Signaling Toolkit Research Articles

Genome-Wide Identification and Expression Profiling of CBL-CIPK Gene Family in Pineapple (Ananas comosus) and the Role of AcCBL1 in Abiotic and Biotic Stress Response.

Ca2+ serves as a ubiquitous second messenger regulating several aspects of plant growth and development. A group of unique calcium sensor proteins, calcineurin B-like (CBL), interact with CBL-interacting protein kinases (CIPKs) to decode the Ca2+ signature inside the cell. Although CBL-CIPK signaling toolkit has been shown to play significant roles in the responses to numerous stresses in different plants, the information about pineapple CBL-CIPK remains obscure. In the present study, a total of eight AcCBL and 21 AcCIPK genes were identified genome-wide in pineapple. The identified genes were renamed on the basis of gene ID in ascending order and phylogenetic analysis divided into five groups. Transcriptomic data analysis showed that AcCBL and AcCIPK genes were expressed differentially in different tissues. Further, the expression analysis of AcCBL1 in different tissues showed significant changes under various abiotic stimuli. Additionally, the ectopic expression of AcCBL1 in Arabidopsis resulted in enhanced tolerance to salinity, osmotic, and fungal stress. The present study revealed the crucial contribution of the CBL-CIPK gene in various biological and physiological processes in pineapple.

Cytosolic Ca2+ Buffers Are Inherently Ca2+ Signal Modulators.

For precisely regulating intracellular Ca2+ signals in a time- and space-dependent manner, cells make use of various components of the "Ca2+ signaling toolkit," including Ca2+ entry and Ca2+ extrusion systems. A class of cytosolic Ca2+-binding proteins termed Ca2+ buffers serves as modulators of such, mostly short-lived Ca2+ signals. Prototypical Ca2+ buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Although initially considered to function as pure Ca2+ buffers, that is, as intracellular Ca2+ signal modulators controlling the shape (amplitude, decay, spread) of Ca2+ signals, evidence has accumulated that calbindin-D28k and calretinin have additional Ca2+ sensor functions. These other functions are brought about by direct interactions with target proteins, thereby modulating their targets' function/activity. Dysregulation of Ca2+ buffer expression is associated with several neurologic/neurodevelopmental disorders including autism spectrum disorder (ASD) and schizophrenia. In some cases, the presence of these proteins is presumed to confer a neuroprotective effect, as evidenced in animal models of Parkinson's or Alzheimer's disease.

Regulation of Membrane Turnover by Phosphatidic Acid: Cellular Functions and Disease Implications.

Phosphatidic acid (PA) is a simple glycerophospholipid with a well-established role as an intermediate in phospholipid biosynthesis. In addition to its role in lipid biosynthesis, PA has been proposed to act as a signaling molecule that modulates several aspects of cell biology including membrane transport. PA can be generated in eukaryotic cells by several enzymes whose activity is regulated in the context of signal transduction and enzymes that can metabolize PA thus terminating its signaling activity have also been described. Further, several studies have identified PA binding proteins and changes in their activity are proposed to be mediators of the signaling activity of this lipid. Together these enzymes and proteins constitute a PA signaling toolkit that mediates the signaling functions of PA in cells. Recently, a number of novel genetic models for the analysis of PA function in vivo and analytical methods to quantify PA levels in cells have been developed and promise to enhance our understanding of PA functions. Studies of several elements of the PA signaling toolkit in a single cell type have been performed and are presented to provide a perspective on our understanding of the biochemical and functional organization of pools of PA in a eukaryotic cell. Finally, we also provide a perspective on the potential role of PA in human disease, synthesizing studies from model organisms, human disease genetics and analysis using recently developed PLD inhibitors.

The Calcium-Signaling Toolkit in Cancer: Remodeling and Targeting.

Processes that are important in cancer progression, such as sustained cell growth, invasion to other organs, and resistance to cell death inducers, have a clear overlap with pathways regulated by Ca2+ signaling. It is therefore not surprising that proteins important in Ca2+ signaling, sometimes referred to as the "Ca2+ signaling toolkit," can contribute to cancer cell proliferation and invasiveness, and the ability of agents to induce cancer cell death. Ca2+ signaling is also critical in other aspects of cancer progression, including events in the tumor microenvironment and processes involved in the acquisition of resistance to anticancer therapies. This review will consider the role of Ca2+ signaling in tumor progression and highlight areas in which a better understanding of the interplay between the Ca2+-signaling toolkit and tumorigenesis is still required.

Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells

BackgroundRemodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors.MethodsIn this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps.ResultsThree distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17β-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein.ConclusionsOur results suggest that the expression of Ca2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.

Characterization of diverse homoserine lactone synthases in Escherichia coli.

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach were still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum signaling synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.

Identification of Ca2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells (NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor (IP3R) type-1 and ryanodine receptor (RyR) type-2 are differentially expressed in cells with neuron- or radial glial-like properties. Furthermore, ATP but not caffeine (IP3R and RyR agonists, respectively), induced the generation of Ca2+ transients in cells exhibiting neuron- or glial-like morphology. These results indicate the differential expression of components of the Ca2+-signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca2+ signaling as a read-out.

MICU3 is a tissue-specific enhancer of mitochondrial calcium uptake.

The versatility and universality of Ca2+ as intracellular messenger is guaranteed by the compartmentalization of changes in [Ca2+]. In this context, mitochondrial Ca2+ plays a central role, by regulating both specific organelle functions and global cellular events. This versatility is also guaranteed by a cell type-specific Ca2+ signaling toolkit controlling specific cellular functions. Accordingly, mitochondrial Ca2+ uptake is mediated by a multimolecular structure, the MCU complex, which differs among various tissues. Its activity is indeed controlled by different components that cooperate to modulate specific channeling properties. We here investigate the role of MICU3, an EF-hand containing protein expressed at high levels, especially in brain. We show that MICU3 forms a disulfide bond-mediated dimer with MICU1, but not with MICU2, and it acts as enhancer of MCU-dependent mitochondrial Ca2+ uptake. Silencing of MICU3 in primary cortical neurons impairs Ca2+ signals elicited by synaptic activity, thus suggesting a specific role in regulating neuronal function.

Glycosylation controls sodium-calcium exchanger 3 sub-cellular localization during cell cycle

The Na+/Ca2+ exchanger (NCX) is a membrane antiporter that has been identified in the plasma membrane, the inner membrane of the nuclear envelope and in the membrane of the endoplasmic reticulum (ER). In humans, three genes have been identified, encoding unique NCX proteins. Although extensively studied, the NCX’s sub-cellular localization and mechanisms regulating the activity of different subtypes are still ambiguous. Here we investigated the subcellular localization of the NCX subtype 3 (NCX3) and its impact on the cell cycle. Two phenotypes, switching from one to the other during the cell cycle, were detected. One phenotype was NCX3 in the plasma membrane during S and M phase, and the other was NCX3 in the ER membrane during resting and interphase. Glycosylation of NCX3 at the N45 site was required for targeting the protein to the plasma membrane, and the N45 site functioned as an on-off switch for the translocation of NCX3 to either the plasma membrane or the membrane of the ER. Introduction of an N-glycosylation deficient NCX3 mutant led to an arrest of cells in the G0/G1 phase of the cell cycle. This was accompanied by accumulation of de-glycosylated NCX3 in the cytosol (that is in the ER), where it transported calcium ions (Ca2+) from the cytosol to the ER. These results, obtained in transfected HEK293T and HeLa and confirmed endogenously in SH-SY5Y cells, suggest that cells can use a dynamic Ca2+ signaling toolkit in which the NCX3 sub-cellular localization changes in synchrony with the cell cycle.

Antibody-Based Fusion Proteins Allow Ca2+ Rewiring to Most Extracellular Ligands.

The Ca2+ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca2+ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca2+ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca2+ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e., nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumor necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state, but instead only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.

De novo assembly of a transcriptome from the eggs and early embryos of Astropecten aranciacus.

