BBA - General Subjects (v.1820, #8)

In recent years, as our understanding of the various roles played by Ca2 + signaling in development and differentiation has expanded, the challenge of imaging Ca2 + dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains.In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca2 + imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell.These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca2 + signaling. The improvement in the spatial resolution that results from specifically targeting a Ca2 + reporter has helped to reveal how a ubiquitous signaling messenger like Ca2 + can regulate coincidental but different signaling events within an individual cell; a Ca2 + signaling paradox that until now has been hard to explain.Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca2 + signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.► Advances in genetics have resulted in improved bioluminescent Ca2 +-imaging methods. ► The genetic expression of apoaequorin in different intact animal models is described. ► The strategies used to load the apoaequorin cofactor, coelenterazine, are outlined. ► We describe the detection and imaging systems used to provide Ca2 + signaling data.
Keywords: Apoaequorin expression; Aequorin luminescence; Coelenterazine; Developmental Ca2 + signaling; Intact embryo and animal;

Optical calcium imaging in the nervous system of Drosophila melanogaster by Thomas Riemensperger; Ulrike Pech; Shubham Dipt; André Fiala (1169-1178).
Drosophila melanogaster is one of the best-studied model organisms in biology, mainly because of the versatility of methods by which heredity and specific expression of genes can be traced and manipulated. Sophisticated genetic tools have been developed to express transgenes in selected cell types, and these techniques can be utilized to target DNA-encoded fluorescence probes to genetically defined subsets of neurons. Neuroscientists make use of this approach to monitor the activity of restricted types or subsets of neurons in the brain and the peripheral nervous system. Since membrane depolarization is typically accompanied by an increase in intracellular calcium ions, calcium-sensitive fluorescence proteins provide favorable tools to monitor the spatio-temporal activity across groups of neurons.Here we describe approaches to perform optical calcium imaging in Drosophila in consideration of various calcium sensors and expression systems. In addition, we outline by way of examples for which particular neuronal systems in Drosophila optical calcium imaging have been used. Finally, we exemplify briefly how optical calcium imaging in the brain of Drosophila can be carried out in practice. Drosophila provides an excellent model organism to combine genetic expression systems with optical calcium imaging in order to investigate principles of sensory coding, neuronal plasticity, and processing of neuronal information underlying behavior. This article is part of a Special Issue entitled Biochemical, Biophysical and Genetic Approaches to Intracellular Calcium Signaling.► Drosophila provides a unique system to investigate defined neurons within a brain. ► DNA-encoded Ca2+ sensors can be used to monitor the activity of defined neurons. ► Progress in Ca2+ sensor development and genetic expression systems is summarized. ► Applications of Ca2+ imaging in Drosophila neurobiology are exemplified. ► Information on how in vivo Ca2+ imaging is performed in Drosophila is provided.
Keywords: Drosophila; Optical calcium imaging; Genetically encoded fluorescence sensor; Calcium sensor protein; Neuronal processing;

Since the 1960s it has been clear that calcium is a key regulator of exocytosis. Early experiments directly showed that the secretory output was calcium dependent. But it has taken improvements in technology and clever experimentation to determine the relationships between the calcium signal and exocytosis. Today controversies still remain because of limitations in our ability to record both the calcium responses within the local domains that control secretion and in the methods used to record exocytosis.Here the techniques used to measure calcium and exocytosis are reviewed with a distinction being drawn between measurements in excitable cells versus measurements in non-excitable cells. The review has a focus on techniques that are relevant to in vitro studies of native tissues and recent in vivo recordings.There are a range of methods used to study the stimulus-secretion pathway. Each presents their own advantages and drawbacks. These are discussed with reference to the latest work determining the factors controlling exocytosis in tissues.Stimulus-secretion coupling is the fundamental step in the control of neurotransmitter release, hormone secretion and protein secretion. Understanding secretory control is therefore important in understanding the physiological regulation of processes ranging from learning and memory to pancreatic secretion. Recent technological advances are now enabling us to study stimulus-secretion coupling within native tissues. This is helping us to understand the physiological complexities of secretory control. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.► Discussion of spatio-temporal control of exocytosis by calcium. ► Methods to determine and manipulate cytosolic calcium in the cell. ► Methods to assay exocytosis and determine secretory output. ► Distinctions in secretion between excitable and non-excitable cells.
Keywords: Calcium; Exocytosis; Secretion;

