As sessile organisms, coordination of development and reproduction in a dynamic, and often stressful, environment presents a particular challenge for plants. Rapid processing of internal and external cues by complex signal transduction pathways leads to stimulus-appropriate physiological responses on an organismal scale. In plants, myriad signaling pathways are mediated by calcium (Ca2+) signals, and it is thought that different stimuli elicit unique patterns of Ca2+ influx into cells (termed Ca2+ ‘signatures’) that encode information important for proper physiological responses. Encoding of information in the form of Ca2+ signatures requires that decoding elements be present in cells to direct downstream cellular processes. This role is filled by Ca2+-binding proteins that serve as Ca2+ sensors. Interestingly, plant genomes encode multiple expanded families of Ca2+ sensors not found in animal genomes. Among these, the calmodulin (CaM)-like proteins (CMLs) are represented by a 50 member family in Arabidopsis. On the basis of structural homology, CMLs are predicted to function like conserved CaM, however, little work has been done to address this question. Biochemical characterization of CML39 indicates that it possesses structural properties consistent with function as a Ca2+ sensor. Analysis of transgenic CML39 loss-of-function (cml39) mutants revealed that CML39 is important for proper seedling establishment in the absence of exogenous metabolisable carbon as cml39 seedlings entered a state of developmental arrest shortly after germination. cml39 mutants also exhibited a conditional ‘de-etiolated’ phenotype when grown in complete darkness and exaggerated hypocotyl elongation under a short-day light regime. Genetic data suggest that CML39 functions in signaling pathways downstream of light perception, and this idea is supported by the observation that CML39 iii is expressed in light-sensing tissues, and that subunit 5 of the COP9 signalosome, a protein critical for photomorphogenesis, was identified as a putative target of CML39. Collectively, results show that CML39 is Ca2+ sensor that serves a critical regulatory role during seedling establishment when sucrose is limited, and importantly, further underscore the pervasiveness of Ca2+ signaling in plant growth and development. / Thesis (Ph.D, Biology) -- Queen's University, 2013-05-29 18:16:25.769

Thimerosal is a mercury-containing component found in many vaccine preparations and used as a preservative. It causes Ca2+ movement across cell membrane in different cells that may be mediated via effects on different receptors and ion channels. A rise in intracellular free Ca2+ concentrations ([Ca2+]i) is a key signal for many pathophysiological processes in cells, including apoptosis and necrosis. Thimerosal increases [Ca2+]i in different cell types. The mechanisms underlying thimerosal-induced Ca2+ signal vary with cell types. The present study evaluated the effects of thimerosal on [Ca2+]i in human prostate cancer cells (PC3). WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of thimerosal. The experimental results may be helpful to understand the pahrmocological and toxicological effects of thimerosal on cells from important organs. Results showed that thimerosal (10¡V200£gM) increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal-induced Ca2+ influx was inhibited by econazole, SKF963656, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, a 200-mM thimerosal-induced [Ca2+]i rise was partly inhibited after pretreatment with 2,5-di-tert-butylhydroquinone (BHQ) (an endoplasmic reticulum Ca2+ pump inhibitor). Thimerosal at 1¡V7£gM induced cell death in a concentration-dependent manner that was not reversed when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N¡¦,N¡¦-tetraacetic acid (BAPTA). Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in PC3 cells, thimerosal induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Thimerosal also induced cell death in a Ca2+-independent apoptotic manner.