Starfish have been instrumental in many fields of biological and ecological research. Oocytes of Astropecten aranciacus, a common species native to the Mediterranean Sea and the East Atlantic, have long been used as an experimental model to study meiotic maturation, fertilization, intracellular Ca2+ signaling, and cell cycle controls. However, investigation of the underlying molecular mechanisms has often been hampered by the overall lack of DNA or protein sequences for the species. In this study, we have assembled a transcriptome for this species from the oocytes, eggs, zygotes, and early embryos, which are known to have the highest RNA sequence complexity. Annotation of the transcriptome identified over 32,000 transcripts including the ones that encode 13 distinct cyclins and as many cyclin-dependent kinases (CDK), as well as the expected components of intracellular Ca2+ signaling toolkit. Although the mRNAs of cyclin and CDK families did not undergo significant abundance changes through the stages from oocyte to early embryo, as judged by real-time PCR, the transcript encoding Mos, a negative regulator of mitotic cell cycle, was drastically reduced during the period of rapid cleavages. Molecular phylogenetic analysis using the homologous amino acid sequences of cytochrome oxidase subunit I from A. aranciacus and 30 other starfish species indicated that Paxillosida, to which A. aranciacus belongs, is not likely to be the most basal order in Asteroidea. Taken together, the first transcriptome we assembled in this species is expected to enable us to perform comparative studies and to design gene-specific molecular tools with which to tackle long-standing biological questions.

Calcium role in human carcinogenesis: a comprehensive analysis and critical review of literature.

The central role played by calcium ion in biological systems has generated an interest for its potential implication in human malignancies. Thus, lines of research, on possible association of calcium metabolism regulation with tumorigenesis, implying disruptions and/or alterations of known molecular pathways, have been extensively researched in the recent decades. This paper is a critical synthesis of these findings, based on a functional approach of the calcium signaling toolkit. It provides strong support that this ubiquitous divalent cation is involved in cancer initiation, promotion, and progression. Different pathways have been outlined, involving equally different molecular and cellular structures. However, if the association between calcium and cancer can be described as constant, it is not always linear. We have identified several influencing factors among which the most relevant are (i) the changes in local or tissular concentrations of free calcium and (ii) the histological and physiological types of tissue involved. Such versatility at the molecular level may probably account for the conflicting findings reported by the epidemiological literature on calcium dietary intake and the risk to develop certain cancers such as the prostatic or mammary neoplasms. However, it also fuels the hypothesis that behind each cancer, a specific calcium pathway can be evidenced. Identifying such molecular interactions is probably a promising approach for further understanding and treatment options for the disease.

Calcium signalling toolkits in astrocytes and spatio-temporal progression of Alzheimer's disease.

Pathological remodelling of astroglia represents an important component of the pathogenesis of Alzheimer's disease (AD). In AD astrocytes undergo both atrophy and reactivity; which may be specific for different stages of the disease evolution. Astroglial reactivity represents the generic defensive mechanism, and inhibition of astrogliotic response exacerbates b-amyloid pathology associated with AD. In animal models of AD astroglial reactivity is different in different brain regions, and the deficits of reactive response observed in entorhinal and prefrontal cortices may be linked to their vulnerability to AD progression. Reactive astrogliosis is linked to astroglial Ca(2+) signalling, this latter being widely regarded as a mechanism of astroglial excitability. The AD pathology evolving in animal models as well as acute or chronic exposure to β-amyloid induce pathological remodelling of Ca(2+) signalling toolkit in astrocytes. This remodelling modifies astroglial Ca(2+) signalling and may be linked to cellular mechanisms of AD pathogenesis.

Looking for answers to L-type calcium channels in the ageing brain (Commentary on Zanos etal.).

Looking for answers to L-type calcium channels in the ageing brain (Commentary on Zanos etal.).

Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage

Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca2+ signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca2+ signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca2+ transporters, combined with manipulations to estimate intracellular Ca2+ storage, we evaluated the contribution of individual transport systems to intracellular Ca2+ homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca2+ homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca2+ uptake and Ca2+ influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca2+ channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca2+ uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca2+ uptake and point to the existence of not-yet identified Ca2+ influx pathways.

