Calcium and Cell Response to Heavy Metals: Can Yeast Provide an Answer? Calcium and Cell Response to Heavy Metals: Can Yeast Provide an Answer?

Despite constant efforts to maintain a clean environment, heavy metal pollution contin - ues to raise challenges to the industrialized world. Exposure to heavy metals is detrimen tal to living organisms, and it is of utmost importance that cells find rapid and efficient ways to respond to and eventually adapt to surplus metals for survival under severe stress. This chapter focuses on the attempts done so far to elucidate the calcium-mediated response to heavy metal stress using the model organism Saccharomyces cerevisiae . The possibilities to record the transient elevations of calcium within yeast cells concomitantly with the heavy metal exposure are presented, and the limitations imposed by interfer ence between calcium and heavy metals are discussed. Ca 2+ influx and mating.


Introduction
Responding to environmental stimuli is a prerequisite for cell adaptation to the ever-changing conditions in the cell surroundings. Stress conditions such as sudden changes of temperature, pH, irradiation, or elevations in various chemicals concentration need to be sensed by the cell in order to respond and adapt to these changes. Calcium ions are one of the most widespread second messengers in the eukaryotic cell, being responsible for triggering many responses to external stress conditions [1]. Various biotic and abiotic stresses induce an increase in cytosolic calcium ions ([Ca 2+ ] cyt ), which in turn activate many proteins involved in signaling pathways, from yeast to humans [2]. Thanks to easy manipulation, rapid growth, genetic amenability and with many genes bearing resemblance with higher eukaryotic genes, the yeast Saccharomyces cerevisiae is one of the widely used model organisms which helped in elucidating a wide variety of molecular mechanisms conserved along evolution, related to cell cycle and cell proliferation, homeostasis, adaptation and survival [3]. Among many others studies, S. cerevisiae was used as a model to investigate the Ca 2+ -mediated responses to a variety of stimuli: hypotonic stress [4][5][6], hypertonic and salt stress [7], cold stress [8], high ethanol [9], β-phenylethylamine [10], glucose [11,12], high pH [13][14][15], amidarone and antifungal drugs [16,17], oxidative stress [18], eugenol [19,20], essential oils [21,22], or heavy metals [23,24]. This chapter focuses on the studies made on S. cerevisiae cells in the effort to understand the role of calcium in cell response to heavy metal exposure.
Heavy metals represent a constant threat to clean environments as they are constantly released in the course of various anthropogenic activities (Figure 1), both industrial (mining, electroplating, smelting, metallurgical processes, nanoparticles, unsafe agricultural practices) and domestic (sewage and waste, metal corrosion), all in the context of rapid industrialization and urbanization [25]. Heavy metals as contaminants are included in the category of persistent pollutants, because they cannot be destroyed or degraded. Being natural components of the earth crust, the environmental contamination becomes serious when heavy metals have the possibility to leach into surface or underground water, or undergo atmospheric deposition and metal evaporation from the water resources [26][27][28]. The ultimate threat imposed by the spread of heavy metals into the environment is their accumulation in the living organisms (Figure 1) via the food chain [29], inducing serious illnesses in animals and humans [30][31][32][33][34]. Some heavy metals (Co 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Ni 2+ , Mo 2+ , and Zn 2+ ) are essential for life, contributing to various biochemical and physiological functions in the living organisms. The nutritional requirements of these elements are generally low and they must be present in food in trace concentrations [35]. However, excessive exposure to higher concentrations is deleterious, representing a threat to living organisms [36]. Other heavy metals (Ag + , Cd 2+ , Pb 2+ , Hg 2+ ) are not essential for life and have no established biological roles, but they are highly toxic because they compete with the essential metals for their biological targets or they simply bind nonspecifically to biomolecules; these metals are able to induce toxicity at low doses [37]. Essential or not, the hazardous heavy metals such as Cd 2+ , Co 2+ , Cu 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Zn 2+ are known to be major threats to the environment [38]. The molecular mechanisms involved in heavy metal transport and homeostasis have been intensively studied in S. cerevisiae [3], along with many aspects regarding their toxicity, tolerance, accumulation, or extrusion [38][39][40][41][42][43][44][45][46][47]. Some of the relevant studies performed in S. cerevisiae correlating heavy metal exposure to calcium-related mechanisms are presented in the following section.