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La survie des cellules T naïves est essentielle au bon fonctionnement du système immunitaire à long terme. Les rats BBDP (Bio-breeding Diabetes prone) sont caractérisés par une haute prédisposition au développement du diabète ainsi que par une diminution significative du nombre de cellules T naïves. Ces rats comportent une mutation de type décalage de lecture dans le gène codant pour «GTPase Immunity-Associated Protein 5» (Gimap5) ce qui entraine l’apoptose des lymphocytes T. Le mécanisme par lequel la déficience de la protéine GIMAP5 conduit les cellules T à la mort est actuellement méconnu. GIMAP5 a également été associée à différentes maladies auto-immunes, ce qui suggère son influence dans l'homéostasie des lymphocytes T. Des résultats antérieurs de notre groupe de recherche ont montré que l'absence de GIMAP5 entraîne une diminution du flux de Ca2+ ainsi qu’une réduction de la capacité mitochondriale à emmagasiner du Ca2+ suite à la stimulation du TCR. Cependant, GIMAP5 n'est pas une protéine mitochondriale. Afin de mieux comprendre le rôle de GIMAP5 dans la biologie des cellules T, au cours de mes études doctorales, je me suis concentré sur la localisation cellulaire de la protéine ainsi que sur son rôle dans l'homéostasie du Ca2+. Comme modèle d’étude, j'ai établi des lignées cellulaires HEK293T stables pour l’expression de GIMAP5, ainsi que pour différents mutants et variantes de la protéine. Ceci m’a permis d’élucider l'importance du domaine transmembranaire (TM) pour la localisation et le rôle physiologique de GIMAP5 ainsi que la différence entre les deux variantes de cette protéine. Mes résultats ont permis de montrer que l'expression de Gimap5 ne semble pas être nécessaire après l’activation des lymphocytes T. En parallèle, j'ai confirmé nos observations antérieures qui démontrent l’influence de GIMAP5 dans l'homéostasie du Ca2+ et sa colocalization avec les microtubules. En outre, j'ai montré que GIMAP5 se trouve dans des structures de type vésiculaire, particulièrement dans la membrane lysosomale où son domaine TM est essentiel à son bon fonctionnement et localisation. Mes résultats suggèrent que les mitochondries exhibent un défaut dans leur capacité à emmagasiner du Ca2+ au niveau basal, ainsi que suite à l’activation du TCR. Enfin, j'ai démontré pour la première fois, que l'influence de GIMAP5 sur le stockage de Ca2+ lysosomal peut avoir un impact sur la survie des lymphocytes T. D’après ces observations, une des fonctions probables de GIMAP5 serait d’empêcher la fermeture prématurée des canaux de relâche calcique. Finalement, GIMAP5 pourrait être engagé dans des mécanismes visant à prolonger et raffiner la signalisation du Ca2+ dans les cellules T. Bref, la régulation du Ca2+ lysosomal médié par GIMAP5 est essentielle à la survie de cellules T naïves. / Abstract: Healthy and long-term survival of naïve T cells is essential for proper functioning of the immune system. In bio-breeding diabetes prone (BBDP) rats, there is a critical decrease in the number of naïve T cells. In these rats, a recessive frameshift mutation in the GTPase of Immune-Associated Protein 5 (Gimap5) gene induces lymphocytes to undergo spontaneous apoptosis. The death of T cells driven by a deficiency of the GIMAP5 is currently not fully understood. Interestingly, different autoimmune diseases have shown an association with perturbations in the Gimap5 gene, which further suggests its influence in basal lymphocyte homeostasis. Previous findings by our group have shown that the absence of GIMAP5 results in a decrease calcium flux following TCR stimulation and an impaired capacity of the mitochondria to buffer calcium entry. However, GIMAP5 is not a mitochondrial protein. During my Ph.D. studies, I focused on clarifying the cellular localization of GIMAP5 as well as its function in Ca2+ homeostasis in order to further understand its role in T cell biology. As a model, I established HEK293T cells stable for the expression of the different mutants and variants of the GIMAP5 protein. Where I uncovered the importance of the transmembrane domain (TM) for GIMAP5 localization and physiological role, as well as the differences between the two variants of GIMAP5. The results obtained show that the expression of Gimap5 is no longer needed after T cells activation. Moreover, our previous observations were confirmed and expanded upon regarding GIMAP5’s influence on Ca2+ homeostasis and colocalization with the cytoskeleton. It was also shown that GIMAP5 localizes to vesicular-like structures, particularly to the lysosomal membrane, where its TM domain is critical for proper functioning and localization. My results suggest that the mitochondria might be impaired to uptake as well as retain Ca2+ at their full capacity in the absence of GIMAP5. Finally, I observed for the first time that GIMAP5’s influence on lysosomal Ca2+ storage could impact lymphocyte survival. These results suggest that GIMAP5 may work as a backup mechanism to prevent premature closure of Ca2+ channels and Ca2+ influx or as a mechanism to prolong and refine Ca2+ signaling in T cells.