Fundamental properties of Ca2 + signals by Kevin Thurley; Alexander Skupin; Rüdiger Thul; Martin Falcke (1185-1194).
Ca2 + is a ubiquitous and versatile second messenger that transmits information through changes of the cytosolic Ca2 + concentration. Recent investigations changed basic ideas on the dynamic character of Ca2 + signals and challenge traditional ideas on information transmission.We present recent findings on key characteristics of the cytosolic Ca2 + dynamics and theoretical concepts that explain the wide range of experimentally observed Ca2 + signals. Further, we relate properties of the dynamical regulation of the cytosolic Ca2 + concentration to ideas about information transmission by stochastic signals.We demonstrate the importance of the hierarchal arrangement of Ca2 + release sites on the emergence of cellular Ca2 + spikes. Stochastic Ca2 + signals are functionally robust and adaptive to changing environmental conditions. Fluctuations of interspike intervals (ISIs) and the moment relation derived from ISI distributions contain information on the channel cluster open probability and on pathway properties.Robust and reliable signal transduction pathways that entail Ca2 + dynamics are essential for eukaryotic organisms. Moreover, we expect that the design of a stochastic mechanism which provides robustness and adaptivity will be found also in other biological systems. Ca2 + dynamics demonstrate that the fluctuations of cellular signals contain information on molecular behavior. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.► We review recent findings on key characteristics of cytosolic Ca2 + dynamics. ► We demonstrate the importance of the hierarchal arrangement of Ca2 + release sites. ► New theoretical concepts exploit emergent behavior of cellular Ca2 + spikes. ► We relate the dynamical regulation of [Ca2 +] to information transmission. ► Stochastic Ca2 + signals are functionally robust and adaptive to changing conditions.
Keywords: Calcium dynamics; Mathematical modeling; Cell signaling; Stochastic process;

Calcium-binding proteins (CBPs) are instrumental in the control of Ca2+ signaling. They are the fastest players within the Ca2+ toolkit responding within microseconds to [Ca2+] changes. The CBPs compete for Ca2+ which plays a direct role in modulating Ca2+ transients and the resulting biochemical message. The kinetic properties of the CBPs have to be known to have a good understanding of Ca2+ signaling.Most techniques used to measure binding kinetics are too slow to accurately determine the fast kinetics of most CBP. Furthermore, many CBPs bind Ca2+ in a cooperative way, which should be incorporated in the kinetic modeling. Here we will review a new ultra-fast in vitro technique for measuring Ca2+ binding properties of CBPs following flash photolysis of caged Ca2+. Compartmental modeling is used to resolve the kinetics of fast cooperative Ca2+ binding to CBPs.Currently this technique has only been used to quantify the kinetics of three CBPs (calbindin, calretinin and calmodulin), but has already provided remarkable insights into the specific role that these kinetics in Ca2+ signaling.The potential to gain novel insights into Ca2+ signaling by quantifying kinetics of other CBPs using this technique is very promising. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.► Kinetics of Ca2+ binding proteins (CBPs) are too fast to gauge with normal methods. ► The fast kinetics of CBPs can be determined using flash photolysis. ► Cooperativity should be included when quantifying binding kinetics of CBPs. ► The binding kinetics of CBPs play a key role in calcium signaling.
Keywords: Calcium binding kinetics; Calcium binding protein; Flash photolysis; Calcium signaling; Cooperative binding;

Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulate synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs) regulate ion channels to control neuronal excitability, and DREAM (KChIP3) is a transcriptional repressor that regulates neuronal gene expression.Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase, NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels.We propose the idea that N-terminal myristoylation is critical for shaping each NCS family member into a unique structure, which upon Ca2 +-induced extrusion of the myristoyl group exposes a unique set of previously masked residues, thereby exposing a distinctive ensemble of hydrophobic residues to associate specifically with a particular physiological target. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.► Structure and target recognition of NCS proteins. ► Ca2 +-binding causes extrusion of N-terminal myristoyl group. ► Myristoylation is critical for shaping each NCS protein into a unique structure.
Keywords: Calcium; EF-hand; Ca2 +-myristoyl switch; NCS-1; Recoverin; GCAP1;