Calcium transport and homeostasis in S. cerevisiae
Intracellular calcium ions are important second messengers in all organisms, including yeast. The mechanisms involved in calcium transport and homeostasis in S. cerevisiae cells have been extensively studied [48][49][50]. Under normal conditions, the [Ca 2+ ] cyt is maintained very low (50-200 nM) at external Ca 2+ concentrations ranging from <1 μM to >100 mM [51,52]. Abrupt changes in the environment can be transduced inside the yeast cells by sudden elevations in [Ca 2+ ] cyt which can be the result of Ca 2+ influx from outside the cell, Ca 2+ release from internal stores (usually vacuole), or both (Figure 2). The yeast plasma membranes contain at least two different Ca 2+ influx systems, the high-affinity Ca 2+ influx system (HACS) and the low-affinity Ca 2+ influx system (LACS), the former being responsible for Ca 2+ influx under stress conditions [50]. The HACS consists of two proteins, Cch1p and Mid1p, which are expressed and colocalize to the plasma membrane. These two subunits form a stable complex that is activated in response to sudden stimulation, boosting the influx of Ca 2+ from the extracellular space. In S. cerevisiae, Cch1p is similar to the pore-forming α1 subunit of mammalian L-type voltagegated Ca 2+ channels (VGCCs) [53], while Mid1p is as a stretch-activated Ca 2 +− permeable cation channel homologous to α2δ subunit of animal VGCCs [54]. HACS is regulated by Ecm7p, a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes the λ subunits of VGCCs. Ecm7p is stabilized by Mid1p, and Mid1p is stabilized by Cch1p under non-signaling conditions [55].
Changes in the cell environment are signaled by a sudden increase in [Ca 2+ ] cyt which can be a consequence of either external Ca 2+ influx via the Cch1p/Mid1p channel on the plasma membrane [4][5][6][7][8][9][10][11][12][13][14]56], release of vacuolar Ca 2+ into the cytosol through the vacuole-located Ca 2+ channel Yvc1p [18,57], or both (Figure 2). After delivering the message, the level of [Ca 2+ ] cyt is restored to the normal very low levels through the action of Ca 2+ pumps and exchangers. Thus, the Ca 2+ -ATPase Pmc1p [58,59] and a vacuolar Ca 2+ /H + exchanger Vcx1p [60,61] independently transport [Ca 2+ ] cyt into the vacuole, while Pmr1p, the secretory Ca 2+ -ATPase, pumps [Ca 2+ ] cyt into endoplasmic reticulum (ER) and Golgi along with Ca 2+ extrusion from the cell [62,63]. These responses are mediated by the universal Ca 2+ sensor protein calmodulin that can bind and activate calcineurin, which inhibits at the post-transcriptional level the function of Vcx1p [60,64,65] and induces the expression of PMC1 and PMR1 genes via activation of the Crz1p transcription factor [64,65]. The release of Ca 2+ from intracellular stores stimulates the extracellular Ca 2+ influx, a process known as capacitative calcium entry [66]. Inversely, the release of vacuolar Ca 2+ via Yvc1p can be further stimulated by the Ca 2+ from outside the cell as well as that released from the vacuole by Yvc1p itself in a positive feedback called Ca 2+induced Ca 2+ release (CICR) [67][68][69][70].

