understood. Although AnxA6 makes up almost 0.25% of total protein in the liver, little is known about its function in hepatocytes. Within the endocytic route, we identified AnxA6 in endosomes and autophagosomes of hepatocytes. Hence, AnxA6 and possibly other annexins might represent new Ca2 + effectors that regulate converging steps of autophagy and endocytic trafficking in hepatocytes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Annexin A6; Autophagosomes; Ca2 +-binding proteins; Hepatocytes; Intracellular trafficking; Liver regeneration;

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2 + by Tomas Luyten; Kirsten Welkenhuyzen; Gemma Roest; Elzbieta Kania; Liwei Wang; Mart Bittremieux; David I. Yule; Jan B. Parys; Geert Bultynck (947-956).
Previous work revealed that intracellular Ca2 + signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2 + signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80 μM, 2 h) was completely abolished in the presence of 10 μM BAPTA-AM, an intracellular Ca2 +-chelating agent. To elucidate the IP3R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2 + or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2 + release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2 + ATPase (SERCA) activity nor the IP3-induced Ca2 + release nor the basal Ca2 + leak from the ER. Also, prolonged (4 h) treatment with 100 μM resveratrol did not affect subsequent IP3-induced Ca2 + release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2 + signals by itself, it acutely decreased the ER Ca2 +-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2 + signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2 + signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Display Omitted
Keywords: Cytosolic Ca2 +;  Inositol 1,4,5-trisphosphate; Inositol 1,4,5-trisphosphate receptor; Autophagy; Resveratrol; HEK 3KO;

High intracellular levels of reactive oxygen species (ROS) cause oxidative stress that results in numerous pathologies, including cell death. Transient potential receptor melastatin-2 (TRPM2), a Ca2 +-permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Here we studied the role and mechanisms of TRPM2-mediated Ca2 + influx on oxidative stress-induced cell death in cancer cells. We found that oxidative stress activated the TRPM2-Ca2 +-CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. On the other hand, TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. Moreover, we found that oxidative stress activated the TRPM2-CaMKII cascade to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondrial membrane potential. In summary, our data demonstrated that oxidative stress activates the TRPM2-Ca2 +-CaMKII-ROS signal loop to inhibit autophagy and induce cell death.
Keywords: TRPM2; Oxidative stress; Ca2 +;  Reactive oxygen species; CaMKII; Autophagy;

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca2+ signaling by Tamara Vervloessem; Hristina Ivanova; Tomas Luyten; Jan B. Parys; Geert Bultynck (968-976).
Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca2+ homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca2+-transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems. Venetoclax (1 μM) did neither trigger Ca2+ release by itself nor affect agonist-induced Ca2+ release in a variety of intact cell models. Among the different cell types, we also studied two Bcl-2-dependent cancer cell models with a varying sensitivity towards venetoclax, namely SU-DHL-4 and OCI-LY-1, both diffuse large B-cell lymphoma cell lines. Acute application of venetoclax did also not dysregulate Ca2+ signaling in these Bcl-2-dependent cancer cells. Moreover, venetoclax-induced cell death was independent of intracellular Ca2+ overload, since Ca2+ buffering using BAPTA-AM did not suppress venetoclax-induced cell death. This study therefore shows that venetoclax does not dysregulate the intracellular Ca2+ homeostasis in a variety of cell types, which may underlie its limited toxicity in human patients. Furthermore, venetoclax-induced cell death in Bcl-2-dependent cancer cells is not mediated by intracellular Ca2+ overload. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Venetoclax; ABT-199; Bcl-2; BH3 mimetic; Calcium; IP3 receptor;