Analysis of IP3 receptors in and out of cells by Ana M. Rossi; Stephen C. Tovey; Taufiq Rahman; David L. Prole; Colin W. Taylor (1214-1227).
Inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP3R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca2 + channels.In this brief review, we first consider a variety of complementary methods that allow the links between IP3 binding and channel gating to be defined. How does IP3 binding to the IP3-binding core in each IP3R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP3, Ca2 + signals and IP3R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP3R signalling.All IP3R are regulated by both IP3 and Ca2 +. This allows them to initiate and regeneratively propagate intracellular Ca2 + signals. The elementary Ca2 + release events evoked by IP3 in intact cells are mediated by very small numbers of active IP3R and the Ca2 +-mediated interactions between them. The spatial organization of these Ca2 + signals and their stochastic dependence on so few IP3Rs highlight the need for methods that allow the spatial organization of IP3R signalling to be addressed with single-molecule resolution.A variety of complementary methods provide insight into the structural basis of IP3R activation and the contributions of IP3-evoked Ca2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.► IP3 receptors are intracellular Ca2+ channels. ► IP3 receptors initiate and propagate Ca2+ signals. ► We review methods that address IP3 receptor behaviour. ► We describe methods to examine structural determinants of IP3 receptor function. ► We describe high-resolution analyses of IP3 receptor activity in intact cells.
Keywords: IP3 receptor; Ca signaling; TIRF microscopy; Patch clamp;

Neuronal calcium sensor proteins represent a subgroup of the family of EF-hand calcium binding proteins. Members of this subgroup are the guanylate cyclase-activating proteins and recoverin, which operate as important calcium sensors in retinal photoreceptor cells. Physiological and biochemical data indicate that these proteins participate in shaping the photoreceptor light response.Biophysical methods have been widely applied to investigate the molecular properties of retinal calcium binding proteins like the guanylate cyclase-activating proteins and recoverin. Properties include the determination of calcium affinities by isotope techniques and spectroscopical approaches. Conformational changes are investigated for example by tryptophan fluorescence emission. A special focus of this review is laid on a new experimental approach to study conformational changes in calcium binding proteins by surface plasmon resonance spectroscopy. In addition this technique has been employed for measuring the calcium-dependent binding of calcium sensors to membranes.Biophysical approaches provide valuable information about key properties of calcium sensor proteins involved in intracellular signalling. Parameters of their molecular properties like calcium binding and conformational changes help to define their physiological role derived from cellular, genetic or physiological studies.Calcium is an important second messenger in intracellular signaling. Calcium signals are propagated via calcium binding proteins that are able to discriminate between incremental differences in intracellular calcium and that regulate their targets with high precision and specificity. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.► Biophysical approaches provide key properties of calcium sensor proteins. ► Conformational changes in proteins investigated by surface plasmon resonance. ► Binding of calcium sensors to membranes probed by surface plasmon resonance.
Keywords: Calcium signaling; Recoverin; Guanylate cyclase-activating protein; Surface plasmon resonance; Conformational change;

Neurotransmitters, neuropeptides and hormones are released from secretory vesicles of nerve terminals and neuroendocrine cells by calcium-activated exocytosis. A key step in this process is the formation of a fusion pore between the vesicle membrane and the plasma membrane. Exocytotic fusion leads to an increase in plasma membrane area that can be measured as a proportional increase in plasma membrane capacitance.High resolution capacitance measurements in single cells, nerve terminals and small membrane patches have become possible with the development of the patch clamp technique. This review discusses the methods of whole cell patch clamp capacitance measurements and their use in conjunction with voltage clamp pulse stimulation and with stimulation by photorelease of caged calcium. It also discusses patch capacitance measurements for the study of single exocytotic events and fusion pore properties in neuroendocrine cells and nerve terminals.Capacitance measurements provide high resolution information on the extent and time course of fusion for the characterization of vesicle pools and the kinetics of exocytosis. They allow the characterization of the mode of fusion including distinction of single vesicle full fusion, transient kiss-and-run fusion or multivesicular compound exocytosis. Furthermore, measurement of fusion pore conductances and their dynamic behavior has enabled the characterization of fusion pore properties in a way that resembles the characterization of ion channel function through single channel recordings.The combination of patch clamp capacitance measurements with pharmacological and molecular manipulations of exocytosis is emerging as a powerful approach to investigate the molecular mechanisms of calcium-activated exocytotic fusion pore formation. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.► Patch clamp capacitance measurement techniques to study Ca2+-activated exocytosis. ► Stimulation pulse protocols and caged Ca2+ reveal vesicle pools and release kinetics. ► Patch capacitance measurements record single exocytotic fusion pore openings. ► Combination with molecular manipulations to investigate molecular fusion mechanism.
Keywords: Exocytosis; Transmitter release; Fusion pore; Patch clamp; Capacitance measurement;