Aequorin, a transgenic molecular tool for detecting [Ca 2+ ] cyt changes in S. cerevisiae
As a second messenger, Ca 2+ triggers a variety of cascade responses by temporarily activating Ca 2+ -binding components of signaling pathways which can lead either to adaptation to influx systems HACS (high-affinity Ca 2+ influx system) and to a lesser extent LACS (low-affinity Ca 2+ influx system) are activated, resulting in a rapid influx of Ca 2+ into the cytosol. Transient increases in intracellular Ca 2+ concentrations may also be due to release from internal compartments, mainly the vacuole, via Yvc1p. Unlike mammalian cells, where the main Ca 2+ stores reside in the endoplasmic reticulum (ER), in yeast the intracellular stores are situated in the vacuole compartment. The increased cytosolic Ca 2+ concentrations ([Ca 2+ ] cyt ) are sensed by calmodulin, activating calcineurin. Activated calcineurin acts on its downstream target Crz1p, inducing its translocation from cytoplasm to nucleus to further induces the expression of a set of Ca 2+ /calcineurin-dependent target genes, including PMC1 and PMR1. Calcineurin also regulates Vcx1p at post-transcriptional level. Subsequently, the [Ca 2+ ] cyt concentration is reduced to basal levels via uptake by organelles, especially vacuole (by means of Pmc1p and Vcx1p) and Golgi (by means of Pmr1p). the environmental changes or to cell death [71]. To determine the [Ca 2+ ] cyt fluctuations during cell exposure to environmental changes, it is necessary to have an system capable to detect the sudden and transient elevations in [Ca 2+ ] cyt . This was made possible by the isolation of aequorin, a Ca 2+ -binding photoprotein, isolated from the luminescent jellyfish, Aequorea victoria. Aequorin consists of two distinct units, the apoprotein apoaequorin (22 kDa) and the prosthetic group, coelenterazine, which reconstitute spontaneously in the presence of molecular oxygen, forming the functional protein [72][73][74]. Aequorin has become a useful instrument for the measurement of intracellular Ca 2+ levels, since it has binding sites for Ca 2+ ions responsible for protein conformational changes that convert through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO 2 ( Figure 3A). As the excited coelenteramide relaxes to the ground state, blue light (λ max 469 nm) is emitted and can be easily detected with a luminometer [75].
The expression of cDNA for apoaequorin in yeast cells and subsequent regeneration of apoaequorin into aequorin provide a noninvasive, nontoxic and effective method to detect the transient variations in yeast [Ca 2+ ] cyt [76]. The yeast strains to be analysed must express the A. victoria apoaequorin, and they need to be reconstituted into fully active aequorin by association with coelenterazine ( Figure 3B). The latter cannot be synthesized by yeast itself; therefore, the way to achieve reconstitution is to incubate the apoaequorin-expressing cells with coelenterazine, prior to Ca 2+ determination. Coelenterazine is a hydrophobic molecule, and therefore, it is easily taken up across yeast cell wall and membrane, making aequorin suitable as a Ca 2+ reporter [52,77]. Aequorin has a number of advantages over other Ca 2+ indicators as follows: because the protein is large, it has a low leakage rate from cells compared to lipophilic dyes and it does not undergo intracellular compartmentalization or sequestration. Also, it does not disrupt cell functions, and the light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so problems with auto-fluorescence are eliminated [78]. The primary limitation of aequorin is that the prosthetic group coelenterazine is irreversibly consumed to produce light. Such issues led to developments of other genetically encoded calcium sensors including the calmodulin-based sensor cameleon, which were less successful in yeast, due to their size [79].
In S. cerevisiae, the reconstituted aequorin is used primarily to detect the Ca 2+ fluctuations in the cytosol [76]; there have been few attempts to obtain apoaequorins targeted to various cell compartment in yeast. One notable example was the construction of a recombinant apoaequorin cDNA whose product localizes in the ER lumen; using this product, a steady state of 10 μM Ca 2+ was detected in the ER lumen of wild type cells, and it was possible to demonstrate that the Golgi pump Pmr1p also controls, at least in part, the ER luminal concentration of Ca 2+ [63]. Nevertheless, no reports on Ca 2+ fluctuation in the ER in response to environmental stress are available in yeast. Surprisingly, no vacuole-targeted aequorin has been reported in yeast, in spite of the fact that the vacuole is the main storage compartment for Ca 2+ in yeast; instead, the vacuolar Ca 2+ traffic was determined indirectly, using genetic approaches (knockout mutants of various Ca 2+ pumps and transporters) [61,80] or blockers of the Ca 2+ influx across the plasma membrane. This latter approach makes use of cell-impermeant Ca 2+ chelators such as 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) [18] or of lanthanide (Ln 3+ ) ions, which are efficient blockers if ion channels due to size similarity between Ca 2+ and Ln 3+ [80]. Of all Ln 3+ , Gd 3+ is the most widely used as Ca 2+ -channel blocker. It was shown that at 1 mM concentration in the medium all the cations from the Ln 3+ series block Ca 2+ entry into cytosol with the exception of La 3+ (lanthanum) and to a lesser extent, Pr 3+ and Nd 3+ [81]. Care must be taken when using Ln 3+ as channel blockers, as it was shown that at low concentrations Ln 3+ may leak into the cytosol via the Cch1p/Mid1p system [82].

