BBA - Molecular Cell Research (v.1833, #7)

The 12th meeting of the European Calcium Society by Jacques Haiech; Claus W. Heizmann; Joachim Krebs (iv-v).

Calcium - a central regulator of pollen germination and tube growth by Leonie Steinhorst; Jörg Kudla (1573-1581).
Pollen tubes grow rapidly by very fast rates and reach extended lengths to bring about fertilization during plant reproduction. The pollen tube grows exclusively at its tip. Fundamental for such local, tip-focused growth are the presence of internal gradients and transmembrane fluxes of ions. Consequently, vegetative pollen tube cells are an excellent single cell model system to investigate cell biological processes of vesicle transport, cytoskeleton reorganization and regulation of ion transport. The second messenger Ca2+ has emerged as a central and crucial modulator that not only regulates but also integrates the coordination each of these processes. In this review we reflect on recent advances in our understanding of the mechanisms of Ca2+ function in pollen tube growth, focusing on its role in basic cellular processes such as control of cell growth, vesicular transport and intracellular signaling by localized gradients of second messengers. In particular we discuss new insights into the identity and role of Ca2+ conductive ion channels and present experimental addressable hypotheses about their regulation. This article is part of a Special Issue entitled:12th European Symposium on Calcium.► A tip-focused calcium gradient is required for pollen tube growth. ► Calcium is involved in regulation of cytoskeleton and vesicle dynamics in pollen. ► We address potential mechanisms for decoding of calcium signals in pollen. ► We hypothesize that calcium-dependent kinases regulate ion channels in pollen. ► We discuss interconnections between different signaling networks in pollen tubes.
Keywords: Pollen tube; Polar growth; Calcium signaling; Ion channels; Vesicular trafficking; Actin dynamics;

Calcium (Ca2 +) is a major second messenger in plant signal transduction mediating stress- and developmental processes. Plant Ca2 +-dependent protein kinases (CDPKs) are mono-molecular Ca2 +-sensor/protein kinase effector proteins, which perceive Ca2 + signals and translate them into protein phosphorylation and thus represent an ideal tool for signal transduction. This review focuses on recent developments in CDPK structural analysis and CDPK in vivo phosphorylation substrate identification. We discuss mechanisms implicated in the in vivo regulation of CDPK activity including Ca2 + binding to the CDPK EF-hands, Ca2 +-triggered intra-molecular conformation changes, and CDPK (auto)-phosphorylation. Moreover, we address regulation and integration into signaling cascades of selected members of the plant CDPK family, for which in vivo function and phosphorylation in abiotic and biotic stress signaling have been demonstrated. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Structure of Ca2 +-dependent protein kinases CDPKs ► Ca2 + and phosphorylation-dependent regulation of CDPKs ► EF-hand as calcium binding side in CDPKs and regulatory motif ► CDPK in vivo functions and implication in signaling networks
Keywords: CDPK; Calcium signaling; EF-hand; Protein kinase; (Auto)-phosphorylation; Stress signal transduction;

Relationships between calcium and sphingolipid-dependent signalling pathways during the early steps of plant–pathogen interactions by Patrice Thuleau; Didier Aldon; Valérie Cotelle; Christian Brière; Benoit Ranty; Jean-Philippe Galaud; Christian Mazars (1590-1594).
An increase in cellular calcium ion (Ca2 +) concentration is now acknowledged to be one of the earliest events occurring during the induction of plant defence responses to a wide variety of pathogens. Sphingoid long-chain bases (LCBs) have also been recently demonstrated to be important mediators of defence-related programmed cell death during pathogen attack. Here, we present recent data highlighting how Ca2 + and LCBs may be interconnected to regulate cellular processes which lead either to plant susceptibility or to resistance mechanisms. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Ca2 + signal is a key early event in plant responses to pathogen recognition. ► LCBs are crucial mediators of plant defence-related PCD during pathogen attack. ► CMLs are important actors of plant innate immunity. ► CPK3, 14-3-3s and MPK6 are regulators of LCB-mediated PCD.
Keywords: Calcium signalling; LCB; Calmodulin; Pseudomonas syringae; CPK;