PURPOSE: Muscle overuse is characterized by inflammation, reduced strength and muscle damage. It has been proposed that calcium (Ca2+) accumulation during muscle contraction, is responsible for muscle damage. Muscle contractile properties are regulated by calcium regulatory excitation contraction coupling mechanisms. Therefore, the main aim of the present study was to investigate the effects of voluntary repetitive tasks during six weeks on the rate of sarcoplasmic reticulum (SR) Ca2+-uptake, and Ca2+-release, in young female Sprague-Dawley rats. Secondly, this study aims to evaluate the effect of the training on muscular strength and the relationship between SR Ca2+-kinetics and grip strength test performance. METHODS: Six rats were trained (EXP), using a well-established model of reaching and handle pulling with the upper extremities (2 hr/day, 3days/week, 6 weeks), six control rats (KON) were included that were not exposed to the task. Grip strength were evaluated using a grip strength meter for rodents, two weeks prior the training was initiated, and two days after the training period was concluded. Tissue samples were obtained from the supraspinatus and trapezius muscle, and the rate of SR Ca2+-uptake and SR Ca2+-release were analysed using the fluorescent Ca2+ indicator indo 1. RESULTS: The analysis revealed that EXP had a significant higher rate of SR Ca2+-uptake, in both supraspinatus (33%, P < 0,05) and trapezius (14%, P < 0,05), compared to KON. However, no significant differences in SR Ca2+-release rate were found between groups, in neither of the muscles. A decline in grip strength were found in both EXP and KON, with no significant differences between groups. No significant correlation between grip strength and the Ca2+ release uptake variables could be found. CONCLUSION: The present results suggests that repetitive voluntary reaching and handle pulling with the upper extremities during six weeks, induce extant changes in SR Ca2+-uptake rate in rats.

Calcium is a highly versatile intracellular signal molecule that can regulate numerous different cellular functions. In filamentous fungi there is evidence for it being involved in regulating various processes, including spore germination, hyphal tip growth, hyphal branching and conidiation. During colony initiation in the filamentous fungus Neurospora crassa, conidia form germ tubes which are involved in colony establishment, and conidial anastomosis tubes (CATs) which are involved in generating fused networks of conidial germlings. The primary research aim of this thesis was to analyze the role of Ca2+-signalling and homeostasis during colony initiation in N. crassa. Removal of Ca2+ from the growth medium showed that external Ca2+ was necessary for CAT fusion and, more specifically, was required for CAT chemoattraction. Two L-type Ca2+ channel blockers (verapamil and diltiazem) with different modes of action were found to inhibit both conidial germination and CAT fusion in wild type strains and CAT fusion was shown to be more sensitive to these two drugs. These channel blockers were additionally found to inhibit Ca2+ uptake by conidial germlings of the wild type expressing the aequorin Ca2+ reporter. However, the channel blockers also, unexpectedly, raised the cytosolic free Ca2+ ([Ca2+]c) resting level in these germlings suggesting that they did not just inhibit L-type Ca2+ activity. The morphological phenotypes (conidial germination, hyphal extension rate, conidiation and hyphal branching) of 22 mutants defective in different components of their Ca2+-signalling and homeostasis machinery were characterized in order to identify their possible roles of Ca2+ during colony initiation and development. The ∆cch-1 mutant lacking the CCH-1 L-type Ca2+ channel gene exhibited a reduction in CAT fusion. CAT fusion was decreased even further in a double mutant (∆cch-1∆mid-1) suggesting that that the CCH-1 and MID-1 proteins operate in combination during this process. Increased extracellular Ca2+ partially restored the phenotypes of the ∆cch-1, ∆mid-1 smco-1 and ∆cch-1∆mid-1 mutants which is consistent with CCH-1 and MID-1 being involved in Ca2+ uptake from the external medium. Calcium signatures following mechanical perturbation were successfully measured in populations of conidial germlings using aequorin expressed in the wild type and in deletion mutants (∆cch-1, ∆yvc-1, ∆fig-1) lacking different Ca2+ channels. The removal of external Ca2+ completely abolished the [Ca2+]c increase in response to mechanical perturbation and CCH-1 was found to partly contribute to this increase in [Ca2+]c. Various Ca2+-sensitive dyes (Oregon green 488, Fluo-4 and Calcium Green-1) were also tested to determine if they can be used to image [Ca2+]c at the single cell and subcellular levels. Only Fluo-4 allowed the measurement of [Ca2+]c in individual cells but the changes in dye fluorescence in response to changes in [Ca2+]c were too small to be useful for imaging [Ca2+]c dynamics at the subcellular level. The other two dyes underwent rapid compartmentalization in organelles when loaded into germlings. The plant antifungal proteins (defensins), MsDef1, MtDef4 and PAF were all found to disrupt Ca2+ signaling/homeostasis in conidial germlings of N. crassa. They all inhibited the [Ca2+]c increase and raised the resting level of [Ca2+]c in response to mechanical perturbation. Analysis of an aequorin expressing mutant that was defective in glucosylceramide synthase (∆gcs) showed that the effects of MsDef1 (but not MtDef4) on [Ca2+]c were mediated by the sphingolipid glucosylceramide. All of the defensins tested were found to exhibit different potencies with regard to their inhibitory effects on conidial germination and CAT fusion.

