Ca2+ influx pathways linked to Ca2+ store depletion and cell signalling

Thesis

Reference

Ca2+ influx pathways linked to Ca2+ store depletion and cell signalling

JOUSSET, Hélène

Abstract

L'élévation de la concentration calcique cytoplasmique est un élément central de la signalisation cellulaire. L'entrée de calcium du milieu extérieur dans le cytosol est un processus essentiel qui assure la pérennité des signaux calciques en permettant le remplissage des stocks calciques intracellulaires situés principalement dans le réticulum endoplasmique (RE). Dans les cellules non excitables, deux différents types d'influx prédominent, l'un activé par la vidange du RE le SOCE (Store-Operated Ca2+ Entry) l'autre activé par des seconds messagers indépendamment du remplissage des stocks calciques le RACE (Receptor-Activated Ca2+ Entry). Dans un premier temps nous avons étudié le processus de remplissage du RE qui accompagne l'influx SOCE ainsi que l'implication de la protéine STIM1 (Stromal Interaction Molecule 1) nouvellement identifiée comme essentielle dans ce processus. Dans un second projet, les différents modes d'entrée calcique (RACE et SOCE) présents dans les cellules endothéliales ont été étudiés.

JOUSSET, Hélène. Ca2+ influx pathways linked to Ca2+ store depletion and cell signalling. Thèse de doctorat : Univ. Genève, 2007, no. Sc. 3928

URN : urn:nbn:ch:unige-6363

DOI : 10.13097/archive-ouverte/unige:636

Available at:

http://archive-ouverte.unige.ch/unige:636

Disclaimer: layout of this document may differ from the published version.

Département de biologie cellulaire FACULTE DES SCIENCES

Professeur J.-C. Martinou

Département de physiologie cellulaire FACULTE DE MEDECINE

et métabolisme Professeur N. Demaurex

_____________________________________________________________________

Ca²⁺ influx pathways linked to Ca²⁺

store depletion and cell signalling

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Hélène JOUSSET de

France

Thèse n°3928 Genève

2007

I Publications of the thesis ___________________________________________ 2 II Résumé en français _______________________________________________ 3 III Introduction ___________________________________________________ 5 A. Ca²⁺ and biology _____________________________________________________5 B. The Ca²⁺ signaling____________________________________________________6 i Ca²⁺ toolkit______________________________________________________________ 6 ii Decoding Ca²⁺ signals _____________________________________________________ 8 C. Ca²⁺ influx __________________________________________________________9 i Stores emptying: Store-Operated Calcium channels (SOCC) _______________________ 9 ii Second messenger interaction: Receptor-activated Ca2+ channels (RACC)___________ 12

IV Aims of the study ______________________________________________ 14 V Experimental tools _______________________________________________ 14 A. Cells ______________________________________________________________14 B. Intracellular Ca²⁺ measurement _______________________________________15 C. Proteins knockdown _________________________________________________17 VI Publication I__________________________________________________ 18 A. Introduction________________________________________________________18 VII Publication II _________________________________________________ 33 A. Introduction________________________________________________________33 VIII Publication III ________________________________________________ 48 A. Introduction________________________________________________________48 IX Discussion and perspectives______________________________________ 85 A. The RACE complexity _______________________________________________85 B. STIM1: an ongoing story _____________________________________________86 X Remerciements __________________________________________________ 88 XI References ___________________________________________________ 89

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My thesis is based on the followed publications:

Publication I

STIM1 knockdown reveals that store-operated Ca²⁺ channels located close to sarco/endoplasmic Ca²⁺ ATPases (SERCA) pumps silently refill the endoplasmic reticulum.

Hélène Jousset, Maud Frieden, and Nicolas Demaurex Journal of Biological Chemistry (2007) 282: 11456-11464

Publication II

Evidence for a receptor-activated Ca²⁺ entry pathway independent from Ca²⁺ store depletion in endothelial cells

Hélène Jousset, Roland Malli, Nathalie Girardin, Wolfgang F. Graier, Nicolas Demaurex, and Maud Frieden.

Cell Calcium (in press)

Publication III

Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion

Fabienne Jaques, Hélène Jousset, Alejandra Tomas, Anne-Lise Prost, Claes B. Wollheim, Jean-Claude Irminger, Nicolas Demaurex and Philippe A. Halban.

Manuscript submitted

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L’élévation de la concentration calcique cytosolique est un élément central de la signalisation cellulaire. Cette augmentation peut provenir de deux origines : le relâchement du Ca²⁺ intracellulaire ou l’entrée de calcium depuis le milieu extracellulaire. En effet, la cellule ayant une concentration calcique très faible dans le cytosol (de l’ordre de 100 nM), elle utilise le Ca²⁺ provenant de deux sources : le réticulum endoplasmique (RE) et le milieu extracellulaire. Le RE constitue le réservoir calcique interne de la cellule avec une concentration avoisinant 200 μM. De plus, le milieu extracellulaire dans lequel la cellule baigne possède une concentration calcique environ 20'000 fois supérieur à celle du cytoplasme (2 mM). Dans les cellules dépourvues de canaux activés par le voltage dites non- excitables, le mécanisme d’entrée calcique le plus étudié est intitulé SOCE (store-operated Ca²⁺ entry). Les SOCC (store-operated Ca²⁺ channel) sont des canaux transmembranaires activés par la vidange calcique des stores intracellulaires. Néanmoins, il existe une deuxième voie d’entrée calcique appelée RACE (receptor-activated Ca²⁺ entry) qui est activée par la génération de seconds messagers. Aussi, contrairement aux canaux SOCC, les canaux RACC sont indépendants du niveau de remplissage calcique du RE. Le but de ma thèse est l’étude des mécanismes responsables de l’entrée calcique principalement dans les cellules non excitables. J’ai tout d’abord étudié le chemin de signalisation aboutissant à l’ouverture des canaux SOCC et plus particulièrement le phénomène de remplissage des stores calciques qui suit cette activation. Dans un deuxième temps j’ai analysé les différents modes d’entrée calcique présents dans les cellules endothéliales ainsi que la possibilité de les différencier.

Finalement, j’ai collaboré avec un groupe de recherche travaillant sur le rôle des contacts intercellulaires des cellules beta du pancréas dans la régulation de la sécrétion d’insuline.