Singularities of calcium signaling in effector T-lymphocytes by Virginie Robert; Emily Triffaux; Magali Savignac; Lucette Pelletier (1595-1602).
CD4+ helper T (Th) lymphocytes orchestrate the immune response and include several types of effectors such as Th1, Th17 and Th2 cells. They fight against intracellular, extracellular pathogens and parasites respectively. They may also cause distinct immunopathological disorders. Th1 and Th17 are implicated in the development of autoimmune diseases while Th2 cells can initiate allergic diseases. These subsets differ by their TCR-associated signaling. In addition, the regulation of intracellular calcium concentration is not the same in Th1, Th2 and 17 cells. Our group showed that Th2 cells selectively overexpressed voltage-activated calcium (Cav1)-related channels. An increasing number of groups report the presence of Cav1-related products in T-lymphocyte subsets. This is a matter of debate since these calcium channels are classically defined as activated by high cell membrane depolarization in excitable cells. However, the use of mice with ablation of some Cav1 subunits shows undoubtedly an immune phenotype raising the question of how Cav1 channels are regulated in lymphocytes. We showed that knocking down Cav1.2 and/or Cav1.3 subunits impairs the functions of Th2 lymphocytes and is beneficial in experimental models of asthma, while it has no effect on Th1 cell functions. Beyond the role of Cav1 channels in T-lymphocytes, the identification of key components selectively implicated in one or the other T cell subset paves the way for the design of new selective therapeutic targets in the treatment of immune disorders while preserving the other T-cell subsets. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Intracellular calcium concentration is differentially regulated in T-cell subsets. ► Cav1 channels contribute to calcium signaling in T-cells. ► The profile of expression of Cav1 channels depends on the state of T-cell activation. ► The singularity of Ca2 + regulation in T-cell subsets is of clinical interest.
Keywords: CD4+; Th1; Th2; Intracellular calcium concentration; Cav1 calcium channels;

Killing cancer cells by cytotoxic T lymphocytes (CTL) and by natural killer (NK) cells is of vital importance. Cancer cell proliferation and apoptosis depend on the intracellular Ca2 + concentration, and the expression of numerous ion channels with the ability to control intracellular Ca2 + concentrations has been correlated with cancer. A rise of intracellular Ca2 + concentrations is also required for efficient CTL and NK cell function and thus for killing their targets, in this case cancer cells. Here, we review the data on Ca2 +-dependent killing of cancer cells by CTL and NK cells. In addition, we discuss emerging ideas and present a model how Ca2 + may be used by CTL and NK cells to optimize their cancer cell killing efficiency. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Cytotoxic T lymphocyte (CTL) and natural killer (NK) cells kill cancer cells. ► Calcium modulates killing of cancer cells by CTL and NK cells. ► Several steps of the actual killing process are calcium dependent. ► A model is discussed how calcium influences the cancer-immune interaction.
Keywords: Calcium; Cancer; Killing; Cytotoxic T lymphocytes; Natural killer cells; ORAI calcium channels;

Regulation of inositol 1,4,5-trisphosphate receptors during endoplasmic reticulum stress by Santeri Kiviluoto; Tim Vervliet; Hristina Ivanova; Jean-Paul Decuypere; Humbert De Smedt; Ludwig Missiaen; Geert Bultynck; Jan B. Parys (1612-1624).
The endoplasmic reticulum (ER) performs multiple functions in the cell: it is the major site of protein and lipid synthesis as well as the most important intracellular Ca2 + reservoir. Adverse conditions, including a decrease in the ER Ca2 + level or an increase in oxidative stress, impair the formation of new proteins, resulting in ER stress. The subsequent unfolded protein response (UPR) is a cellular attempt to lower the burden on the ER and to restore ER homeostasis by imposing a general arrest in protein synthesis, upregulating chaperone proteins and degrading misfolded proteins. This response can also lead to autophagy and, if the stress can not be alleviated, to apoptosis. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and IP3-induced Ca2 + signaling are important players in these processes. Not only is the IP3R activity modulated in a dual way during ER stress, but also other key proteins involved in Ca2 + signaling are modulated. Changes also occur at the structural level with a strengthening of the contacts between the ER and the mitochondria, which are important determinants of mitochondrial Ca2 + uptake. The resulting cytoplasmic and mitochondrial Ca2 + signals will control cellular decisions that either promote cell survival or cause their elimination via apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► ER stress is a condition of disturbed ER function. ► ER stress can lead to autophagy or apoptosis. ► Autophagy and apoptosis are affected by intracellular Ca2 + signaling. ► The activity of IP3Rs and other Ca2 +-handling proteins is modulated during ER stress. ► Ca2 + signals participate in cell-fate determination subsequently to ER stress.
Keywords: ER stress; IP3 receptor; Ca2 + signaling; Unfolded protein response; Apoptosis; Autophagy;