Mag-Fluo4 in T cells: Imaging of intra-organelle free Ca2 + concentrations by Björn-Philipp Diercks; Ralf Fliegert; Andreas H. Guse (977-986).
Ca2 + signaling is a major signal transduction pathway involved in T cell activation, but also in apoptosis of T cells. Since T cells make use of several Ca2 +-mobilizing second messengers, such as nicotinic acid adenine dinucleotide phosphate, d-myo-inositol 1,4,5-trisphosphate, and cyclic ADP-ribose, we intended to analyze luminal Ca2 + concentration upon cell activation. Mag-Fluo4/AM, a low-affinity Ca2 + dye known to localize to the endoplasmic reticular lumen in many cell types, showed superior brightness and bleaching stability, but, surprisingly, co-localized with mito-tracker, but not with ER-tracker in Jurkat T cells. Thus, we used Mag-Fluo4/AM to monitor the free luminal mitochondrial Ca2 + concentration ([Ca2 +]mito) in these cells. Simultaneous analysis of the free cytosolic Ca2 + concentration ([Ca2 +]i) and [Ca2 +]mito upon cell stimulation revealed that Ca2 + signals in the majority of mitochondria were initiated at [Ca2 + ]i  ≥ approx. 400 to 550 nM.In primary murine CD4+ T cells, Mag-Fluo4 showed two different localization patterns: either co-localization with mito-tracker, as in Jurkat T cells, or with ER-tracker. Thus, in single primary murine CD4+ T cells, either decreases of [Ca2 + ]ER or increases of [Ca2 + ]mito were observed upon cell stimulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Display Omitted
Keywords: Ca2 + signaling; Mag-Fluo4; Endoplasmic reticular Ca2 + concentration; Mitochondrial Ca2 + concentration; T cell; Signal transduction;

Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium by Abhishek Aggarwal; Herbert Schulz; Teresa Manhardt; Martin Bilban; Rajesh V Thakker; Enikö Kallay (987-996).
Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca2+]o) in colon cancer cells.Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24 h with 2 mM [Ca2+]o identified significant changes in expression of 1571 probe sets (ANOVA, p  < 10− 5). The main biological processes affected by [Ca2+]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca2+]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet.We show that among the mechanisms behind the chemopreventive effect of [Ca2+]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.
Keywords: Calcium; Colorectal cancer; DNA replication licensing; Minichromosome maintenance complex; Calcium-sensing receptor;

Identification of residues that control Li+ versus Na+ dependent Ca2 + exchange at the transport site of the mitochondrial NCLX by Soumitra Roy; Kuntal Dey; Michal Hershfinkel; Ehud Ohana; Israel Sekler (997-1008).
The Na+/Ca2 +/Li+ exchanger (NCLX) is a member of the Na+/Ca2 + exchanger family. NCLX is unique in its capacity to transport both Na+ and Li+, unlike other members, which are Na+ selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li+ or Na+ selective Ca2 + transport and map their putative location on NCLX cation transport site.We combined molecular modeling to map transport site of NCLX with euryarchaeal H+/Ca2 + exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2 + efflux of transport site mutants of NCLX in permeabilized cells.Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca2 + exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2 + exchange activity but a reduced Na+/Ca2 + exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2 + exchange, but Na+/Ca2 + exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2 + binding residues was required to completely abolish both Na+/Ca2 + and Li+/Ca2 + exchange activities.We identified distinct Na+ and Li+ selective residues in the NCLX transport site. We propose that functional segregation in Li+ and Na+ sites reflects the functional properties of NCLX required for Ca2 + exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane.The results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: NCLX; Na+/Ca2 + exchange; Li+/Ca2 + exchange; Permeabilized cells; 3D modelling;