Dans ce troisième projet mon rôle fut de d’examiner l’impact de la perte de connections intercellulaires sur l’homéostasie calcique.

Le premier projet traite donc de l’influx SOC et plus précisément du processus de remplissage des stocks calciques qui accompagne cette entrée calcique. Récemment, et suite à 20 ans d’investigations, le mécanisme liant la déplétion du RE à l’activation de l’influx SOC a été clarifié par l’identification de la protéine STIM1. STIM1 (STromal Interaction Molecule 1) est une protéine transmembranaire localisée dans la membrane du RE. Son rôle est d’activer les canaux SOC à la membrane lors de la vidange des réservoirs calciques intracellulaires. STIM1 est sensible à la concentration en Ca²⁺ l’entourant et, lorsque le niveau de Ca²⁺ dans le RE diminue, STIM1 est recruté à la membrane pour activer les canaux SOC.

L’invalidation de STIM1 dans les cellules HeLa (diminution de 73% de l’ARNm) entraine une réduction de l’entrée SOC de 73 % quand les pompes SERCA (sarco/endoplasmic Ca²⁺

ATPases) sont inhibées par la thapsigargin. Lorsque les pompes SERCA sont actives, nous avons observé un remplissage fonctionnel du RE bien qu’aucun signal calcique ne soit détecté dans le cytosol. Les mesures calciques à l’intérieur du RE révèlent que l’activité basale des pompes SERCA n’est pas affectée dans les cellules avec un niveau de STIM1 réduit. De plus, malgré une cinétique plus lente dans les cellules invalidées pour STIM1, la concentration initiale en Ca²⁺ libre dans le RE est récupérée en 2 minutes de remplissage. En conséquence,

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intracellulaires de manière silencieuse, c'est-à-dire sans élévation calcique au niveau du cytosol. En outre l’activité des mitochondries n’est pas utilisée pour effectuer ce remplissage

« silencieux ». Nos expériences démontrent donc que le remplissage du RE est largement préservé en dépit de l’extrême réduction de l’entrée calcique SOC induit par l’invalidation de la protéine STIM1. Ces résultats sont cohérents avec la formation d’agrégats contenant des canaux SOC au niveau de la membrane plasmique ainsi que des senseurs STIM1 dans la membrane du RE. Ces zones de contact entre membrane plasmique et membrane du réticulum constituent de par la présence de SERCA des domaines privilégiés de remplissage en Ca²⁺ du RE (Hélène Jousset et al. J Biol. Chem. 2007, 282:11456-64).

Mon deuxième projet est consacré à la différenciation de l’entrée calcique RACE par rapport à celle du SOCE. Pour cela, nous avons utilisé des stimulations différentes, soit l’addition d’agoniste (histamine 100 μM), soit d’un inhibiteur de la pompe SERCA (10 µm thapsigargin) ce qui permet d’activer préférentiellement l’influx RACE ou l’influx SOCE respectivement. Après différents tests pharmacologiques nous avons démontré que le lanthanum à une concentration de 10 μM est un inhibiteur permettant de discriminer les deux influx. En effet, le lanthanum bloque presque entièrement l’influx SOCE mais n’a aucun effet sur l’influx RACE. De plus les mesures de la variation du Ca²⁺ libre dans la lumière du RE ont confirmé que la déperdition de Ca²⁺ dans le RE était très faible lors de la stimulation des cellules avec l’histamine. Ce qui signifie que la voie de signalisation SOCE est très peu impliquée lors de la stimulation des cellules endothéliales avec un agoniste. Par ailleurs l’inhibition des mitochondries a aussi un effet discriminatoire sur les deux influx, car elle diminue le SOCE sans affecter le RACE. Ces résultats indiquent qu’en condition de stimulation physiologique le RACE est la voie de signalisation prédominante et que le SOCE dans les cellules endothéliales représente probablement un système de sécurité responsable du maintient de la forte concentration calcique du RE (Hélène Jousset et al. Cell Calcium, in press).

La troisième publication discute du rôle des jonctions intercellulaires des cellules beta du pancréas dans la régulation de la sécrétion d’insuline. Contrairement aux cellules cultivées de manière confluente, les cellules beta isolées révèlent une dérégulation majeure de l’exocytose d’insuline. Les cellules isolées présentent une augmentation de la sécrétion basale d’insuline ainsi qu’une diminution de la réponse au glucose. Dans les cellules confluentes, l’invalidation de la protéine E-cadherine, essentielle à la formation des jonctions adhérentes entre cellules, provoque des dérégulations similaires. Au niveau basal, c'est-à-dire en présence de bas glucose, les cellules isolées présentent une activité calcique augmentée, un cytosquelette dépolymérisé ainsi qu’une activation des enzymes ERK1/2. Les élévations calciques mesurées dans le cytosol sont inhibées par la suppression transitoire du Ca²⁺ du milieu extracellulaire démontrant que ces élévations calciques proviennent de l’entrée de Ca²⁺. De plus, l’augmentation de la sécrétion basale d’insuline observée dans les cellules dispersées est elle aussi corrigée par la suppression du Ca²⁺ extracellulaire. En conclusion, l’augmentation basale de la sécrétion d’insuline des cellules dispersées est engendrée par une entrée calcique spontanée (Fabienne Jaques et al. Manuscrit submited)

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A. Ca²⁺ and biology

The famous Oxford dictionary defines calcium as “a chemical element, one of the ‘metals of the alkaline earths’, being the basis of lime; though one of the most abundant of elements, it is found in nature only in composition, and was first separated by Davy in 1808, as a light yellow metal, ductile and malleable, about as hard as gold, which rapidly oxidizes in air containing moisture, and forms ‘quick-lime’”. This designation highlights the chemical properties of calcium and its occurrence in nature. However it does not stress that calcium is essential for living organisms, particularly in cell physiology.

Ca²⁺ is one of the most frequent elements on earth crust. And together with sodium, chloride, magnesium and potassium, Ca²⁺ was one of the 5 ions contained in the early ocean.