Pathology of the brain is caused by the deficiency in tissue homeostasis. As the main homeostatic element of the mammalian nervous system is represented by astrocytes, these glial cells are involved in many, if not all, brain disorders. Diseased astrocytes undergo a variety of morphological and functional changes, including deregulation of calcium dynamics. To rectify undesirable changes in astrocytes and/or neurones that occur in disease, we postulate the future use of nanotechnology-based therapeutics. Carbon nanotubes emerged as one of the most promising advanced nanomaterials for use in neuroprosthesis. Recently, they have been used to affect morpho-functional characteristics of astrocytes. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Description of general pathophysiology of neuroglia ► Discussion on astroglial calcium signalling in neurological diseases ► Discussion on astrodegeneration and astroglial atrophy in neurological diseases ► Description of carbon nanotubes as ion channel modulators
Keywords: Astrocyte; Calcium; Carbon nanotube; Morphology; Nanotechnology; Nanomedicine;

Different optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP–aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca2+-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca2+-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca2+-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca2+-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► In-vivo bioluminescence recording of induced or spontaneous activity in the brain ► We have recorded odor-induced Ca2+-activity in the mushroom-bodies. ► Two peaks of spontaneous Ca2+-activity in the mushroom-bodies occur during the night. ► Recording overall (pan-neuronal) spontaneous brain activity during long term period ► Recorded spontaneous activity in glial cells is cell autonomous.
Keywords: Bioluminescence; Calcium; Drosophila; GFP–aequorin; Glial cell; Mushroom-body; Pan-neuronal;

Characterization of Ca2 + signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development by Michael Y.F. Yuen; Sarah E. Webb; Ching Man Chan; Bernard Thisse; Christine Thisse; Andrew L. Miller (1641-1656).
Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca2 + reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca2 + waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~ 4.5 h post-fertilization: hpf), and ending at the shield stage (~ 6.0 hpf) these distinct classes of waves propagated at mean velocities of ~ 50 and ~ 4 μm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca2 + wave propagation occurred only when this was <~ 8 μm; an IND >~ 8 μm was coincidental with wave termination at shield stage. Treatment with the IP3R antagonist, 2-APB, the Ca2 + buffer, 5,5′-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca2 + waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca2 + waves are generated mainly via Ca2 + release from IP3Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► A series of Ca2 + waves is propagated within the E-YSL of zebrafish embryos. ► Wave propagation starts at dome stage to 30% epiboly when the E-YSN form clusters. ► Waves are generated via Ca2 + release from IP3Rs, possibly in the perinuclear ER. ► At shield stage, increasing inter-nuclear distances resulted in wave termination. ► Ca2 + waves never propagated in the I-YSL or between the E- and I-YSL.
Keywords: Ca2 + wave; Blastula/gastrula period; Nuclei clustering; Perinuclear endoplasmic reticulum; Yolk syncytial layer; Zebrafish;

Capturing single L-type Ca2 + channel function with optics by Matthew A. Nystoriak; Madeline Nieves-Cintrón; Manuel F. Navedo (1657-1664).
Advances in imaging technology have allowed optical analysis of Ca2 +-permeable ion channel activity. Here, we briefly review novel developments in optical recording of L-type voltage-dependent Ca2 + channel (LTCC) function with high spatial and temporal resolution. Underlying principles supporting the use of total internal reflection fluorescence (TIRF) microscopy for optical measurement of channel activity and new functional characteristics of LTCCs revealed by application of this approach are discussed. Visualization of Ca2 + influx through single LTCCs (“LTCC sparklets”) has demonstrated that channel activity is regionally heterogeneous and that clustered channels are capable of operating in a cooperative, or “coupled” manner. In light of these findings, we describe a current molecular model for the local control of LTCC activity and coupled gating in physiological and pathological contexts. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Advances of imaging technology have allowed optical analysis of L-type Ca2 + channels with high resolution. ► L-type Ca2 + channel activity varies regionally throughout the plasmalemma of excitable cells. ► A subpopulation of L-type Ca2 + channels can operate in a coupled manner with profound implications in health and disease. ► Emerging Ca2 + indicators are promising developments in understanding nano- and microdomain Ca2 + signals.
Keywords: Calcium sparklet; Coupled gating; TIRF;