Pathological consequences of MICU1 mutations on mitochondrial calcium signalling and bioenergetics by Gauri Bhosale; Jenny A. Sharpe; Amanda Koh; Antonina Kouli; Gyorgy Szabadkai; Michael R. Duchen (1009-1017).
Loss of function mutations of the protein MICU1, a regulator of mitochondrial Ca2 + uptake, cause a neuronal and muscular disorder characterised by impaired cognition, muscle weakness and an extrapyramidal motor disorder. We have shown previously that MICU1 mutations cause increased resting mitochondrial Ca2+ concentration ([Ca2 +]m). We now explore the functional consequences of MICU1 mutations in patient derived fibroblasts in order to clarify the underlying pathophysiology of this disorder. We propose that deregulation of mitochondrial Ca2+ uptake through loss of MICU1 raises resting [Ca2+]m, initiating a futile Ca2+ cycle, whereby continuous mitochondrial Ca2+ influx is balanced by Ca2+ efflux through the sodium calcium exchanger (NLCXm). Thus, inhibition of NCLXm by CGP-37157 caused rapid mitochondrial Ca2+ accumulation in patient but not control cells. We suggest that increased NCLX activity will increase sodium/proton exchange, potentially undermining oxidative phosphorylation, although this is balanced by dephosphorylation and activation of pyruvate dehydrogenase (PDH) in response to the increased [Ca2+]m. Consistent with this model, while ATP content in patient derived or control fibroblasts was not different, ATP increased significantly in response to CGP-37157 in the patient but not the control cells. In addition, EMRE expression levels were altered in MICU1 patient cells compared to the controls. The MICU1 mutations were associated with mitochondrial fragmentation which we show is related to altered DRP1 phosphorylation. Thus, MICU1 serves as a signal–noise discriminator in mitochondrial calcium signalling, limiting the energetic costs of mitochondrial Ca2+ signalling which may undermine oxidative phosphorylation, especially in tissues with highly dynamic energetic demands. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Mitochondria; Calcium; MICU1; PDH;

Bisacodyl and its cytotoxic activity on human glioblastoma stem-like cells. Implication of inositol 1,4,5-triphosphate receptor dependent calcium signaling by Jihu Dong; Francisco J. Aulestia; Suzana Assad Kahn; Maria Zeniou; Luiz Gustavo Dubois; Elias A. El-Habr; François Daubeuf; Nassera Tounsi; Samuel H. Cheshier; Nelly Frossard; Marie-Pierre Junier; Hervé Chneiweiss; Isabelle Néant; Marc Moreau; Catherine Leclerc; Jacques Haiech; Marie-Claude Kilhoffer (1018-1027).
Glioblastoma is the most common malignant brain tumor. The heterogeneity at the cellular level, metabolic specificities and plasticity of the cancer cells are a challenge for glioblastoma treatment. Identification of cancer cells endowed with stem properties and able to propagate the tumor in animal xenografts has opened a new paradigm in cancer therapy. Thus, to increase efficacy and avoid tumor recurrence, therapies need to target not only the differentiated cells of the tumor mass, but also the cancer stem-like cells. These therapies need to be effective on cells present in the hypoxic, slightly acidic microenvironment found within tumors. Such a microenvironment is known to favor more aggressive undifferentiated phenotypes and a slow-growing “quiescent state” that preserves the cells from chemotherapeutic agents, which mostly target proliferating cells. Based on these considerations, we performed a differential screening of the Prestwick Chemical Library of approved drugs on both proliferating and quiescent glioblastoma stem-like cells and identified bisacodyl as a cytotoxic agent with selectivity for quiescent glioblastoma stem-like cells. In the present study we further characterize bisacodyl activity and show its efficacy in vitro on clonal macro-tumorospheres, as well as in vivo in glioblastoma mouse models. Our work further suggests that bisacodyl acts through inhibition of Ca2 + release from the InsP3 receptors.
Keywords: Glioblastoma; Cancer stem-like cells; Macro-tumorospheres; Bisacodyl; Calcium; InsP3R;