However, in contrast with the other ions, Ca²⁺ is the only one holding important biological properties that are inherent to its ambivalence. On the one hand, high Ca²⁺ induces proteins and nucleic acids aggregation, membrane alteration and phosphate precipitation. Therefore, high Ca²⁺ concentration impedes the accurate course of fundamental biological events and thus is incompatible with life. On the other hand, Ca²⁺ has particular physical and chemical properties allowing rapid interaction with biological molecules. When the first cell appeared, creation of membranes separated the intra-cellular compartment from the extra-cellular milieu tallies with the generation of a huge Ca²⁺ gradient corresponding to a Ca²⁺ concentration

~10’000-20’000 times lower inside the cell than outside. Hence, in the protected intra-cellular environment, life became possible. With time, nature evolved to adapt to what was at the beginning a threat, and profited of this extremely high electro-chemical gradient to build a versatile and ubiquitous Ca²⁺ based signaling machinery [1].

In the human body, more than 99 % of total amount of Ca²⁺ is trapped into the skeleton or teeth. But the small remaining fraction (0.7%) of circulating Ca²⁺ is fundamental for cellular physiology and, trough evolution, calcium divalent cation (Ca²⁺) became a keystone of cellular biology signaling. To communicate with each other cells decode stimuli received at their surface. Initially, the cells transduce the external signals into the production of intra- cellular second messengers. Subsequently, second messengers trigger the activation of a biological cascade leading to a cellular response. Ca²⁺ ion is an ubiquitous and universal second messenger involved in numerous cellular processes. The first evidence of Ca²⁺

implication in a biological process was discovered fortuitously by S. Ringer in 1883 [2].

Working on heart physiology, Ringer discovered that Ca²⁺ is necessary for heart beatings. The use of tap water instead of distilled water to prepare the experimental solution is at the origin of this fundamental discovery. S. Ringer could make an isolated frog heart beat when bathed in a specific solution. However trying to repeat this experiment, he could not reproduce the beating phenomenon. He thus realized that the first successful experiment has been done accidentally in tap water solution and that beating could not occur in standard distilled water solution. S. Ringer deduced that an element present in the tap water is essential for the contraction of heart. In order to find out which element of tap water was responsible for the

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the addition of calcium was responsible for the contractions observed. Thus he concluded that external Ca²⁺ is indispensable for the beating process. Since then, Ca²⁺ was found to be involved in an immeasurable number of physiological functions. It even appears impossible to find a biological mechanism that does not imply Ca²⁺ [3]. Among the various processes that require Ca²⁺ signaling, fertilization, cell proliferation and differentiation, gene transcription, neurotransmission, muscle contraction, secretion/exocytosis, and cell death are the most emblematic Ca²⁺-regulated functions [4].

B. The Ca²⁺ signaling i Ca²⁺ toolkit

Cytosolic Ca²⁺ increasing above the resting concentration generates Ca²⁺ signals that regulate, as mentioned above, a plethora of important cellular processes. Those signals result from the coordination of a complex machinery composed of Ca²⁺ pumps, exchangers and channels. Each cell type expresses a unique set of Ca²⁺ players responsible for the specific Ca²⁺ signaling pattern of a cell that properly regulates precise cellular functions [4]. In this paragraph, a description of this important Ca²⁺ signaling toolkit will be depicted.

The cytosolic Ca²⁺ concentration is actively maintained around 100 nM and continuously exposed to extra-cellular Ca²⁺ overflow where its concentration is around 2 mM. Low cytosolic Ca²⁺ is upholded by PMCA (Plasma Membrane Ca²⁺ ATPase) that actively extrudes Ca²⁺ outside the cell. The extrusion activity of the PMCA pump is vital for the cell and the two housekeeping genes coding for PMCA1 and 4 isoforms are expressed in every single cell.

In addition to PMCA, a Na⁺/Ca²⁺ exchanger located at the plasma membrane is also able to drive Ca²⁺ ions out [5].

Ca²⁺ elevations can originate either from internal Ca²⁺ stores or from extra-cellular space.

With a free Ca²⁺ concentration of about 400 μM, the endoplasmic reticulum (ER) constitute the major internal Ca²⁺ store and massive Ca²⁺ release from the ER is a central process of Ca²⁺ signaling. The high luminal calcium concentration is maintained by SERCA pumps (sarcoplasmic endoplasmic reticulum calcium ATPase) located in the ER membrane that actively transport Ca²⁺ against the chemical gradient from the cytosol to the lumen of the ER.

Hence, the SERCA pump activity is essential to prevent cytosolic Ca²⁺ overload and to maintain a high concentration of Ca²⁺ into the lumen of the ER [5]. In the ER, Ca²⁺ is trapped by Ca²⁺-binding proteins, creating equilibrium between bound and free Ca²⁺. This free luminal Ca²⁺ constitute an essential on-demand Ca²⁺ pool ready to be released during Ca²⁺

signaling. Ca²⁺ release can be mediated by the opening of different channels such as inositol 1,4,5-trisphosphate receptor (IP3R), or Ryanodine receptor (RyR) [6]. The opening of IP3R results from the generation of the second messenger IP3 (inositol 1,4,5-trisphosphate).

Stimulation of cells with an agonist induces the activation of phospholipase C that hydrolyses Phosphatidylinositol (4,5)-bisphosphate leading to the production of diacylglycerol (DAG) and IP3. On the contrary to DAG, IP3 is able to diffuse in the cytosol and provokes IP3R opening. Similarly to IP3R, RyR are also activated by a second messenger: Ca²⁺ itself. By diffusing to the neighboring RyR, Ca²⁺ provokes supplementary Ca²⁺ release from

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induce Ca²⁺ release (CICR) and is responsible for Ca²⁺ waves recorded in muscle for example [8]. Besides, other less known messengers such as cyclic adenosine 5'-diphosphate ribose (cADP ribose) and nicotinic acid–adenine dinucleotide phosphate (NAADP) appear to stimulate Ca²⁺ release from stores. cADP ribose induce RyR opening, whereas the mechanism of NAADP- induced Ca²⁺ release has not been clearly defined, but appears to involved acidic stores [9, 10].