Numerous Ca2 + signaling events have been associated with early development of vertebrate embryo, from fertilization to organogenesis. In Xenopus laevis, Ca2 + signals are key regulators in the earliest steps of the nervous system development. If neural determination is one of the best-characterized examples of the role of Ca2 + during embryogenesis, increasing literature supports a determining role of organogenesis and differentiation. In blastula the cells of the presumptive ectoderm (animal caps) are pluripotent and can be induced toward neural fate with an intracellular increase of free Ca2 + triggered by caffeine. To identify genes that are transcribed early upon Ca2 + stimuli and involved in neural determination, we have constructed a subtractive cDNA library between neuralized and non-neuralized animal caps. Here we present the expression pattern of three new Ca2 +-sensitive genes: fus (fused in sarcoma), brd3 (bromodomain containing 3) and wdr5 (WD repeat domain 5) as they all represent potential regulators of the transcriptional machinery. Using in situ hybridization we illustrated the spatial expression pattern of fus, brd3 and wdr5 during early developmental stages of Xenopus embryos. Strikingly, their domains of expression are not restricted to neural territories. They all share a specific expression throughout renal organogenesis which has been found to rely also on Ca2 + signaling. This therefore highlights the key function of Ca2 + target genes in specific territories during early development. We propose that Ca2 + signaling through modulation of fus, brd3 and wdr5 expressions can control the transcription machinery to achieve proper embryogenesis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.►A subtractive library identifies fus, brd3 and wdr5 as new calcium target genes. ► We examine the expression patterns of these early calcium-sensitive target genes. ► fus, brd3 and wdr5 expressions are spatially and temporally restricted in Xenopus. ► We report the expressions of fus, brd3 and wdr5 in the developing kidney.
Keywords: Calcium-dependent target gene; Development; Neurogenesis; Pronephros; Kidney; Xenopus;

Both synaptic N-methyl-d-aspartate (NMDA) receptors and voltage-operated calcium channels (VOCCs) have been shown to be critical for nuclear calcium signals associated with transcriptional responses to bursts of synaptic input. However the direct contribution to nuclear calcium signals from calcium influx through NMDA receptors and VOCCs has been obscured by their concurrent roles in action potential generation and synaptic transmission. Here we compare calcium responses to synaptically induced bursts of action potentials with identical bursts devoid of any synaptic contribution generated using the pre-recorded burst as the voltage clamp command input to replay the burst in the presence of blockers of action potentials or ionotropic glutamate receptors. Synapse independent replays of bursts produced nuclear calcium responses with amplitudes around 70% of their original synaptically generated signals and were abolished by the L-type VOCC blocker, verapamil. These results identify a major direct source of nuclear calcium from local L-type VOCCs whose activation is boosted by NMDA receptor dependent depolarization. The residual component of synaptically induced nuclear calcium signals which was both VOCC independent and NMDA receptor dependent showed delayed kinetics consistent with a more distal source such as synaptic NMDA receptors or internal stores. The dual requirement of NMDA receptors and L-type VOCCs for synaptic activity-induced nuclear calcium dependent transcriptional responses most likely reflects a direct somatic calcium influx from VOCCs whose activation is amplified by synaptic NMDA receptor-mediated depolarization and whose calcium signal is boosted by a delayed input from distal calcium sources mostly likely entry through NMDA receptors and release from internal stores. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► We used ‘replay of synaptic activity’ to study nuclear calcium signaling. ► We analyzed the role of different calcium entry sites to nuclear calcium signaling. ► We observed a dual requirement for NMDA receptors and L-type calcium channels.
Keywords: NMDA receptor; L-type voltage-operated calcium channel; Nuclear calcium; Synapse-to-nucleus communication; Neuronal calcium signaling; Intracellular calcium store;

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca2 + and Na+ ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na+ via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na+, the membrane blebbing was completely inhibited, but Ca2 + influx did not change under these conditions. Na+ influx was modulated by the intracellular Ca2 + concentration ([Ca2 +]i). Elevation of [Ca2 +]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca2 +, capsaicin caused only little increase in [Ca2 +]i indicating that the increase in [Ca2 +]i observed after capsaicin application is derived essentially from extracellular Ca2 + and not from internal Ca2 + stores. In the absence of extracellular Ca2 + also the process of cell swelling was considerably slower. Calretinin is a Ca2 + buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca2 + signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca2 + and Na+ influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca2 +-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.► Influx of Ca2 + and Na+ via TRPV1 causes cell swelling and membrane bleb formation. ► Elevation of [Ca2 +]i sensitizes/activates TRPV1 channels augmenting cell swelling. ► Calretinin expression diminishes capsaicin-induced cell volume increases. ► Acute capsaicin toxicity results from impaired cell membrane integrity, not apoptosis.
Keywords: TRPV1; Membrane bleb formation; Calretinin; Calcium-binding proteins; Sensory neurons;