Correlations between calcium and heavy metal exposure as seen in S. cerevisiae cells
When grown in media contaminated with heavy metals, the yeast cell wall is the first to get in contact with the surplus cations present in the cell surroundings. If the contamination is not excessive, the cations would probably get stuck at this level, due to the mannoproteins that compose the outer layer of the cell wall (alongside of β-glucans and chitin) which are heavily phosphorylated and carboxylated, decorating the cell façade with a negatively charged shield prone to bind to positively charged species, such as the metal cations [83]. Excess metal ions which escape the negatively charged groups on the cell wall surface penetrate the porous cell  [72][73][74]. Cells expressing apo-aequorin are first incubated with the cell-permeant coelenterazine to produce functional aequorin. When Ca 2+ binds to aequorin, the protein undergoes a conformational change leading to the destabilization of the peroxide group (-O-O-), linking apoaequorin to coelenterazine, decomposing it to to coelenteramide and CO 2 ; the coelenteramide, which is in an excited state, generates blue light (λmax = 469 nm). B. Schematic representation of Ca 2+ -induced bioluminescence of yeast cells expressing reconstituted aequorin in the cytosol. When cells are exposed to an insult (e. g., environmental stress) the secondary messenger Ca 2+ ions enter the cytosol and bind to aequorin, rendering the cell luminescent. Luminescence traces indicate the intensity and the duration of the [Ca 2+ ] cyt wave [75,76].
wall and reach the membrane to exert their toxic effect by disrupting the lipid bilayer or by assaulting the membrane transporters.
Several heavy metals (Co 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Ni 2+ , and Zn 2+ ) are essential for life in their ionic forms, acting mainly as cofactors for a variety of enzymes. They are necessary only in minute amounts inside the cell (hence their denomination as "trace" elements); if their concentration goes beyond the physiological threshold they become toxic by nonspecifically binding to any biomolecule bearing a negative charge or a metal-chelator fragment. The bipolar nature of trace metals determined the development of intricate cellular systems dedicated to their uptake, buffering, sequestration, intracellular trafficking, compartmentalization and excretion. As in many other directions of study, S. cerevisiae brought a considerable contribution to the understanding of the molecular mechanism involved in trace metal transport and homeostasis [3,[38][39][40][41][42][43][44][45][46][47]. Several heavy metal transporters were identified at the plasma membrane level (Figure 4A), with both high and low affinity. For example, Ctr1p, Smf1p and Zrt1p are involved in the high-affinity uptake of Cu + , Mn 2+ and Zn 2+ , respectively [84][85][86]. Low-affinity plasma membrane transporters are more numerous and less specific: Fet4p for Fe 2+ , but also for Cu 2+ , Cd 2+ , Mn 2+ , and Zn 2+ ; Zrt2p for Zn 2+ , but also for Fe 2+ , Co 2+ , Cu 2+ , Cd 2+ , Mn 2+ [87,88]. Transporters for phosphate or amino acids were also shown to participate in the low-affinity transport of Cd 2+ , Co 2+ , Cu 2+ , Mn 2+ , and Ni 2+ [89,90]. All these transporters are likely to be assaulted by surplus metals (Figure 4B) when cells are exposed to contaminated environments [91].
To have any chance of survival under heavy metal stress, the cell needs to be one step ahead of the "villain" ions and to get prepared for defense by using various strategies. The attempts Figure 4. Toxicity of heavy metal exposure. A. Schematic representation of transporters involved in the uptake of essential metals under normal conditions. B. Under high surplus of heavy metals, the transporters will carry the excess cations into the cell, where they bind non-specifically to biomolecules, altering their structure and functionality [91]. to understand the role of calcium in preparing the yeast cell to resist the heavy metal attack are summarized in the following sections.