In addition to Ca²⁺ stores, cytosolic Ca²⁺ elevations can also originate from plasma membrane channel openings and the description of such channels will be discussed in the next chapter entitled “Ca²⁺ influx”. In addition to the endoplasmic reticulum, mitochondria are also involved in Ca²⁺ homeostasis [11]. On top of their implication in energy production, mitochondria play an important role in the shaping of cytosolic Ca²⁺ signals. Mitochondria can rapidly accumulate and release Ca²⁺ and thereby are able to buffer local Ca²⁺. Two players are involved in Ca²⁺ mitochondrial Ca²⁺ buffering: the mitochondrial Ca²⁺ uniporter (MCU) which is responsible for mitochondrial Ca²⁺ uptake and the mitochondrial Na⁺/Ca²⁺

exchanger (mNCX) that extrude Ca²⁺ from mitochondria [12]. The molecular identity of both players remained something of a mystery. However, recently, the mitochondrial uncoupling proteins (UCP), UCP2 and UCP3 where shown to be indispensable for mitochondrial Ca²⁺

uptake [13]. Still, whether UCP proteins themselves form the channel or regulate its activity remains to be determined.

Figure 1| Cellular Ca²⁺ homeostasis players.

Cells possess number of proteins to regulate Ca²⁺

signaling. This toolkit is composed of channels, pumps and exchangers embedded in diverse membranes. High Ca²⁺ concentration in the endoplasmic reticulum is maintained by the pumping of the sarco-endoplasmic Ca²⁺ ATPase (SERCA). Moreover, Ca²⁺ release from stores arise from either Inositol-1,4,5 trisphosphate receptor (IP3R) or Ryanodine receptor (RyR) activated respectively by IP3 or Ca²⁺. Mitochondria buffer cytosolic Ca²⁺ elevation by taking up and releasing Ca²⁺ through mitochondrial Ca²⁺ uniporter (MCU) and mitochondrial Na⁺/Ca²⁺ exchanger (mNCX), respectively. Finally, both the plasma membrane Ca²⁺ ATPase (PMCA) and the Na⁺/Ca²⁺ exchanger (NCX) extrudes Ca²⁺ out of the cells, and various channels are involved in Ca²⁺ entry/influx.

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This complex Ca²⁺ signaling toolkit allows to encode Ca²⁺ signals in space, time, frequency and amplitude [4]. Spatial coding of Ca²⁺ signals results in local Ca²⁺ elevations called microdomains. High Ca²⁺ microdomain allows a spatial regulation of specific cellular processes. Microdomains often result from elementary Ca²⁺ events generated by the opening of Ca²⁺ channels. Those microdomains have been given different names depending on the channel they derive from, such as sparklet, spark, or puff originating respectively from voltage-gated calcium channels (VGCCs), ryanodine receptor, and inositol triphosphate receptor opening [8]. Frequency is another important aspect of Ca²⁺ signals versatility.

Physiological stimulations are often transduced into cytosolic Ca²⁺ oscillations. Frequencies of such Ca²⁺ oscillations can be decoded as discriminating signals that activate specific transcription factors [14]. Thus, in one given cell, different patterns of Ca²⁺ signals drive the activation of diverse cellular functions [4].

Ca²⁺ signals are then decoded by Ca²⁺ sensors that bind Ca²⁺ and initiate the activation of specific signaling pathway. Three types of Ca²⁺ binding proteins exist: proteins containing EF-hand domains, protein containing C2 domains, gelsolin and annexins [3]. Among those Ca²⁺ sensors, EF-hand proteins are the one that have been best characterized and calmodulin is its most studied member. When bound to Ca²⁺, calmodulin undergoes a conformational change which allows the interaction with target protein. Subsequently, a second conformational change induces the activation of target proteins such as enzymes. Calmodulin is used to regulate many signaling cascade ending with the accomplishment of specific cellular processes such as contraction, gene transcription, ion channel modulation, etc… [4].

For example when T cells are activated by the binding of an antigen, it triggers the production of the IP3 leading to ER Ca²⁺ release induced by IP3R opening. This ER Ca²⁺ depletion leads to store-operated Ca²⁺ channels opening at the plasma membrane. The following cytosolic Ca²⁺ elevation elicits the conformational change of the calmodulin/calcineurin complex that dephosphorylates the transcription factor NF-AT. When dephosphorylated, NF-AT is recruited to the nucleus where it initiates the transcription of various gene involved in T cell proliferation [4].

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The signals produced by Ca²⁺ release from Ca²⁺ stores are limited by the capacity of the ER. Thus, a lot of Ca²⁺ signals are supported by Ca²⁺ influx that results from the opening of Ca²⁺ channels located at the plasma membrane. The activity of Ca²⁺ channels can be regulated by three types of stimuli: plasma membrane voltage, ligand or second messenger interaction, and stores emptying [15] (Figure 2).

Voltage gated Ca²⁺ channels (VGCC) located in the plasma membrane are activated by membrane depolarization and mediate Ca²⁺ influx. Their expression by a cell is sufficient to characterize this cell type as “excitable”. My work being focused on Ca²⁺ entry in non- excitable cells, I will not discuss VGCC in this introduction. Instead, Store-operated Ca²⁺

entry (SOCE) and receptor-activated Ca²⁺ entry (RACE) stimulated by the interaction with intra-cellular second messenger will be detailed.

Figure 2| The different Ca²⁺ entry channels.

Ca²⁺ entry channels are located in the plasma membrane and support Ca²⁺

influx from the extra-cellular space to the cytosol. They are classified depending on their mode of activation: plasma membrane voltage, ER Ca²⁺ filling state and second messenger.

VGCC: Voltage-Gated Ca²⁺ Channels SOCC: Store-Operated Ca²⁺ Channels RACC: Receptor-Activated Ca²⁺

Channels

i Stores emptying: Store-Operated Calcium channels (SOCC)

In non-excitable cells, the main route of Ca²⁺ entry is the store-operated Ca²⁺ entry (SOCE) pathway. As implied by their name store-operated Ca²⁺ channels (SOCC) are regulated by Ca²⁺ stores and more specifically by the amount of Ca²⁺ sequestered into ER stores. When the cellular Ca²⁺ pools are depleted, SOCC located at the plasma membrane are activated leading to Ca²⁺ influx into the cytosol and Ca²⁺ store refilling. This ubiquitous process is highly conserved among species from paramecia to human [16] and could be assimilated to an emergency mechanism preserving Ca²⁺ stores filling state. The theory of store-operated Ca²⁺ entry was first proposed by James W Putney in 1986, a mechanism that he called capacitative Ca²⁺ entry. This concept emerged from the observation that Ca²⁺ stores reload after cytosolic Ca²⁺ entry so that Ca²⁺ release and entry were part of the same

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stimulations that trigger Ca²⁺ release from stores through IP3 receptor. However, the discovery of thapsigargin completely changed the way SOCE was experimentally activated [17]. Thapsigargin is an inhibitor of the SERCA pump that induces Ca²⁺ stores emptying.