Alzheimer's disease (AD) is the most widespread, age-related neurodegenerative disorder. Its two subtypes are sporadic AD (SAD) of unknown etiology and genetically encoded familial AD (FAD). The onset of AD is often preceded by mild cognitive impairment (MCI). Calcium dynamics were found to be dysregulated in FAD models, but little is known about the features of calcium dynamics in SAD. To explore calcium homeostasis during the early stages of SAD, we investigated store-operated calcium entry (SOCE) and inositol triphosphate receptor (IP3R)-mediated calcium release into the cytoplasm in unmodified B lymphocytes from MCI and SAD patients and compared them with non-demented subjects (NDS). Calcium levels in the endoplasmic reticulum and both the rising and falling SOCE slopes were very similar in all three groups. However, we found that SAD and MCI cells were more prone to IP3R activation than NDS cells, and increases in calcium levels in the cytoplasm were almost twice as frequent in SAD cells than in NDS cells. MCI cells and SAD cells exhibited an enhanced magnitude of calcium influx during SOCE. MCI cells but not SAD cells were characterized by higher basal cellular calcium levels than NDS cells. In summary, perturbed calcium homeostasis was observed in peripheral cells from MCI and SAD patients. Thus, lymphocytes obtained from MCI subjects may be promising in the early diagnosis of individuals who will eventually develop SAD. However, no conclusions are made regarding SAD due to the limited number patients. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► In lymphocytes from MCI and SAD patients calcium homeostasis is perturbed. ► Basal calcium levels and SOCE influx are higher in MCI than in non-demented people. ► Response to IP3R stimulation is increased in SAD and MCI cells.
Keywords: Alzheimer's disease; Calcium; Inositol triphosphate receptor; B lymphocytes; Mild cognitive impairment; Store-operated calcium entry;

Disruption of the annexin A1/S100A11 complex increases the migration and clonogenic growth by dysregulating epithelial growth factor (EGF) signaling by Michaela Poeter; Susanne Radke; Meryem Koese; Florian Hessner; Anika Hegemann; Agnes Musiol; Volker Gerke; Thomas Grewal; Ursula Rescher (1700-1711).
Endocytosis of activated growth factor receptors regulates spatio-temporal cellular signaling. In the case of the EGF receptor, sorting into multivesicular bodies (MVBs) controls signal termination and subsequently leads to receptor degradation in lysosomes. Annexin A1, a Ca2 +-regulated membrane binding protein often deregulated in human cancers, interacts with the EGF receptor and is phosphorylated by internalized EGF receptor on endosomes. Most relevant for EGF receptor signal termination, annexin A1 is required for the formation of internal vesicles in MVBs that sequester ligand-bound EGF receptor away from the limiting membrane. To elucidate the mechanism underlying annexin A1-dependent EGF receptor trafficking we employed an N-terminally truncated annexin A1 mutant that lacks the EGF receptor phosphorylation site and the site for interaction with its protein ligand S100A11. Overexpression of this dominant-negative mutant induces a delay in EGF-induced EGF receptor transport to the LAMP1-positive late endosomal/lysosomal compartment and impairs ligand-induced EGF receptor degradation. Consistent with these findings, EGF-stimulated EGF receptor and MAP kinase pathway signaling is prolonged. Importantly, depletion of S100A11 also results in a delayed EGF receptor transport and prolonged MAP kinase signaling comparable to the trafficking defect observed in cells expressing the N-terminally truncated annexin A1 mutant. These results strongly suggest that the function of annexin A1 as a regulator of EGF receptor trafficking, degradation and signaling is critically mediated through an N-terminal interaction with S100A11 in the endosomal compartment. This interaction appears to be essential for lysosomal targeting of the EGF receptor, possibly by providing a physical scaffold supporting inward vesiculation in MVBs. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► We interfered with AnxA1/S100A11 complex formation in HeLa cells ► Delayed EGFR transport to late endosomes and degradation was observed ► This was associated with prolonged EGF-induced EGFR and MAPK signaling ► These cells showed enhanced cell migration and clonogenicity ► We conclude that EGF signal termination is balanced by the AnxA1/S100A11 complex
Keywords: Annexin A1; S100A11; Epidermal growth factor receptor; Endocytosis; Late endosomes; MAP kinase signaling;