Systems Approaches to Mitochondrial Calcium Signaling

Mitochondria are essential hubs of calcium-mediated signaling networks. The organelle can take up, buffer, and release calcium ions to effectively shape intracellular calcium transients, stimulate ATP production and regulate cell death. Although, the basic mechanisms of mitochondrial calcium homeostasis have been firmly established for decades, the molecular identity of the mitochondrial calcium signaling toolkit has evaded classical bottom-up approaches. Our previous studies (1,2) have provided a compelling example of the power of systems approaches applied to mitochondrial calcium signaling to discover hitherto unknown molecular components of the calcium uniporter. Currently, we are developing computational and experimental frameworks for a systematic reconstruction of calcium-dependent signal transduction cascades in mitochondria. By combining evolutionary genomics and loss-of-function genetic and chemical screens, our systems approach holds the potential to shed light on yet unanswered questions in the field of mitochondrial calcium signaling.1. Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y, Bao XR, Strittmatter L, Goldberger O, Bogorad RL, Koteliansky V, Mootha VK (2011). Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature. 476(7360):341-5.2. Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, Palmer AE, Mootha VK (2010). MICU1 encodes a mitochondrial EF hand protein required for calcium uptake. Nature. 467(7313):291-6.

Module 1: Introduction

The aim of this website is to describe cell signalling within its biological context. There has been an explosion in the characterization of signalling components and pathways. The next major challenge is to understand how cells exploit this large signalling toolkit to assemble the specific signalling pathways they require to communicate with each other. The primary focus is the biology of cell signalling. The emerging information on cell signalling pathways is integrated and presented within the context of specific cell types and processes. The beauty of cell signalling is the way different pathways are combined and adapted to control a diverse array of cellular processes in widely different spatial and temporal domains. The first half of the website characterizes the components and properties of the major cell signalling pathways, with special emphasis on how they are switched on and off. Attention is also focused on the spatial and temporal aspects that determine how information is encoded and distributed to precise cellular locations. The second half of the website deals with the way these different signalling pathways are employed to control the life history of cells from their birth during the process of cell proliferation, their differentiation into specific cell types to carry out different cell functions, and finally their death through processes such as apoptosis. Cell signalling orchestrates all these cellular processes. Many of the same signalling systems that control development come into play again to regulate a wide range of specific processes in adult cells, such as contraction, secretion, metabolism, proliferation, information processing in neurons and sensory perception. These examples illustrate how cell signalling pathways are adapted and co-ordinated to regulate many different cellular processes. This intimate relationship between cell signalling and biology is providing valuable insights into the underlying genetic and phenotypic defects responsible for many of the major human diseases.

Decoding calcium signaling across the nucleus.

Calcium (Ca(2+)) is an important multifaceted second messenger that regulates a wide range of cellular events. A Ca(2+)-signaling toolkit has been shown to exist in the nucleus and to be capable of generating and modulating nucleoplasmic Ca(2+) transients. Within the nucleus, Ca(2+) controls cellular events that are different from those modulated by cytosolic Ca(2+). This review focuses on nuclear Ca(2+) signals and their role in regulating physiological and pathological processes.

Differential deregulation of astrocytic calcium signalling by amyloid-β, TNFα, IL-1β and LPS

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca2+ deregulation. Recently, we proposed a mechanism by which nanomolar Aβ42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca2+ signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aβ42 on Ca2+ signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1β, LPS). Here we report that Aβ42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca2+ signals and store operated Ca2+ entry (SOCE) were augmented in Aβ42-treated cells due to up-regulation of a set of Ca2+ signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1β and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aβ42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aβ42 on astrocytic Ca2+ signalling differ from and may contrast to the effects of pro-inflammatory agents.

Observing the Dynamics of Luminal and Cytosolic Calcium During IP3R-Mediated Calcium Signals

Ca2+ signaling is ubiquitous across cell types. Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) is a key component of the Ca2+ signaling toolkit. The role of cytosolic calcium on the kinetics of IP3R's and on the dynamics of the evoked signals has been studied at large both experimentally and by modeling. The role of luminal calcium has not been investigated with that much detail although it has been found that it is relevant for signal termination in the case of Ca2+ release through Ryanodine Receptors. In this work we present the results of observing the dynamics of luminal and cytosolic Ca2+ simultaneously in Xenopus Laevis oocytes using two dyes that have their peaks of emission at different wavelengths. Through a continuous photorelease of caged IP3 we are able to evoke a series of global and localized signals in this system. The analysis of such signals allows a characterization of the extent to which distant regions can be coupled via the dynamics of luminal calcium. In this way we expect to advance into answering whether luminal calcium can generate the global feedback mechanism that is necessary to explain the robustness of IP3R-mediated calcium spikes as a signaling tool in spite of the inter-spike interval randomness.