Indeed, when the SERCA pump is inhibited the ER Ca²⁺ leak could not be balanced leading to the passive depletion of ER Ca²⁺ stores. This tool allowed to demonstrate a direct link between Ca²⁺ entry channels and the ER without the involvement of any upstream intracellular pathway such as phospholipase C activation. The Ca²⁺ entry elicited by SERCA inhibition triggers an ubiquitous and extremely robust cytosolic Ca²⁺ increase that facilitates the characterization of the associated current. One current supporting SOCE was first described by Hoth and Penner in mast cells and named ICRAC (calcium released-activated Ca²⁺

current) [18]. ICRAC is a very small inward rectifying current highly selective for Ca²⁺ [19] and with a single channel conductance estimated by noise analysis at 24 fS. However, SOC currents can be very heterogeneous depending of the cell type studied and in addition to ICRAC, that has been mainly studied in blood cells, many others SOC currents with different electrophysiological properties can be recorded.

Despite intense investigations, the mode of activation of SOCE remained elusive for a long time. Over the years, many theories were proposed but not conclusively demonstrated.

Among those models, three types of signals from the ER to the plasma membrane were suggested: the existence of a diffusible factor [23], the fusion of vesicles [21], and the involvement of a macromolecular conformational coupling [22]. In 1993, the purification of a Ca²⁺ influx factor (CIF) which is released upon Ca²⁺ store depletion and activates SOCE was the first evidence to support the diffusible factor model [23]. However, the molecular identity of this factor is still unknown and the isolated product appears to vary from one laboratory to another. Consequently and despite recent work on CIF involvement in ICRAC stimulation [24], the direct role of CIF in SOCE activation is still controversial. The vesicle fusion theory implies the insertion of store-operated channels to the plasma membrane following store depletion via the fusion of channels-containing vesicles. However, this vesicle fusion model appears to address the question of SOCE amplitude regulation rather than the mechanism of SOCE activation. Effectively, insertion of additional channels is a frequent process to increase the current amplitude. Nevertheless, this argument does not rule out the possibility that the fusion of vesicle-containing channels could activate SOCE. The conformational coupling model was proposed in analogy with the conformational coupling-induced Ca²⁺ signaling occurring in skeletal muscle. M. Berridge suggest that [25] similarly to dihydropyridine channels and ryanodine receptor, IP3 receptors located in the ER could be physically linked to SOC channels at the plasma membrane and that the IP3R conformational change induced by stores emptying leads to SOC activation.

Recently, major breakthroughs were achieved by the molecular identification of two SOCE key players: STIMs (Stromal interaction molecule) and ORAIs proteins. STIM1 (also named GOK) was cloned in 1996 [26] and described as a glycosylated protein of the plasma membrane [27]. The STIM gene family is ubiquitous and evolved from one gene in C-elegans into two genes in human (STIM1 and STIM2) [28]. STIM1 was first suggested to be a tumor

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of STIM1 (11p15.5 locus) maps to a region involved in many cancers. However in 2005, STIM1 proteins identified by RNA interference screens were demonstrated to be essential for SOCE [29, 30]. STIM1 is a single membrane-spanning domain protein inserted in the ER membrane. Thanks to its EF-hand domain located in the lumen of the ER, STIM1 seems to be able to sense the ER Ca²⁺ filling state. Following Ca²⁺ stores emptying, STIM1 aggregates forming punctate structures anchored in the ER that re-localize near the plasma membrane [30, 31]. STIM1 puncta formation precedes the activation of Ca²⁺ entry taking place in the aggregates. Furthermore, the proximity of STIM1 puncta to the plasma membrane (10-15nM) is close enough to allow physical interaction with proteins anchored in the plasma membrane [32]. Hence, STIM1 functions as an ER Ca²⁺ sensor that regulates the SOCC opening.

Evidences of STIM1 mode of action further support the conformational coupling model of SOCE activation and this conjecture was reinforced by the identification of STIM1 SOC channel partner: ORAI1.

Independently of STIM1 investigations, ORAI1 protein was discovered by genetic linkage in patients suffering from severe combined immunodeficiency (SCID). SCID patients lack functional CRAC in T lymphocytes, and this defect was genetically associated to mutations in the gene coding for ORAI1 protein [33]. Concurrently, Vig and colleagues cloned ORAI1 protein (named CRACM1 in their study) thanks to a genome-wide RNA interference screen in Drosophila where ORAI1 knock-down clearly inhibits store-operated Ca²⁺ entry [34]. The ORAI family is composed of three homologues; ORAI1, ORAI2, and ORAI3 that are each composed of four trans-membrane domains.

Figure 3| Model of store-operated Ca²⁺ entry.

At rest, ER Ca²⁺ stores are filled and STIM1 EF-hand is bound to Ca²⁺. STIM1 and ORAI are dispersed in the ER and plasma membranes, respectively (A). Upon ER Ca²⁺ store depletion, STIM1 EF-hand sense the ER Ca²⁺

decrease, moves underneath the plasma membrane and forms clusters. STIM1 clusters co-localize with adjacent ORAI1 clusters in the plasma membrane (B). This interaction precedes the initiation of Ca²⁺ influx through activated ORAI1 channels (C). Ca²⁺ entry allow the Ca²⁺ refilling of the stores which terminates the process so that cells return to resting state (D).

