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Background14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial.AimsIn this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5.Methods and ResultsAmongst the seven 14-3-3 isoforms, only 14-3-3η (encoded byYWHAH) co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The amplitude of Nav1.5-mediated sodium peak current density significantly increased upon co-expression with difopein.ConclusionsOur findings illustrate that the direct implication of 14-3-3 in regulating Nav1.5 is not evident in the heterologous expression system used here. Potentiation of Nav1.5-mediated current by difopein could be explained by the inhibition of 14-3-3/ligand interactions with other partner proteins of the channel complex (e.g., PKA, PKC, CaMKII) and should be further investigated.

Cytosolic Ca2+ oscillations provide signaling input to several effector systems of the cell. These include neuronal development, migration and networking. Although similar signaling events are hijacked by highly aggressive cancer cells, the complexity of the ‘neuron-like’ remodeling in metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques we show that strongly metastatic prostate cancer cells acquire specific Na+/Ca2+ signature required for persistent invasion. We identify the ‘neuronal’ Na+ leak channel, NALCN, at the hot spots of the Ca2+ wave initiation and invadopodia formation. Mechanistically, NALCN associates functionally with plasmalemmal and mitochondrial Na+/Ca2+ exchangers, reactive oxygen species and store-operated channels to generate intracellular Ca2+ oscillations. In turn, this stimulates the activity of protooncogene Src kinase co-localized with NALCN, actin remodeling and secretion of proteolytic enzymes, thus increasing an invasive potential of the cancer cells and metastatic lesions in vivo (accessed in pre-clinical models). Overall, our findings provide new insight into the signaling pathway specific for metastatic cells where NALCN plays the role of the persistent invasion “launcher and controller”.

Intracellular calcium (Ca2+ ) signals are key regulators of multiple cellular functions, both healthy and physiopathological. It is therefore unsurprising that several cancers present a strong Ca2+ homeostasis deregulation. Among the various hallmarks of cancer disease, a particular role is played by metastasis, which has a critical impact on cancer patients' outcome. Importantly, Ca2+ signalling has been reported to control multiple aspects of the adaptive metastatic cancer cell behaviour, including epithelial-mesenchymal transition, cell migration, local invasion and induction of angiogenesis (see Abstract Figure). In this context Ca2+ signalling is considered to be a substantial intracellular tool that regulates the dynamicity and complexity of the metastatic cascade. In the present study we review the spatial and temporal organization of Ca2+ fluxes, as well as the molecular mechanisms involved in metastasis, analysing the key steps which regulate initial tumour spread.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca

International audience; Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8(TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca(2+)release channels.

Senescence is one of the primary responses to the activation of oncoproteins or down-regulation of tumor suppressors in normal cells and is therefore considered as being anti-tumorigenic but the mechanisms controlling this process are still much unknown. Calcium (Ca2+) plays a major role in many cellular processes and calcium channels control many of the “hallmarks of cancer” but their involvement in tumor initiation is poorly understood and remains unclear.Therefore, in this article we review some striking senescence-associated characteristics and their potential regulation by Ca2+. The main aim is to produce plausible hypothesis on how calcium homeostasis may participate in cancer-related senescence. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Calcium (Ca

Intracellular calcium (Ca2+) signals are key regulators of multiple cellular functions, both healthy and physiopathological. It is therefore unsurprising that several cancers present a strong Ca2+ homeostasis deregulation. Among the various hallmarks of cancer disease, a particular role is played by metastasis, which has a critical impact on cancer patients’ outcome. Importantly, Ca2+ signalling has been reported to control multiple aspects of the adaptive metastatic cancer cell behaviour, including epithelial–mesenchymal transition, cell migration, local invasion and induction of angiogenesis (see Abstract Figure). In this context Ca2+ signalling is considered to be a substantial intracellular tool that regulates the dynamicity and complexity of the metastatic cascade. In the present study we review the spatial and temporal organization of Ca2+ fluxes, as well as the molecular mechanisms involved in metastasis, analysing the key steps which regulate initial tumour spread.

Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.

International audience; Sodium (Na+) ions are known to regulate many signaling pathways involved in both physiological and pathological conditions. In particular, alterations in intracellular concentrations of Na+ and corresponding changes in membrane potential are known to be major actors of cancer progression to metastatic phenotype. Though the functionality of Na+ channels and the corresponding Na+ currents can be investigated using the patch-clamp technique, the latter is rather invasive and a technically difficult method to study intracellular Na+ transients compared to Na+ fluorescence imaging. Despite the fact that Na+ signaling is considered an important controller of cancer progression, only few data using Na+ imaging approaches are available so far, suggesting the persisting challenge within the scientific community. In this study, we describe in detail the approach for application of Na+ imaging technique to measure intracellular Na+ variations in human prostate cancer cells. Accordingly, we used three Na+-specific fluorescent dyes-Na+-binding benzofuran isophthalate (SBFI), CoroNa™ Green (Corona) and Asante NaTRIUM Green-2 (ANG-2). These dyes have been assessed for optimal loading conditions, dissociation constant and working range after different calibration methods, and intracellular Na+ sensitivity, in order to determine which probe can be considered as the most reliable to visualize Na+ fluctuations in vitro.