Human S100A3 tetramerization propagates Ca2 +/Zn2 + binding states by Kenji Kizawa; Yuji Jinbo; Takafumi Inoue; Hidenari Takahara; Masaki Unno; Claus W. Heizmann; Yoshinobu Izumi (1712-1719).
The S100A3 homotetramer assembles upon citrullination of a specific symmetric Arg51 pair on its homodimer interface in human hair cuticular cells. Each S100A3 subunit contains two EF-hand-type Ca2 +-binding motifs and one (Cys)3His-type Zn2 +-binding site in the C-terminus. The C-terminal coiled domain is cross-linked to the presumed docking surface of the dimeric S100A3 via a disulfide bridge. The aim of this study was to determine the structural and functional role of the C-terminal Zn2 +-binding domain, which is unique to S100A3, in homotetramer assembly. The binding of either Ca2 + or Zn2 + reduced the α-helix content of S100A3 and modulated its affinity for the other cation. The binding of a single Zn2 + accelerated the Ca2 +-dependent tetramerization of S100A3 while inducing an extensive unfolding of helix IV. The Ca2 + and Zn2 + binding affinities of S100A3 were enhanced when the other cation bound in concert with the tetramerization of S100A3. Small angle scattering analyses revealed that the overall structure of the S100A3 tetramer bound both Ca2 + and Zn2 + had a similar molecular shape to the Ca2 +-bound form in solution. The binding states of the Ca2 + or Zn2 + to each S100A3 subunit within a homotetramer appear to be propagated by sensing the repositioning of helix III and the rearrangement of the C-terminal tail domain. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► We explored functional role of the C-terminal Zn2 +-binding domain unique to S100A3. ► Affinity to Ca2 + and Zn2 + of S100A3 was enhanced when the other cation bound. ► A single Zn2 + binding accelerates Ca2 +-dependent S100A3 tetramerization. ► Overall shape of an S100A3 tetramer is predicted by small angle scattering analyses. ► Cation binding states to each S100A3 subunit propagate within a homotetramer.
Keywords: Calcium; EF-hand; Citrullination; S100 protein; Small angle scattering; Zinc;

A general framework to characterize inhibitors of calmodulin: Use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains by Emilie Audran; Rania Dagher; Sophie Gioria; Philipp O. Tsvetkov; Alexandra A. Kulikova; Bruno Didier; Pascal Villa; Alexander A. Makarov; Marie-Claude Kilhoffer; Jacques Haiech (1720-1731).
The prominent role of Ca2 + in cell physiology is mediated by a whole set of proteins involved in Ca2 +-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells.In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM.Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent.The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► General strategy finding and characterizing antagonists of protein–protein interactions ► Use of fluorescence polarization to study CaM/target peptide interactions ► CaM antagonists differentiate CaM target peptides complexes. ► New avenue for CaM pharmacology
Keywords: Fluorescence polarization; Fluorescence anisotropy; High-throughput screening; CaM antagonists; CaM inhibitors; CaM pharmacology;

Calcium sensing receptor signalling in physiology and cancer by Sarah C. Brennan; Ursula Thiem; Susanne Roth; Abhishek Aggarwal; Irfete Sh. Fetahu; Samawansha Tennakoon; Ana Rita Gomes; Maria Luisa Brandi; Frank Bruggeman; Romuald Mentaverri; Daniela Riccardi; Enikö Kallay (1732-1744).
The calcium sensing receptor (CaSR) is a class C G-protein-coupled receptor that is crucial for the feedback regulation of extracellular free ionised calcium homeostasis. While extracellular calcium (Ca2 + o) is considered the primary physiological ligand, the CaSR is activated physiologically by a plethora of molecules including polyamines and l-amino acids. Activation of the CaSR by different ligands has the ability to stabilise unique conformations of the receptor, which may lead to preferential coupling of different G proteins; a phenomenon termed ‘ligand-biased signalling’. While mutations of the CaSR are currently not linked with any malignancies, altered CaSR expression and function are associated with cancer progression. Interestingly, the CaSR appears to act both as a tumour suppressor and an oncogene, depending on the pathophysiology involved. Reduced expression of the CaSR occurs in both parathyroid and colon cancers, leading to loss of the growth suppressing effect of high Ca2 + o. On the other hand, activation of the CaSR might facilitate metastasis to bone in breast and prostate cancer. A deeper understanding of the mechanisms driving CaSR signalling in different tissues, aided by a systems biology approach, will be instrumental in developing novel drugs that target the CaSR or its ligands in cancer. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► The calcium-sensing receptor (CaSR) is a key controller for Ca2 + o levels ► The CaSR is associated with both anti- and pro-proliferative signals ► Altered CaSR expression/signalling is associated with some forms of cancer ► A systems biology approach may help unravel CaSR-mediated signalling/interactions ► Targeting the CaSR may lead to novel cancer therapeutics
Keywords: Parathyroid adenoma; Colorectal cancer; Breast cancer; Prostate cancer; Systems biology; Ligand-biased signalling;