Voltage-gated (Cav) Ca2 + channels are multi-subunit complexes that play diverse roles in a wide variety of tissues. A fundamental mechanism controlling Cav channel function involves the Ca2 + ions that permeate the channel pore. Ca2 + influx through Cav channels mediates feedback regulation to the channel that is both negative (Ca2 +-dependent inactivation, CDI) and positive (Ca2 +-dependent facilitation, CDF).This review highlights general mechanisms of CDI and CDF with an emphasis on how these processes have been studied electrophysiologically in native and heterologous expression systems.Electrophysiological analyses have led to detailed insights into the mechanisms and prevalence of CDI and CDF as Cav channel regulatory mechanisms. All Cav channel family members undergo some form of Ca2 +-dependent feedback that relies on CaM or a related Ca2 + binding protein. Tremendous progress has been made in characterizing the role of CaM in CDI and CDF. Yet, what contributes to the heterogeneity of CDI/CDF in various cell-types and how Ca2 +-dependent regulation of Cav channels controls Ca2 + signaling remain largely unexplored.Ca2 + influx through Cav channels regulates diverse physiological events including excitation–contraction coupling in muscle, neurotransmitter and hormone release, and Ca2 +-dependent gene transcription. Therefore, the mechanisms that regulate channels, such as CDI and CDF, can have a large impact on the signaling potential of excitable cells in various physiological contexts. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.► Electrophysiological analysis have revealed detailed mechanisms of Cav channel modulation. ► Cav channels undergo CDI/CDF that relies on Ca2+ binding proteins like calmodulin. ► The heterogeneity of CDI/CDF in various cell-types remains largely unexplored.
Keywords: Calcium; Cellular excitability;

Genetic analysis of IP3 and calcium signalling pathways in C. elegans by Howard A. Baylis; Rafael P. Vázquez-Manrique (1253-1268).
The nematode, Caenorhabditis elegans is an established model system that is particularly well suited to genetic analysis. C. elegans is easily manipulated and we have an in depth knowledge of many aspects of its biology. Thus, it is an attractive system in which to pursue integrated studies of signalling pathways. C. elegans has a complement of calcium signalling molecules similar to that of other animals.We focus on IP3 signalling. We describe how forward and reverse genetic approaches, including RNAi, have resulted in a tool kit which enables the analysis of IP3/Ca2 + signalling pathways. The importance of cell and tissue specific manipulation of signalling pathways and the use of epistasis analysis are highlighted. We discuss how these tools have increased our understanding of IP3 signalling in specific developmental, physiological and behavioural roles. Approaches to imaging calcium signals in C. elegans are considered.A wide selection of tools is available for the analysis of IP3/Ca2 + signalling in C. elegans. This has resulted in detailed descriptions of the function of IP3/Ca2 + signalling in the animal's biology. Nevertheless many questions about how IP3 signalling regulates specific processes remain.Many of the approaches described may be applied to other calcium signalling systems. C. elegans offers the opportunity to dissect pathways, perform integrated studies and to test the importance of the properties of calcium signalling molecules to whole animal function, thus illuminating the function of calcium signalling in animals. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling. ► C. elegans has many advantages for integrative studies of calcium signalling. ► A tool-kit of genetic and related approaches has been used to dissect IP3 signalling. ► Roles for IP3 in development, defecation, ovulation and other processes are discussed. ► Genetic interactions and transgenic tools enable us to define signalling networks. ► In vivo calcium imaging reveals links between proteins, signals and animal function.
Keywords: Caenorhabditis elegans; Inositol 1,4,5-trisphosphate (IP3) receptor; Calcium signalling; Phospholipase C; RNAi; Biological rhythm;