Phospholipids and calmodulin modulate the inhibition of PMCA activity by tau by María Berrocal; Isaac Corbacho; M. Rosario Sepulveda; Carlos Gutierrez-Merino; Ana M. Mata (1028-1035).
The disruption of Ca2 + signaling in neurons, together with a failure to keep optimal intracellular Ca2 + concentrations, have been proposed as significant factors for neuronal dysfunction in the Ca2 + hypothesis of Alzheimer's disease (AD). Tau is a protein that plays an essential role in axonal transport and can form abnormal structures such as neurofibrillary tangles that constitute one of the hallmarks of AD. We have recently shown that plasma membrane Ca2 +-ATPase (PMCA), a key enzyme in the maintenance of optimal cytosolic Ca2 + levels in cells, is inhibited by tau in membrane vesicles. In the present study we show that tau inhibits synaptosomal PMCA purified from pig cerebrum, and reconstituted in phosphatidylserine-containing lipid bilayers, with a Ki value of 1.5 ± 0.2 nM tau. Noteworthy, the inhibitory effect of tau is dependent on the charge of the phospholipid used for PMCA reconstitution. In addition, nanomolar concentrations of calmodulin, the major endogenous activator of PMCA, protects against inhibition of the Ca2 +-ATPase activity by tau. Our results in a cellular model such as SH-SY5Y human neuroblastoma cells yielded an inhibition of PMCA by nanomolar tau concentrations and protection by calmodulin against this inhibition similar to those obtained with purified synaptosomal PMCA. Functional studies were also performed with native and truncated versions of human cerebral PMCA4b, an isoform that has been showed to be functionally regulated by amyloid peptides, whose aggregates constitutes another hallmark of AD. Kinetic assays point out that tau binds to the C-terminal tail of PMCA, at a site distinct but close to the calmodulin binding domain. In conclusion, PMCA can be seen as a molecular target for tau-induced cytosolic calcium dysregulation in synaptic terminals.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Ca2 +; PMCA; Tau; Calmodulin; Phospholipid;

Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca2 + channel in neuromuscular function and regeneration by John D. Chan; Dan Zhang; Xiaolong Liu; Magdalena Zarowiecki; Matthew Berriman; Jonathan S. Marchant (1036-1045).
The robust regenerative capacity of planarian flatworms depends on the orchestration of signaling events from early wounding responses through the stem cell enacted differentiative outcomes that restore appropriate tissue types. Acute signaling events in excitable cells play an important role in determining regenerative polarity, rationalized by the discovery that sub-epidermal muscle cells express critical patterning genes known to control regenerative outcomes. These data imply a dual conductive (neuromuscular signaling) and instructive (anterior-posterior patterning) role for Ca2 + signaling in planarian regeneration. Here, to facilitate study of acute signaling events in the excitable cell niche, we provide a de novo transcriptome assembly from the planarian Dugesia japonica allowing characterization of the diverse ionotropic portfolio of this model organism. We demonstrate the utility of this resource by proceeding to characterize the individual role of each of the planarian voltage-operated Ca2 + channels during regeneration, and demonstrate that knockdown of a specific voltage operated Ca2 + channel (Ca v 1B) that impairs muscle function uniquely creates an environment permissive for anteriorization. Provision of the full transcriptomic dataset should facilitate further investigations of molecules within the planarian voltage-gated channel portfolio to explore the role of excitable cell physiology on regenerative outcomes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Display Omitted
Keywords: Voltage-operated Ca2 + channels; Neuromuscular signaling; Regeneration; Transcriptome;

Many cells in an organism are exposed to constant and acute mechanical stress that can induce plasma membrane injuries. These plasma membrane wounds have to be resealed rapidly to guarantee cell survival. Plasma membrane resealing in response to mechanical strain has been studied in some detail in muscle, where it is required for efficient recovery after insult. However, less is known about the capacity of other cell types and tissues to perform membrane repair and the underlying molecular mechanisms. Here we show that vascular endothelial cells, which are subject to profound mechanical burden, can reseal plasma membrane holes inflicted by laser ablation. Resealing in endothelial cells is a Ca2 +-dependent process, as it is inhibited when cells are wounded in Ca2 +-free medium. We also show that annexin A1 (AnxA1), AnxA2 and AnxA6, Ca2 +-regulated membrane binding proteins previously implicated in membrane resealing in other cell types, are rapidly recruited to the site of plasma membrane injury. S100A11, a known protein ligand of AnxA1, is also recruited to endothelial plasma membrane wounds, albeit with a different kinetic. Mutant expression experiments reveal that Ca2 + binding to AnxA2, the most abundant endothelial annexin, is required for translocation of the protein to the wound site. Furthermore, we show by knock-down and rescue experiments that AnxA2 is a positive regulator of plasma membrane resealing. Thus, vascular endothelial cells are capable of active, Ca2 +-dependent plasma membrane resealing and this process requires the activity of AnxA2.
Keywords: Annexin; Calcium; Endothelium; Membrane;