The role of calcium in VDAC1 oligomerization and mitochondria-mediated apoptosis by Nurit Keinan; Hadas Pahima; Danya Ben-Hail; Varda Shoshan-Barmatz (1745-1754).
The voltage-dependent anion channel (VDAC), located at the outer mitochondria membrane (OMM), mediates interactions between mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca2 +, and metabolites. Substantial evidence points to VDAC1 as being a key player in apoptosis, regulating the release of apoptogenic proteins from mitochondria, such as cytochrome c, and interacting with anti-apoptotic proteins. Recently, we demonstrated that VDAC1 oligomerization is a general mechanism common to numerous apoptogens acting via different initiating cascades and proposed that a protein-conducting channel formed within a VDAC1 homo/hetero oligomer mediates cytochrome c release. However, the molecular mechanism responsible for VDAC1 oligomerization remains unclear. Several studies have shown that mitochondrial Ca2 + is involved in apoptosis induction and that VDAC1 possesses Ca2 +-binding sites and mediates Ca2 + transport across the OMM. Here, the relationship between the cellular Ca2 + level, [Ca2 +]i, VDAC1 oligomerization and apoptosis was studied. Decreasing [Ca2 +]i using the cell-permeable Ca2 + chelating reagent BAPTA-AM was found to inhibit VDAC1 oligomerization and apoptosis, while increasing [Ca2 +]i using Ca2 + ionophore resulted in VDAC1 oligomerization and apoptosis induction in the absence of apoptotic stimuli. Moreover, induction of apoptosis elevated [Ca2 +]i, concomitantly with VDAC1 oligomerization. AzRu-mediated inhibition of mitochondrial Ca2 + transport decreased VDAC1 oligomerization, suggesting that mitochondrial Ca2 + is required for VDAC1 oligomerization. In addition, increased [Ca2 +]i levels up-regulate VDAC1 expression. These results suggest that Ca2 + promotes VDAC1 oligomerization via activation of a yet unknown signaling pathway or by increasing VDAC1 expression, leading to apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Keywords: Apoptosis; Ca2 +; Mitochondria; Oligomerization; VDAC1;

Non-apoptotic roles of Bcl-2 family: The calcium connection by Benjamin Bonneau; Julien Prudent; Nikolay Popgeorgiev; Germain Gillet (1755-1765).
The existence of the bcl-2 (B-cell lymphoma-2) gene was reported nearly 30 years ago. Yet, Bcl-2 family group of proteins still surprises us with their structural and functional diversity. Since the discovery of the Bcl-2 family of proteins as one of the main apoptosis judges, the precise mechanism of their action remains a hot topic of intensive scientific research and debates. Although extensive work has been performed on the role of mitochondria in apoptosis, more and more studies point out an implication of the endoplasmic reticulum in this process. Interestingly, Bcl-2 family proteins could be localized to both the mitochondria and the endoplasmic reticulum highlighting their crucial role in apoptosis control. In particular, in these organelles Bcl-2 proteins seem to be involved in calcium homeostasis regulation although the mechanisms underlying this function are still misunderstood. We now assume with high degree of certainty that the majority of Bcl-2 family members take part not only in apoptosis regulation but also in other processes important for the cell physiology briefly denominated as “non-apoptotic” functions. Drawing a complete and comprehensive image of Bcl-2 family requires the understanding of their implications in all cellular processes. Here, we review the current knowledge on the control of calcium homeostasis by the Bcl-2 family at the endoplasmic reticulum and at the mitochondria. Then we focus on the non-apoptotic functions of the Bcl-2 proteins in relation with the regulation of this versatile intracellular messenger. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.Display Omitted► Bcl-2 proteins are acknowledged to be the key regulators of apoptosis. ► The versatile second messenger calcium can induce apoptosis by different pathways. ► Bcl-2 proteins regulate Ca2 + homeostasis at the ER and at the mitochondria. ► Beyond apoptosis, Bcl-2 proteins are involved in other cellular processes. ► Ca2 + homeostasis regulation may be a common mechanism for these non-apoptotic roles.
Keywords: Bcl-2; Calcium; Endoplasmic reticulum; Mitochondria; Non-apoptotic; Apoptosis;