Cd 2+
Cd 2+ is one of the most studied non-essential heavy metals as it is a global environmental pollutant present in soil, air, water, and food, representing a major hazard to human health [92]. External Cd 2+ was shown to unequivocally induce the [Ca 2+ ] cyt elevations in S. cerevisiae, as recorded in aequorin-expressing cells, which responded through a sharp increase in the [Ca 2+ ] cyt , just a few seconds after being exposed to high Cd 2+ [23]. Interestingly, the chemically similar Zn 2+ and Hg 2+ failed to elicit [Ca 2+ ] cyt elevations under the same conditions [23]. The response to high Cd 2+ depended mainly on external Ca 2+ (transported through the Cch1p/ Mid1p channel) and to a lesser extent on the vacuolar Ca 2+ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd 2+ was influenced by perturbations in Ca 2+ homeostasis in that the tolerance to Cd 2+ often correlated with sharp Cd 2+ -induced [Ca 2+ ] cyt pulses ( Figure 5A, B), while the Cd 2+ sensitivity was accompanied by the incapacity to rapidly restore the low levels of [Ca 2+ ] cyt [23] ( Figure 5C).
It had been suggested that Cd 2+ toxicity was a direct consequence of Cd 2+ accumulation in the ER and that Cd 2+ does not inhibit disulphide bond formation (which could account for the lack of response in the case of Zn 2+ and Hg 2+ ) but perturbs calcium metabolism. Cd 2+ activates the calcium channel Cch1/Mid1 under low external Ca 2+ , which also contributes to Cd 2+ entry into the cell [93]; the protective effect of Ca 2+ may be the result of competitive uptake between the two cations at the plasma membrane. In this line of evidence, it was shown that excess concentration of extracellular Ca 2+ attenuates the Cd 2+ -induced ER stress [94]. It was Figure 5. determined that divalent Cd 2+ and Ca 2+ have very similar physical properties, with ionic radii of Ca 2+ (0.97 Å) and Cd 2+ (0.99 Å) giving similar charge/radius ratios, meaning that these ions are able to exert strong electrostatic forces on biological macromolecules [95]. Under such circumstances, the Cd 2+ -induced aequorin luminescence observed could also be the result of aequorin binding to Cd 2+ instead of Ca 2+ . This was not the case though: when measuring the Cd 2+ accumulation in yeast cells, it was revealed that the Cd 2+ -induced aequorin luminescence occurred significantly faster than the Cd 2+ uptake, indicating that the luminescence produced was the result of increase in [Ca 2+ ] cyt [23].

Cu 2+
Cu 2+ is one of the most important essential metals: a variety of enzymes require copper as a cofactor for electron transfer reactions [96]. Nevertheless, when in excess, Cu 2+ is very toxic in the free form because of its ability to produce free radicals when cycling between oxidized Cu 2+ and reduced Cu + . Studies correlating Ca 2+ with Cu 2+ toxicity in yeast are scarce, but it had been known that the inhibitory effect of Cu 2+ on glucose-dependent H + efflux from S. cerevisiae could be alleviated by Ca 2+ [97]. The role of Ca 2+ in mediating the cell response to high concentrations of Cu 2+ was investigated in parallel with Cd 2+ , and it was noted that exposure to high Cu 2+ determined broad and prolonged [Ca 2+ ] cyt waves which showed a different pattern from the [Ca 2+ ] cyt pulses induced by high Cd 2+ [23]. In contrast to Cd 2+ , Ca 2+ − mediated responses to high Cu 2+ depend predominantly on internal Ca 2+ stores [24] (Figure 6A).
It was found that the cell exposure to high Cu 2+ -induced broad Ca 2+ waves into the cytosol which were accompanied by elevations in cytosolic Ca 2+ with patterns that were influenced by the Cu 2+ concentration but also by the oxidative state of the cell [18,24]. When Ca 2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca 2+ pool under Cu 2+ stress was the vacuolar Ca 2+ channel, Yvc1p, also activated by the Cch1pmediated Ca 2+ influx (Figure 6). Using yeast mutants defective in the Cu 2+ transport across the plasma membrane, it was found that the Cu 2+ -dependent Ca 2+ elevation could correlate with the accumulated metal, but also with the Cu 2+ − induced oxidative stress and the overall oxidative status. Moreover, it was revealed that Cu 2+ and H 2 O 2 acted in synergy to induce Ca 2+ -mediated responses to external stress [24]. Interestingly, other redox active metals such as Mn 2+ or Fe 2+ were inactive in inducing [Ca 2+ ] cyt waves ( [23], unpublished observations), probably because these metals are less redox-reactive than the Cu 2+ /Cu + couple ( Figure 6D) under aerobic conditions [98].

