The organisation and functions of local Ca²⁺ signals

Calcium (Ca2+) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in space, time and amplitude. To generate and interpret the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling 'toolkit', which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology. Recent studies have demonstrated the importance of local Ca2+ signals in defining the specificity of the interaction of Ca2+ with its targets. Furthermore, local Ca2+ signals are the triggers and building blocks for larger global signals that propagate throughout cells

Loading fluorescent Ca²⁺ indicators into living cells

Small-molecule fluorescent Ca2+ reporters are the most widely used tools in the field of Ca2+ signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca2+ as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca2+ indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca2+ indicators have carboxylic acid groups for binding of Ca2+. Because these “free-acid” forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxymethyl [AM] or acetate esters). The ester versions of the indicators permeate the plasma membrane. The Ca2+-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases

Fundamentals of Cellular Calcium Signaling: A Primer

Ionized calcium (Ca2+) is the most versatile cellular messenger. All cells use Ca2+ signals to regulate their activities in response to extrinsic and intrinsic stimuli. Alterations in cellular Ca2+ signaling and/or Ca2+ homeostasis can subvert physiological processes into driving pathological outcomes. Imaging of living cells over the past decades has demonstrated that Ca2+ signals encode information in their frequency, kinetics, amplitude, and spatial extent. These parameters alter depending on the type and intensity of stimulation, and cellular context. Moreover, it is evident that different cell types produce widely varying Ca2+ signals, with properties that suit their physiological functions. This primer discusses basic principles and mechanisms underlying cellular Ca2+ signaling and Ca2+ homeostasis. Consequently, we have cited some historical articles in addition to more recent findings. A brief summary of the core features of cellular Ca2+ signaling is provided, with particular focus on Ca2+ stores and Ca2+ transport across cellular membranes, as well as mechanisms by which Ca2+ signals activate downstream effector systems

An update on nuclear calcium signalling

Over the past 15 years or so, numerous studies have sought to characterise how nuclear calcium (Ca2+) signals are generated and reversed, and to understand how events that occur in the nucleoplasm influence cellular Ca2+ activity, and vice versa. In this Commentary, we describe mechanisms of nuclear Ca2+ signalling and discuss what is known about the origin and physiological significance of nuclear Ca2+ transients. In particular, we focus on the idea that the nucleus has an autonomous Ca2+ signalling system that can generate its own Ca2+ transients that modulate processes such as gene transcription. We also discuss the role of nuclear pores and the nuclear envelope in controlling ion flux into the nucleoplasm

Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation

Neural stem cells can generate in vitro progenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes in these cells are poorly understood. Here we tested the hypothesis that Ca2+ signaling controls differentiation of neural precursors. We found low-frequency global and local Ca2+ transients occurring predominantly during early stages of differentiation. Spontaneous Ca2+ signals in individual precursors were not synchronized with Ca2+ transients in surrounding cells. Experimentally induced changes in the frequency of local Ca2+signals and global Ca2+ rises correlated positively with neurite outgrowth and the onset of GABAergic neurotransmitter phenotype, respectively. NMDA receptor activity was critical for alterations in neuronal morphology but not for the timing of the acquisition of the neurotransmitter phenotype. Thus, spontaneous Ca2+ signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency may specify neuronal morphology and acquisition of neurotransmitter phenotype

Oncogenic K-Ras suppresses IP₃-dependent Ca²⁺ release through remodeling of IP₃Rs isoform composition and ER luminal Ca²⁺ levels in colorectal cancer cell lines

The GTPase Ras is a molecular switch engaged downstream of G-protein coupled receptors and receptor tyrosine inases that controls multiple cell fate-determining signalling athways. Ras signalling is frequently deregulated in cancer underlying associated changes in cell phenotype. Although Ca2+ signalling pathways control some overlapping functions with Ras, and altered Ca2+ signalling pathways are emerging as important players in oncogenic transformation, how Ca2+ signalling is remodelled during transformation and whether it has a causal role remains unclear. We have investigated Ca2+ signalling in two human colorectal cancer cell lines and their isogenic derivatives in which the mutated K-Ras allele (G13D) has been deleted by homologous recombination. We show that agonist-induced Ca2+ release from intracellular stores is enhanced by loss of K-RasG13D through an increase in the ER store content and a modification of IP3R subtype abundance. Consistently, uptake of Ca2+ into mitochondria and sensitivity to apoptosis was enhanced as a result of KRasG13D loss. These results suggest that suppression of Ca2+ signalling is a common response to naturally occurring levels of K-RasG13D that contributes to a survival advantage during oncogenic transformation

Alzheimer’s disease-associated peptide Aβ₄₂ mobilizes ER Ca²⁺ via InsP₃R-dependent and -independent mechanisms

Dysregulation of Ca2+ homeostasis is considered to contribute to the toxic action of the Alzheimer’s Disease (AD) associated Amyloid β-peptide (Aβ). Ca2+ fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca2+ fluxes induced by Aβ42. Here, we investigated the contribution of Ca2+ release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca2+ homeostasis and the mechanism by which Aβ42 elicited these effects. Consistent with previous reports, application of soluble oligomeric forms of Aβ42 exhibited Ca2+ mobilizing properties. The Aβ42-stimulated Ca2+ signals persisted in the absence of extracellular Ca2+ indicating a significant contribution of Ca2+ release from the ER Ca2+ store to the generation of these signals. Moreover, inositol 1,4,5-trisphosphate (InsP3) signaling contributed to Aβ42-stimulated Ca2+ release. The Ca2+ mobilizing effect of Aβ42 was also observed when applied to permeabilized cells deficient in InsP3 receptors revealing an additional direct effect of internalized Aβ42 upon the ER, and a mechanism for induction of toxicity by intracellular Aβ42

Deleterious effects of calcium indicators within cells; an inconvenient truth

The study of cellular Ca⁠2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca⁠⁠2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca⁠⁠2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes

Second Messengers

Second messengers are small molecules and ions that relay signals received by cell-surface receptors to effector proteins. They include a wide variety of chemical species and have diverse properties that allow them to signal within membranes (e.g., hydrophobic molecules such as lipids and lipid derivatives), within the cytosol (e.g., polar molecules such as nucleotides and ions), or between the two (e.g., gases and free radicals). Second messengers are typically present at low concentrations in resting cells and can be rapidly produced or released when cells are stimulated. The levels of second messengers are exquisitely controlled temporally and spatially, and, during signaling, enzymatic reactions or opening of ion channels ensure that they are highly amplified. These messengers then diffuse rapidly from the source and bind to target proteins to alter their properties (activity, localization, stability, etc.) to propagate signaling