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time to connect the two stories. Two months after ORAI1 cloning, it was demonstrated that the co-expression of STIM1 together with ORAI1 is sufficient to reconstitute store-operated Ca²⁺ entry as well as ICRAC current [35-37]. Besides, mutagenesis studies revealed that ORAI1 is a subunit of SOC channel supporting Icrac [38-40]. Between the first and the second trans- membrane domains, ORAI1 contains a conserved acidic loop that when mutated altered the ICRAC current characteristics: ionic selectivity, rectification, pharmacology [38, 39]. Hence, the ORAI1 acidic loop forms the ICRAC Ca²⁺ channel permeation pore.

Both STIM1 and ORAI are essential for CRAC channel fonction, but do they interact directly with each other and if yes how? The most likely mechanism is that STIM1 and ORAI1 are physically connected or belong to a common macromolecular complex that associates at the puncta formation site. Several evidences support the direct coupling model.

First, following store depletion, ORAI1 in the plasma membrane is recruited adjacently to the ER-located STIM1 clusters and defines a constrained spatial domain were Ca²⁺ enters the cells [41] (Figure 3). Moreover, Yeromin et al. demonstrated that the Drosophila homologues of STIM1 and ORAI co-immunoprecipitate and that this association was increased by Ca²⁺

store emptying [39]. However, no firm results clearly demonstrated a direct interaction between STIM1 and ORAI1 and the presence of additional molecular components within the STIM1-Orai1 complex should also be considered [42].

In addition to ORAI1 channels that specifically support ICRAC, members of transient receptor potential (TRP) channel family are also good candidate for SOC channels. This channel family regroups 28 members divided in 7 subfamilies: TRPC, TRPV, TRPM, TRPN, TRPA, TRPP and TRPML. It is clear that some of the TRPCs are involved in store-operated mechanism. However, the SOC current measured after TRPC over-expression does not recpitulate to ICRAC properties in term of selectivity and conductance size. Recent findings demonstrated that STIM1 can not only associate with ORAIs channels but can also interact with endogenous hTRPC (TRPC1, 2, 4, 5), thus STIM1 activates other SOC channels distinct from CRAC channels [43]. And among these alternative pathways, receptor-activated Ca²⁺

entry (RACE) is the most studied one.

ii Second messenger interaction: Receptor-activated Ca2+ channels (RACC) In non excitable cells it becomes more and more evident that some Ca²⁺ entry pathways take place independently of Ca²⁺ stores filling state. Receptor-activated Ca²⁺ entry (RACE) pathways that signal via the production of second messengers and activate Ca²⁺

channels located at the plasma membrane [44]. Since the late eighties, Ca²⁺ entry were uncovered artificially with a protocol were cells were Ca²⁺ depleted in Ca²⁺ free medium using thapsigargin, and next Ca²⁺ was added back to the medium to monitor a “pure” Ca²⁺

influx dissociated from Ca²⁺ release. Similar two phase protocols were also used with agonist- induced Ca²⁺ depletion where Ca²⁺ entry was observed subsequently by Ca²⁺ re-addition.

These largely used protocols cause the complete depletion of Ca²⁺ stores, and thus mainly activate store-operated Ca²⁺ entry, possibly hiding other Ca²⁺ entry pathways. A lot of second messengers were shown to stimulate Ca²⁺ entry independently of ER Ca²⁺ store depletion [45]

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(DAG) [49]…. To date the best characterized non-store-operated Ca²⁺ entry is the arachidonic acid-activated Ca²⁺ selective current (ARC) [50]. This channel is exclusively activated by low concentrations of agonists that induce the production of arachidonic acid and the activation of Ca²⁺ entry without previous ER Ca²⁺ store depletion. ARC channels support an inward rectifying, highly Ca²⁺-selective current which is inhibited by La³⁺ and Gd³⁺. Recently, Shuttleworth and colleagues demonstrated that the plasma membrane pool of STIM1 was essential for ARC channels activation, independently of Ca²⁺ store depletion [51]. Before its role in SOCE was discovered, STIMI was previously described as a glycosylated protein anchored in the plasma membrane. However, some recent studies localize STIM1 only to the ER membrane and other to both the ER and the plasma membrane. Biotinylation studies demonstrated that a small fraction of STIM1 also localized to the plasma membrane.

However, the functional YFP-STIM1 fusion protein able to potentiate SOCE when co- expressed with ORAI1 could not be identified at the plasma membrane [52]. Nevertheless the most accepted consensus state that a minor fraction of STIM1 is present at the plasma membrane but is not involved in SOC pathway. The most obvious explanation is that the small amount of plasma membrane STIM1 doest not play an active role in SOCE, but rather participates in Ca²⁺ entry not gated by stores filling state such as ARC pathway [53].

Therefore, STIM1 appears to have multiple functions not exclusively restricted to SOCE regulation.

In addition to ARC channels, numerous store-independent Ca²⁺ influx pathways exist.

On the contrary to ARC, the other non-SOC entry Ca²⁺ channels are generally non Ca²⁺

selective, and thus differ significantly from ICRAC [45]. Except for their electrophysiological properties, the different characteristics of these channels are still obscure. First, the pharmacological characterization of RACE remains elusive as no specific blockers are available. Second, the molecular identity of RACE is unresolved and may be variable from one cell type to another. Although, TRP channels are the most likely candidate to support RACE as some TRP channel current are clearly gated by second messengers [54], and neither the over-expression nor the invalidation experiences clearly demonstrated a specific and ubiquitous involvement of one TRP in RACE or SOCE pathway. The results obtained are often controversial certainly reflecting the differential expression pattern of TRPs among cell types and also the ability of TRPs to from heteromultimers with different properties [55].

Considering their mode of activation some TRP channels have been described as SOC or RAC channels. For example, TRPC1 and TRPC4 have been described as gated by store content and thus behaving like SOCC [20]. Whereas TRPC3 [56], TRPC6 [57] and TRPC7 [58] were shown to be activated by second messenger and seem rather involved in RACE.

During physiological stimulations of non-excitable cells, both the production of second messenger and the IP3-induced Ca²⁺ store depletion occurs simultaneously. Thus, both RACE and SOCE pathways are often activated concurrently. That is why, the two pathways are so difficult to differentiate. When Ca²⁺ entry is recorded, the prevalence of SOCE versus RACE could only be considered by evaluating the ER Ca²⁺ store depletion. However, the ER Ca²⁺ content is rarely directly assessed, and the unique involvement of SOCE pathway is

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favor of SOCE. In conclusion, it is of great importance to better characterize RACE as it may constitute an essential pathway to transduce physiological signals.