Mn 2+
High manganese failed to elicit Ca 2+ elevations irrespective of the magnitude of the insult applied ( [23]; unpublished observations). The response was monitored over a wide range of concentrations (from the quasi-physiological 0.5 mM to the super lethal 50 mM) and times (up to 60 min of exposure

Fe 2+
Fe 2+ toxicity can be the result of direct ionic effect, but the indirect effect of catalyzing Fenton reactions, in which highly reactive oxygen species arise, represents the main concern raised  [24]. The cell behavior described in A-C is similar to the Ca 2+ -mediated response to oxidative stress [18], suggesting that the Cu 2+ -induced [Ca 2+ ] cyt changes may be indirectly mediated by the formation of reactive oxygen species during copper shuffling between oxidative states Cu 2+ -Cu + (D).
by Fe 2+ surplus. As in the case of Mn 2+ , excess Fe 2+ did not elicit sudden elevations in [Ca 2+ ] cyt upon exposure [23]. It had been reported that yeast strains lacking the components of the Cch1p/Mid1p plasma membrane channel were hypersensitive to Fe 2+ . When measuring the relative Ca 2+ accumulation, it was noted that iron stress also increased the residual Ca 2+ uptake in the cch1Δ mid1Δ double knockout mutant [8]. As the Ca 2+ measurements in this study were done radiometrically, there must have been a considerable lag between application of the stimulus and Ca 2+ measurement (unlike aequorin determinations, which allow Ca 2+ detection simultaneously with stimulus application), and the mutant's sensitivity towards Fe 2+ might have been caused by Ca 2+ lingering in the cytosol, as in the case of Cd 2+ -sensitive mutants [23].

Other metals
The surplus of heavy metals such as Ni 2+ , Co 2+ , Pb 2+ , Hg 2+ , and Ag + did not have the ability to rapidly induce elevations in [Ca 2+ ] cyt . In some cases, (Ni 2+ and Co 2+ ) exogenous Ca 2+ alleviated the toxicity of the metal ions, but this effect was rather related to the inhibition of Co 2+ or Ni 2+ uptake by Ca 2+ [103].

Concluding remarks
In this chapter, we attempted to highlight the studies made in S. cerevisiae which correlate the exposure to high concentrations of heavy metals with the Ca 2+ -mediated cellular responses. S. cerevisiae is a very good model to study the cell response to sudden changes of metal concentration in the environment; such studies were greatly facilitated by the ease of obtaining yeast cells expressing aequorin in the cytosol, thus allowing the realtime detection of [Ca 2+ ] cyt fluctuations. By combining Ca 2+ monitoring under metal stress with the genetic approaches that make use of mutants with perturbed heavy metal or Ca 2+ homeostasis, important aspects related to cell adaptation or cell death under heavy metal stress have been elucidated. Using yeast cells expressing aequorin in the cytosol provides answers regarding the immediate Ca 2+ -mediated responses, which are crucial for deciding the cell fate. Nevertheless, to understand the Ca 2+ -mediated cell responses which occur at later phases, developing sensitive Ca 2+ sensors targeted to specific compartments is still a desiderate for future studies.