The genetics of calcium signaling in Drosophila melanogaster by Tetyana Chorna; Gaiti Hasan (1269-1282).
Genetic screens for behavioral and physiological defects in Drosophila melanogaster, helped identify several components of calcium signaling of which some, like the Trps, were novel. For genes initially identified in vertebrates, reverse genetic methods have allowed functional studies at the cellular and systemic levels.The aim of this review is to explain how various genetic methods available in Drosophila have been used to place different arms of Ca2 + signaling in the context of organismal development, physiology and behavior.Mutants generated in genes encoding a range of Ca2 + transport systems, binding proteins and enzymes affect multiple aspects of neuronal and muscle physiology. Some also affect the maintenance of ionic balance and excretion from malpighian tubules and innate immune responses in macrophages. Aspects of neuronal physiology affected include synaptic growth and plasticity, sensory transduction, flight circuit development and function. Genetic interaction screens have shown that mechanisms of maintaining Ca2 + homeostasis in Drosophila are cell specific and require a synergistic interplay between different intracellular and plasma membrane Ca2 + signaling molecules.Insights gained through genetic studies of conserved Ca2 + signaling pathways have helped understand multiple aspects of fly physiology. The similarities between mutant phenotypes of Ca2 + signaling genes in Drosophila with certain human disease conditions, especially where homologous genes are causative factors, are likely to aid in the discovery of underlying disease mechanisms and help develop novel therapeutic strategies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.► A genetic model for studying cellular and systemic roles of signaling pathways. ► Identification of Ca2+ signaling in specific cells at specific developmental stages. ► A mix and match of Ca2+ signaling components controls tissue specific physiology. ► Mutants illustrate conservation of Ca2+ signaling function with vertebrates. ► Conserved pathways for human diseases can help identify new therapeutic targets.
Keywords: Voltage gated Ca2 + channel; Orai; TRPs; RyR; InsP3R; CamKII;

Analysis of calcium signaling pathways in plants by Oliver Batistič; Jörg Kudla (1283-1293).
Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Ca2 + signals are represented by stimulus-specific spatially and temporally defined Ca2 + signatures. These Ca2 + signatures are detected, decoded and transmitted to downstream responses by a complex toolkit of Ca2 + binding proteins that function as Ca2 + sensors.This review will reflect on advancements in monitoring Ca2 + dynamics in plants. Moreover, it will provide insights in the extensive and complex toolkit of plant Ca2 + sensor proteins that relay the information presented in the Ca2 + signatures into phosphorylation events, changes in protein–protein interaction or regulation of gene expression.Plants' response to signals is encoded by different Ca2 + signatures. The plant decoding Ca2 + toolkit encompasses different families of Ca2 + sensors like Calmodulins (CaM), Calmodulin-like proteins (CMLs), Ca2 +-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs). These Ca2 + sensors are encoded by complex gene families and form intricate signaling networks in plants that enable specific, robust and flexible information processing.This review provides new insights about the biochemical regulation, physiological functions and of newly identified target proteins of the major plant Ca2 + sensor families. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.► Plants contain a unique calcium signalling toolkit. ► Novel calcium reporter proteins advance our understanding of plant calcium signals. ► Plant calcium sensor proteins regulate diverse cellular functions.
Keywords: Calcium; Cameleon; Calmodulin; Ca2 +-dependent protein kinase; Calcineurin B-like protein; CBL-interacting protein kinase;

Cytosolic Ca2 + buffers are members of the large family of Ca2 +-binding proteins and are essential components of the Ca2 + signaling toolkit implicated in the precise regulation of intracellular Ca2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca2 + buffers or mice with ectopic expression.In this review, results obtained with knockout mice for the three most prominent Ca2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.The absence of Ca2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca2 + buffer-specific changes in intracellular Ca2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca2 + buffers, but have revealed that the absence of Ca2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.The complex phenotype of Ca2 + buffer knockout mice arises from the direct effect of these proteins on Ca2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca2 + signaling, a global view on Ca2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.
Keywords: Calcium-binding protein; Calcium buffer; Calcium homeostasome; Parvalbumin; Calbindin-D28k; Calretinin;