IV Aims of the study

The purpose of my thesis was to study the different pathways of Ca²⁺ entry. The three articles presented herein correspond to three independent projects all dealing with Ca²⁺ influx.

I studied the three types of Ca²⁺ entry mechanism, store-operated Ca²⁺ entry (SOCE), receptor activated Ca²⁺ entry (RACE) and voltage-gated Ca²⁺ entry (VGCE).

In the first project, I focused on SOCE pathway and more particularly on the ER refilling mechanism. To appreciate the capacity of the cell to preserve Ca²⁺ stores level, we investigated the impact of extremely reduced SOCE on ER Ca²⁺ refilling. The aim of the study was to assess the route used by Ca²⁺ to reach internal Ca²⁺ stores, and to define the different players involved.

In the second project, we investigated whether different Ca²⁺ entry pathways co-exist in endothelial cells. The purpose of this study was to discriminate SOCE and RACE and to determine the specific pharmacological profile of each pathway.

Finally, in the third project, I collaborated with F. Jaques and colleagues to work on excitable secretary β-cells. Their work addresses the role of β-cells contact in insulin secretion mechanism. In this study, my objective was to consider the impact of defects in cell-cell contacts on cytosolic Ca²⁺ homeostasis.

V Experimental tools

A. Cells

For the three different projects, I used three different cellular models. We took advantage of Hela cells that are simple to handle genetically, to study SOCE pathway and ER Ca²⁺ refilling mechanism. Practically, HeLa cells are easily cultivated and transfected allowing robust protein over-expression or invalidation. HeLa cells are derived from epithelial cervical adenocarcinoma and thus immortal. Besides, HeLa cells display standard Ca²⁺ signals of non-excitable cells such as the ubiquitous SOCE pathway. To discriminate between SOCE and RACE we used an endothelial cell line: EA.hy926 [59]. EA.hy926 derived from human umbilical vein endothelial cells and have been immortalized by the fusion with A549 lung carcinoma cells [59]. EA. hy926 hybridomes possess most of the differentiated features characteristic of endothelial cell such as: factor VIII expression, secretion of von-Willebrand factors, Nitric oxide (NO) production, etc… Cytosolic Ca²⁺ concentration regulates many of these important endothelial functions. And different Ca²⁺ entry such as RACE or SOCE could be a way for endothelial cells to regulate distinct functions. Finally, we studied the link between β-cells contact and Ca²⁺ homeostasis in the subclone B1 of the MIN6 cell line [60].

MIN6 cell line derived from mouse pancreatic β-cells. The B1 sublone is an interesting model

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function of cell confluence.

B. Intracellular Ca²⁺ measurement

The experimental basis of my thesis is the recording of free Ca²⁺ concentration variations in living cells. For this purpose we took advantage of fluorescent Ca²⁺ sensors.

Using microscopes, we imaged fluorescent changes which correlate with Ca²⁺ concentration variations. In biological imaging experiments, two types of Ca²⁺ indicators can be used, the chemical and the genetically encoded probes.

Chemical Ca²⁺ sensors are Ca²⁺ sensitive fluorophores whose emission spectra changes upon calcium binding. We used only one type of synthetic Ca²⁺ sensors: Fura-2 AM.

Fura-2 is a widely used UV-excitable fluorescent calcium indicator derived from EGTA (ethylene glycol tetraacetic acid), a Ca²⁺ chelator [61]. Measurements were done by a dual excitation at 340 and 380 nn and emission was acquired at 510 nm. When Ca²⁺ binds to the indicator, the two wavelength intensities move in an anti-parallel manner, allowing a convenient ratiometric measurement ( , A). The affinity of Fura-2 for Ca²⁺ is high with a Kd of 224 nm, this affinity range is ideal for free cytosolic Ca²⁺ measurement fluctuating approximately from 50 nM up to 1 μM. To overcome the problem of Fura-2 cellular impermeability, ester loading techniques have been used. On the contrary to the original, the AM (acétoxy-méhyl ester) indicators are hydrophobe and can go through the plasma membrane. Once in the intracellular compartment, cytosolic esterases cleave the AM group and trap the fura-2 inside the cell. However the intracellular localization of synthetic dyes is difficult to control, fura-2 for instance can accumulate in diverse organelles such as lysosomes or mitochondria. One option to overcome this problem of compartmentalization is to use Ca²⁺ sensitive fluorescent proteins.

Figure 4

Genetically-encoded Ca²⁺ probes are human-engineered fluorescent proteins that when expressed in a cell, sense free Ca²⁺ concentration variations. The huge advantage of such probes is that they can be targeted to diverse organelles [62]. Indeed, transfection of constructs that fused Ca²⁺ probes cDNA with specific addressing sequences results in the expression of the indicator in the expected organelle. These Ca²⁺ indicators are based on two types of protein, both discovered in medusa Aequorea Victoria: aequorin (AEQ) and green fluorescent protein (GFP) [63]. AEQ possesses three Ca²⁺ binding sites and becomes luminescent when at least two sites are bound. Both jellyfish proteins enables great advance in life science research. However, as natural GFP does not interact with Ca²⁺, the first application of GFP did not concern Ca²⁺ recordings but rather protein localization or gene expression measurements. Nonetheless, the group of R. Tsien succeeded to engineer recombinant fluorescent Ca²⁺-sensitive protein derived from GFP [64]. Most of the GFP- based Ca²⁺ probes sense Ca²⁺ thanks to the insertion of calmodulin and calmodulin-binding peptide motifs that change conformation upon Ca²⁺ binding and modify the fluorescent properties of the indicator. Three types of GFP-based Ca²⁺ sensor were developed: cameleons, camgaroos and pericams. In this study we took advantage of cameleons Ca²⁺ probes to follow free Ca²⁺ changes in different compartments. Unlike the others GFP-based Ca²⁺ sensors,