During excitation-contraction coupling, Ca2+ transient induced by the depolarization of membrane potential is the trigger of mechanical contraction in cardiac myocytes, which is responsible for the pumping function of the heart. However, mechanisms underlying intracellular Ca2+ regulation and the coupling between Ca2+ transient and membrane potential are not completely understood. Abnormalities in intracellular Ca2+ regulation have been observed during heart failure and cardiac arrhythmias, such as intracellular Ca2+ alternans and T-tubule disorganization. In this project, intracellular Ca2+ dynamics in different types of cardiac myocytes were investigated by using computer modelling. For atrial myocytes, a biophysically detailed computer model was developed to describe the observations of Ca2+ alternans and Ca2+ wave propagation in cardiac myocytes lacking T-tubules. The model was validated by its ability to reproduce experimental observed Ca2+ wave propagation under normal condition and the influences on spatial Ca2+ distribution by modifying various aspects of Ca2+ cycling, such as Ca2+ influx, SR Ca2+ uptake and SR Ca2+ release in cardiac myocytes lacking T-tubules. Mechanisms underlying the genesis of Ca2+ alternans in this type of cell were investigated by the model. Furthermore, a spontaneous second Ca2+ release was observed in response to a single voltage stimulus pulse with enhanced Ca2+ influx as well as SR Ca2+ overload. For the ventricular myocytes, an existing canine model was used to study the genesis of APD and intracellular Ca2+ alternans under various conditions. The genesis of Ca2+ alternans was investigated by analyzing the relationship between systolic Ca2+ concentration and SR Ca2+ content. On the other side, the roles of SR Ca2+ regulation and action potential restitution in the genesis of intracellular Ca2+ and APD alternans were also examined under various conditions. In addition, it was shown that spatially discordant Ca2+ alternans was generated when the Ca2+-dependent inactivation of ICa,L was strong. It tended to be concordant for weak Ca2+-dependent inactivation of ICa,L. For the sinoatrial node cells, a mathematical model was developed to simulate stochastic opening of unitary L-type Ca2+ channel and single RyR channel, thereby reproducing experimental observed local Ca2+ release during diastolic depolarization phase of the action potential. Simulation results of ionic channel block and modifications of SR Ca2+ regulation suggested a limited role of intracellular Ca2+ in the automaticity of central SA node cells.