Calcium/calmodulin-mediated regulation of plant immunity by Cécilia Cheval; Didier Aldon; Jean-Philippe Galaud; Benoît Ranty (1766-1771).
Calcium is a universal messenger involved in the modulation of diverse developmental and adaptive processes in response to various physiological stimuli. Ca2 + signals are represented by stimulus-specific Ca2 + signatures that are sensed and translated into proper cellular responses by diverse Ca2 + binding proteins and their downstream targets. Calmodulin (CaM) and calmodulin-like (CML) proteins are primary Ca2 + sensors that control diverse cellular functions by regulating the activity of various target proteins. Recent advances in our understanding of Ca2 +/CaM-mediated signalling in plants have emerged from investigations into plant defence responses against various pathogens. Here, we focus on significant progress made in the identification of CaM/CML-regulated components involved in the generation of Ca2 + signals and Ca2 +-dependent regulation of gene expression during plant immune responses. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► Ca2 + signals play critical roles in regulating plant defense responses against pathogens. ► Calmodulin and calmodulin-like proteins are key regulators of plant immune responses. ► Calmodulin targets regulate pathogen-induced changes in gene expression.
Keywords: Calcium signalling; Signal transduction; Calmodulin-binding proteins; Defence responses; Plant-microbe interactions; Transcriptional regulation;

IP3, a small molecule with a powerful message by Elke Decrock; Marijke De Bock; Nan Wang; Ashish K. Gadicherla; Mélissa Bol; Tinneke Delvaeye; Peter Vandenabeele; Mathieu Vinken; Geert Bultynck; Dmitri V. Krysko; Luc Leybaert (1772-1786).
Research conducted over the past two decades has provided convincing evidence that cell death, and more specifically apoptosis, can exceed single cell boundaries and can be strongly influenced by intercellular communication networks. We recently reported that gap junctions (i.e. channels directly connecting the cytoplasm of neighboring cells) composed of connexin43 or connexin26 provide a direct pathway to promote and expand cell death, and that inositol 1,4,5-trisphosphate (IP3) diffusion via these channels is crucial to provoke apoptosis in adjacent healthy cells. However, IP3 itself is not sufficient to induce cell death and additional factors appear to be necessary to create conditions in which IP3 will exert proapoptotic effects. Although IP3-evoked Ca2 + signaling is known to be required for normal cell survival, it is also actively involved in apoptosis induction and progression. As such, it is evident that an accurate fine-tuning of this signaling mechanism is crucial for normal cell physiology, while a malfunction can lead to cell death. Here, we review the role of IP3 as an intracellular and intercellular cell death messenger, focusing on the endoplasmic reticulum-mitochondrial synapse, followed by a discussion of plausible elements that can convert IP3 from a physiological molecule to a killer substance. Finally, we highlight several pathological conditions in which anomalous intercellular IP3/Ca2 + signaling might play a role. This article is part of a Special Issue entitled:12th European Symposium on Calcium.► Gap junctions (GJs) provide a direct pathway to expand cell death. ► IP3 transfer through GJs is crucial to provoke bystander apoptosis. ► Additional factors are necessary to convert IP3 into a toxic molecule. ► Various GJ-permeable factors could sensitize cells to IP3-induced apoptosis. ► Intercellular IP3/Ca2 + signaling can play a role in different pathologies.
Keywords: Inositol 1,4,5-trisphosphate; Calcium; Intercellular communication; Cell death; Connexin; Gap junction;

Genetically encoded Ca2 + indicators: Properties and evaluation by Vadim Pérez Koldenkova; Takeharu Nagai (1787-1797).
Genetically encoded calcium ion (Ca2 +) indicators have become very useful and widely used tools for Ca2 + imaging, not only in cellular models, but also in living organisms. However, the in vivo and in situ characterization of these indicators is tedious and time consuming, and it does not provide information regarding the suitability of an indicator for particular experimental environments. Thus, initial in vitro evaluation of these tools is typically performed to determine their properties. In this review, we examined the properties of dynamic range, affinity, selectivity, and kinetics for Ca2 + indicators. Commonly used strategies for evaluating these properties are presented. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.► In vivo evaluation of genetically encoded Ca2 + indicators (GECIs) is an important but time-taking procedure. ► Usually, a preliminary in vitro evaluation is performed, to determine their properties. ► These properties of GECIs, dynamic range, affinity, selectivity, kinetics, are described.
Keywords: Genetically encoded calcium ion indicators; Ca2 + imaging; Fluorescent proteins; FRET; Ratiometric; Intensiometric;