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calmodulin-binding peptide motifs. The two fluorescent proteins have different spectral properties so that their close interaction generates fluorescence resonance energy transfer (FRET) [63]. FRET is a physical mechanism by which an excited donor chromophore is able to transfer its emission energy to an acceptor chromophore. The transfer of energy occurs only when the two chromophore are in close proximity (<10 nm) which gives rise to the acceptor chromophore emission. Generally, cyan and yellow fluorescent proteins are used as donor and acceptor chromophore respectively. When Ca²⁺ binds to the calmodulin-containing cameleon it induces the association of the calmodulin with its partner, bringing the two fluorescent proteins in close proximity and increasing FRET (Figure 4, B). Thus, upon Ca²⁺ concentration augmentation, the cyan fluorescence intensity decreases and the yellow fluorescence intensity increases. Therefore, cameleons are ratiometric probes that undergo FRET in function of free Ca²⁺ concentration. Since 1997, cameleons have greatly evolved in term of Ca²⁺ affinity, ratio dynamic and maturation of the probe. The last cameleons progress consists in the design of synthetic partners to replace the calmodulin-peptide pair in order to prevent the interference with endogenous calmodulin [65, 66]. Precisely, to assess the free Ca²⁺ changes into the endoplasmic reticulum we used one of this new generation cameleons targeted to the ER:

D1ER (Figure 4, C).

B) Scheme of cameleons probe principles.

CFP: cyan fluorescent protein, YFP: yellow fluorescent protein, CaM: calmodulin, M13: synthetic peptide derived from calmodulin-binding domain of skeletal muscle myosin light chain kinase, FRET: fluorescence Figure 4| Ca²⁺ indicators

A) Fura-2 excitation spectra. 340 nm intensity increases with Ca²⁺ concentration, on the contrary to 380 nm that decreases.

resonance energy transfer.

C) Confocal image of HeLa cells transfected with the ER-targeted cameleon probe: D1ER

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One way to study the function of a gene is to inhibit its cellular expression.

Conception of mouse knockout is certainly the most conclusive experimental technique.

However, such experiments are fastidious and time consuming. In 2001, the elaboration of a new technique that silence gene expression in cultured cells really revolutionized loss of function studies in cell physiology [67]. This technique is based on short RNAs that interfered with complementary mRNA and prevent their translation, those RNAs were called small interfering RNA (siRNA). siRNA technique is a powerful approach to knockdown genes of interest whose sequence is known [68].

The invalidation of proteins of interest was performed by lipid transfection of siRNA.

Double stranded RNA (dsRNA) were directly transfected into the cells without the use of siRNA expression vector. Hence, the selection of transfected cells could not be achieved by drug resistance or fluorescent protein expressed by the vector. To overcome this problem, we co-transfected dsRNA together with plasmids coding for fluorescent Ca²⁺ probes assuming that one cell received both constructs or nothing. And Ca²⁺ responses were measured at the single-cell level by Ca²⁺ imaging, so that recorded fluorescent cells were also silenced for the protein of interest.

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A. Introduction

Maintaining a high Ca²⁺ concentration into the lumen of the endoplasmic reticulum is vital for the cell fate. In one hand, some fundamental cellular processes taking place in the ER require high Ca²⁺ concentration. For instance, the accurate folding of secreted and trans- membrane proteins necessitate a precise ER Ca²⁺ concentration as a lot of ER resident chaperones are Ca²⁺ dependent [69]. On the other hand Ca²⁺ stores enable cells to respond to Ca²⁺ release stimuli and a rapid replenishment of the stores is indispensable to guarantee long term Ca²⁺ signaling and repetitive Ca²⁺ responses such as oscillations. After the discharge of Ca²⁺ from ER, the cell has two options to refill its store: pump back the cytosolic Ca²⁺ that has been released or use the extra-cellular Ca²⁺ source. However, recycling of released Ca²⁺ is generally not sufficient to replenish the store as a substantial fraction of Ca²⁺ is extruded out of the cytosol by plasma membrane Ca²⁺ ATPases (PMCA). To compensate this loss, cytosolic Ca²⁺ entry is required and is supported by the activation of Store-operated Ca²⁺ entry (SOCE).

As presented in the introduction, SOC channels (SOCC) are controlled by ER Ca²⁺

store content, and are stimulated upon ER Ca²⁺ depletion. Conversely, their inactivation is triggered by the complete refilling of Ca²⁺ stores [70]. However, others mechanisms such as high a Ca²⁺ concentration close to the cytosolic side of SOCC are also able to inhibit Ca²⁺

entry [19]. Mitochondria play an important role in preventing this Ca²⁺ inhibition mechanism as sub-plasmalemmal mitochondria are able to take up Ca²⁺ at the mouth of SOCC channels [71]. This buffering process is important to sustain long lasting Ca²⁺ entry essential for store replenishment [72].

The understanding of the mechanism by which SOCC are regulated were greatly improved in May 2005 with the identification of STIM1 ER Ca²⁺ sensors. STIM1 is a type one ER membrane protein that senses the ER Ca²⁺ level and regulates SOCC at the plasma membrane. The implication of STIM1 in SOCE activation was clearly demonstrated by knockdown and over-expression studies. STIM1 silencing intensely reduces SOCE[29, 30], and conversely, over-expression of STIM1 together with its partner ORAI1 channels increases SOCE [35-37]. Upon ER Ca²⁺ store depletion, STIM1 re-localizes into plasma- membrane adjacent puncta in order to activate SOC channels [32]. The interaction of STIM1 and ORAI1 channels at the interface between ER and plasma membranes delimits a spatially restricted zone where Ca²⁺ entry occurs [41]. Hence, the Ca²⁺ influx supported by SOCC takes place in an extremely confined space generating high Ca²⁺ microdomains.

In previous STIM1 knockdown studies, thapsigargin-induced Ca²⁺ release and resting cytosolic Ca²⁺ concentrations were preserved [29, 30], suggesting that Ca²⁺ store content were conserved although SOCE were drastically inhibited. What’s more, similar results were observed in STIM1-deficient DT40 B cells [73], implying that the complete absence of STIM1 does not strongly impede ER Ca²⁺ homeostasis.

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abundance of STIM1 protein is reduced. We address the role of SERCA and mitochondria as active members of replenishment or as Ca²⁺ buffering elements. We also consider STIM1 cluster domains as a possible location of preferential ER refilling and tested whether SERCA participate is this ER-membrane structure.

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