A major intracellular calcium (Ca2 +) uptake pathway in both excitable and non-excitable eukaryotic cells is store-operated Ca2 + entry (SOCE). SOCE is the process by which endoplasmic reticulum (ER)-stored Ca2 + depletion leads to activation of plasma membrane Ca2 + channels to provide a sustained increase in cytosolic Ca2 + levels that mediate a plethora of physiological processes ranging from the immune response to platelet aggregation. Stromal interaction molecule-1 (STIM1) is the principal regulator of SOCE and responds to changes in ER stored Ca2 + through luminal sensing machinery composed of EF-hand and SAM domains (EFSAM). The EFSAM domain can undergo N-glycosylation at Asn131 and Asn171 sites; however, the precise role of EFSAM N-glycosylation in the Ca2 + sensing mechanism of STIM1 is unclear. By establishing a site-specific chemical approach to covalently linking glucose to EFSAM and examining α-helicity, thermal stability, three dimensional atomic-resolution structure, Ca2 + binding affinity and oligomerization, we show that N-glycosylation of the EFSAM domain enhances the properties that promote STIM1 activation. This augmentation occurs through changes in structure localized near the Asn131 and Asn171 sites that together permeate through the protein core and lead to decreased Ca2 + binding affinity, reduced stability and enhanced oligomerization. Congruently, Ca2 + influx via SOCE in HEK293 cells co-expressing Orai1 and STIM1 was diminished when N-glycosylation was blocked by introducing Asn131Gln and Asn171Gln mutations. Collectively, our data suggests that N-glycosylation enhances the EFSAM destabilization-coupled oligomerization in response to ER Ca2 + depletion thereby augmenting the role of STIM1 as a robust ON/OFF regulator of SOCE. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Display Omitted
Keywords: EFSAM; Stromal interaction molecule-1; Store-operated calcium entry; N-glycosylation; Nuclear magnetic resonance spectroscopy; Stability;

Store-operated Ca2 + entry (SOCE) is a major mechanism for the regulation of intracellular Ca2 + homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca2 + imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Orai1; Orai2; Calcium entry; Proliferation; Expression; Migration; HL60 cells;

Overexpression of STIM1 in neurons in mouse brain improves contextual learning and impairs long-term depression by Łukasz Majewski; Filip Maciąg; Paweł M. Boguszewski; Iga Wasilewska; Grzegorz Wiera; Tomasz Wójtowicz; Jerzy Mozrzymas; Jacek Kuznicki (1071-1087).
STIM1 is an endoplasmic reticulum calcium sensor that is involved in several processes in neurons, including store-operated calcium entry. STIM1 also inhibits voltage-gated calcium channels, such as Cav1.2 and Cav3.1, and is thus considered a multifunctional protein. The aim of this work was to investigate the ways in which transgenic neuronal overexpression of STIM1 in FVB/NJ mice affects animal behavior and the electrophysiological properties of neurons in acute hippocampal slices. We overexpressed STIM1 from the Thy1.2 promoter and verified neuronal expression by quantitative reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry. Mature primary hippocampal cultures expressed STIM1 but exhibited no changes in calcium homeostasis. Basal synaptic transmission efficiency and short-term plasticity were comparable in slices that were isolated from transgenic mice, similarly as the magnitude of long-term potentiation. However, long-term depression that was induced by the glutamate receptor 1/5 agonist (S)-3,5-dihydroxyphenylglycine was impaired in STIM1 slices. Interestingly, transgenic mice exhibited a decrease in anxiety-like behavior and improvements in contextual learning. In summary, our data indicate that STIM1 overexpression in neurons in the brain perturbs metabotropic glutamate receptor signaling, leading to impairments in long-term depression and alterations in animal behavior. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: STIMs; ORAIs; SOCE; LTD; mGluR; Behavior;

Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization by Azra Alajbegovic; Karolina M. Turczyńska; Tran Thi Hien; Pilar Cidad; Karl Swärd; Per Hellstrand; Alessandro Della Corte; Amalia Forte; Sebastian Albinsson (1088-1098).
The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA.In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve.By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs.In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying vascular disease states, such as aortic aneurysm, as well as novel ideas regarding therapeutic strategies. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Vascular disease; Smooth muscle; Phenotype; Actin polymerization; MRTF-A; MicroRNA;

Calcium, oxidative stress and connexin channels, a harmonious orchestra directing the response to radiotherapy treatment? by Elke Decrock; Delphine Hoorelbeke; Raghda Ramadan; Tinneke Delvaeye; Marijke De Bock; Nan Wang; Dmitri V Krysko; Sarah Baatout; Geert Bultynck; An Aerts; Mathieu Vinken; Luc Leybaert (1099-1120).
Although radiotherapy is commonly used to treat cancer, its beneficial outcome is frequently hampered by the radiation resistance of tumor cells and adverse reactions in normal tissues. Mechanisms of cell-to-cell communication and how intercellular signals are translated into cellular responses, have become topics of intense investigation, particularly within the field of radiobiology. A substantial amount of evidence is available demonstrating that both gap junctional and paracrine communication pathways can propagate radiation-induced biological effects at the intercellular level, commonly referred to as radiation-induced bystander effects (RIBE). Multiple molecular signaling mechanisms involving oxidative stress, kinases, inflammatory molecules, and Ca2+ are postulated to contribute to RIBE. Ca2+ is a highly versatile and ubiquitous second messenger that regulates diverse cellular processes via the interaction with various signaling cascades. It furthermore provides a fast system for the dissemination of information at the intercellular level. Channels formed by transmembrane connexin (Cx) proteins, i.e. hemichannels and gap junction channels, can mediate the cell-to-cell propagation of increases in intracellular Ca2+ by ministering paracrine and direct cell-cell communication, respectively. We here review current knowledge on radiation-induced signaling mechanisms in irradiated and bystander cells, particularly focusing on the contribution of oxidative stress, Ca2+ and Cx channels. By illustrating the tight interplay between these different partners, we provide a conceptual framework for intercellular Ca2+ signaling as a key player in modulating the RIBE and the overall response to radiation.
Keywords: Ionizing radiation; Calcium; Oxidative stress; Connexin hemichannel; Gap junction; Bystander effect;

Shear stress enhances diastolic and systolic Ca2 + concentration in ventricular myocytes. Here, using confocal Ca2 + imaging in rat ventricular myocytes, we assessed the effects of shear stress (~ 16 dyn/cm2) on the frequency of spontaneous Ca2 + sparks and explored the mechanism underlying shear-mediated Ca2 + spark regulation. The frequency of Ca2 + sparks was immediately increased by shear stress (by ~ 80%), and increased further (by ~ 150%) during prolonged exposure (20 s). The 2-D size and duration of individual sparks were increased by shear stimulation. Inhibition of nitric oxide synthase (NOS) only partially attenuated the prolonged shear-mediated enhancement in spark frequency. Pretreatment with antioxidants significantly attenuated the short- and long-term effects of shear on spark frequency. Microtubule or nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) inhibition abolished the immediate shear-induced increase in spark frequency and suppressed the effects of prolonged exposure to shear stress by ~ 70%. Scavenging of mitochondrial reactive oxygen species (ROS) and mitochondrial uncoupling also abolished the effect of short-term shear on spark occurrence, and markedly reduced (by ~ 80%) the effects of prolonged shear. Mitochondrial ROS levels increased under shear; this was eliminated by blocking Nox2. Sarcoplasmic reticulum Ca2 + content was increased only by prolonged shear. Our data suggest that shear stress enhances ventricular spark frequency mainly via ROS generated from mitochondria through Nox2, and that NOS and higher sarcoplasmic reticulum Ca2 + concentrations may also contribute to the enhancement of Ca2 + sparks under shear stress. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Keywords: Shear stress; Ca2 + spark; Ventricular myocytes; Reactive oxygen species; Mitochondria; Nox2;