Dans les cardiomyocytes, la concentration intracellulaire de Ca2+ doit être finement régulée pour maintenir l’homéostasie calcique. La protéine Stromal interaction molecule 1 (STIM1), qui joue un rôle important dans le maintien des niveaux intracellulaires de Ca2+ des cellules non excitables en opérant l’entrée capacitative de Ca2+ (SOCE), est aussi présente dans les cardiomyocytes. Plusieurs études démontrent que STIM1 et le SOCE jouent un rôle important dans le développement de l’hypertrophie des cardiomyocytes. De plus, récemment, un nouveau rôle de STIM1 a commencé à émerger. STIM1 pourrait moduler l’expression de certains canaux calciques à la membrane plasmique en régulant leur trafic intracellulaire. Le but de l’étude était d’identifier des partenaires d’interaction de STIM1 dans l’optique de révéler le mécanisme par lequel STIM1 induit l’internalisation d’un canal calcique voltage-dépendant. Les protéines recueillies par le pull-down à partir des lysats de coeurs de rats avec une colonne d’affinité composée du domaine ERM de STIM1, ont été analysées en spectrométrie de masse. La protéine Muscle related coiled-coil (MURC), une protéine de la famille des Cavin, a été retenue comme partenaire d’interaction potentiel de STIM1. Comme elle est exprimée dans les cardiomyocytes et dans les cellules musculaires squelettiques, qui sont des cellules où la régulation de la signalisation calcique est primordiale pour le bon fonctionnement des tissus et qu’elle semble interagir avec STIM1, qui est un acteur important de la signalisation calcique, notre objectif s’est élargi et nous avons investigué sur l’implication de MURC dans la signalisation calcique en général. Nous avons donc confirmé par co-immunoprécipitation que le domaine ERM de STIM1 interagissait avec MURC. Puis, par des essais d’imagerie calcique, nous avons démontré que la surexpression de MURC pouvait provoquer différentes réponses dans différents types cellulaires en fonction de l’activation de la mobilisation calcique. En effet, nous avons observé une augmentation du SOCE qui est indépendante de la voie RhoA/ROCK dans les cellules HEK293T, une diminution de l’entrée de calcium médiée par un récepteur (ROCE) qui est pourrait être dépendante de la voie RhoA/ROCK dans les cellules T6.11 et une diminution de l’activation de RhoA de façon dépendante de l’activation du SOCE dans les cardiomyocytes HL-1. Nous avons aussi montré que MURC pouvait interagir à la membrane plasmique avec les protéines Orai1, qui sont les protéines formant les canaux CRAC (Ca2+ release-activated Ca2+) du SOCE et ce de façon dépendante de l’activation de STIM1. Enfin, les résultats de cette étude suggèrent que MURC est un partenaire d’interaction de STIM1 impliqué dans la signalisation calcique. En effet, MURC peut moduler l’activation de RhoA, ce qui pourrait induire l’internalisation de canaux calciques. De plus, son interaction avec STIM1 et Orai1 pourrait notamment faire un pont facilitant l’interaction entre STIM1 et Orai1 ce qui aurait pour effet d’augmenter le SOCE et possiblement contribuer à augmenter l’hypertrophie des cardiomyocytes.

Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

Information is transmitted from the cell surface to various specific targets in the cell via several cellular signaling pathways. Cytosolic free calcium (Ca2+)is one of the most versatile and ubiquitous intracellular messengers since it is able to regulate diverse number of functions such as proliferation, secretion, fertilization, metabolism, learning and memory. In the last couple of years, evidence has been accumulating that Ca2+ ion is able to integrate information from multiple signaling pathways and convert this information into a code which regulates events ranging from contraction to modification of gene expression (Berridge et al. 1998). It was shown that Ca2+ concentration displays oscillatory behavior in response to agonist stimulation in a variety of cells(Goldbeter 1996) and the frequency of these oscillations increases with the concentration of agonist, a behavior called frequency encoding which has led to the concept that many Ca2+-regulated processes are controlled by these codes(Berridge 1998). Although the presence of Ca2+ oscillations and the sources of Ca2+ pools involved is known in many cell types, it is yet not known how the various frequencies of Ca2+ oscillations are converted into codes that regulate the numerous cellular events. Recently a number of cellular targets that decode Ca2+ signals and are tuned to the frequency of Ca2+ oscillations have been identified. Prominent among them are calcium-calmodulin kinase II (CAM II) and protein kinase C (PKC). The objective of this work is to study and mathematically model the oxytocin and vasopressin-induced Ca2+ oscillations in cells of normal rat liver (Clone 9) and cells of pregnant human myometrium. The proposed model accounts for the receptor-controlled Ca2+ oscillations involving positive feedback leading to activation of phospholipase C (PLC) and negative feedback from PKC onto G-proteins which simulates many of the features of observed intracellular Ca2+. The model also incorporates the concept that coordinated Ca2+ signals in a group of hepatocytes require both effective gap junctions and the presence of agonist at each cell surface. Another objective of this research is to understand the relevance of frequency-encoded signals by performing an analysis of frequencies of Ca2+ oscillations using the Fast Fourier Transform and the Wavelet Transform. The validity of the model was confirmed by using statistical tests to check if the frequencies and amplitudes of the experimental Ca2+ oscillations match with those of the modelled oscillations.