STIM1 translocation to the nucleus protects cells from DNA damage.

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.

Internalized Amyloid-ß (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells.

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-ß peptides (Aß) in AD. In recent works, we have proposed that Aß-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 µM Aß(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aß(1-42), respectively. Internalized Aß(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aß. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aß(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aß(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aß(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 µM Aß(1-42) oligomers. We conclude that Aß(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

STIM1 Deficiency Leads to Specific Down-Regulation of ITPR3 in SH-SY5Y Cells.

STIM1 is an endoplasmic reticulum (ER) protein that modulates the activity of a number of Ca2+ transport systems. By direct physical interaction with ORAI1, a plasma membrane Ca2+ channel, STIM1 activates the ICRAC current, whereas the binding with the voltage-operated Ca2+ channel CaV1.2 inhibits the current through this latter channel. In this way, STIM1 is a key regulator of Ca2+ signaling in excitable and non-excitable cells, and altered STIM1 levels have been reported to underlie several pathologies, including immunodeficiency, neurodegenerative diseases, and cancer. In both sporadic and familial Alzheimer's disease, a decrease of STIM1 protein levels accounts for the alteration of Ca2+ handling that compromises neuronal cell viability. Using SH-SY5Y cells edited by CRISPR/Cas9 to knockout STIM1 gene expression, this work evaluated the molecular mechanisms underlying the cell death triggered by the deficiency of STIM1, demonstrating that STIM1 is a positive regulator of ITPR3 gene expression. ITPR3 (or IP3R3) is a Ca2+ channel enriched at ER-mitochondria contact sites where it provides Ca2+ for transport into the mitochondria. Thus, STIM1 deficiency leads to a strong reduction of ITPR3 transcript and ITPR3 protein levels, a consequent decrease of the mitochondria free Ca2+ concentration ([Ca2+]mit), reduction of mitochondrial oxygen consumption rate, and decrease in ATP synthesis rate. All these values were normalized by ectopic expression of ITPR3 in STIM1-KO cells, providing strong evidence for a new mode of regulation of [Ca2+]mit mediated by the STIM1-ITPR3 axis.

RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence.

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

Correction to: STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

The original publication of this paper contains errors.

Role of STIM1 in neurodegeneration.

STIM1 is an endoplasmic reticulum (ER) protein with a key role in Ca2+ mobilization. Due to its ability to act as an ER-intraluminal Ca2+ sensor, it regulates store-operated Ca2+ entry (SOCE), which is a Ca2+ influx pathway involved in a wide variety of signalling pathways in eukaryotic cells. Despite its important role in Ca2+ transport, current knowledge about the role of STIM1 in neurons is much more limited. Growing evidence supports a role for STIM1 and SOCE in the preservation of dendritic spines required for long-term potentiation and the formation of memory. In this regard, recent studies have demonstrated that the loss of STIM1, which impairs Ca2+ mobilization in neurons, risks cell viability and could be the cause of neurodegenerative diseases. The role of STIM1 in neurodegeneration and the molecular basis of cell death triggered by low levels of STIM1 are discussed in this review.

STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

STIM1 is an endoplasmic reticulum protein with a role in Ca2+ mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer's disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues (medium frontal gyrus) of pathologically confirmed Alzheimer's disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitochondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, demonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SH-SY5Y cell death. KEY MESSAGES: STIM1 protein expression decreases with the progression of neurodegeneration in Alzheimer's disease. STIM1 is essential for cell viability in differentiated SH-SY5Y cells. STIM1 deficiency triggers voltage-regulated Ca2+ entry-dependent cell death. Mitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.

Store-operated calcium entry is dispensable for the activation of ERK1/2 pathway in prostate cancer cells.

STIM1, the endoplasmic reticulum Ca2+ sensor that modulates the activity of plasma membrane Ca2+ channels, becomes phosphorylated at ERK1/2 target sites during Ca2+ store depletion triggered by thapsigargin or epidermal growth factor (EGF). This ERK1/2-dependent phosphorylation regulates STIM1 localization and dissociation from microtubules, and it is known that enhances the binding to ORAI1, a store-operated Ca2+ entry (SOCE) channel, leading to the activation of this Ca2+ influx pathway. However, there remained some evidence of a role for SOCE in the activation of ERK1/2, and here we assessed the contribution of SOCE to ERK1/2 activation by generating a STIM1-deficient cell line by CRISPR/Cas9 genome editing of the STIM1 locus in prostate cancer PC3 cells. The genomic modification consisted of a 16 base-pair insertion in exon 5 of both alleles, therefore abrogating STIM1 synthesis. STIM1-KO cells did show a striking decrease in Ca2+ influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca2+ entry in PC3 cells during stimulation with EGF. Moreover, identical levels of total ERK1/2 were found in STIM1-KO cells and the parental cell line, and ERK1/2 activation was fully activated in KO cells, both in the presence and in the absence of extracellular Ca2+, a result that supports that STIM1 and SOCE are not required for ERK1/2 activation. This activation was sensitive to Src kinase inhibition, but not to CAMKII nor PKC inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.

Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains.

Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immunoprecipitated with endogenous CTTN. This latter result, in addition to the KO cells' phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling by the Ca2+-channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling.

Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells.

STIM1 is a Ca(2+) sensor of the endoplasmic reticulum (ER) that triggers the activation of plasma membrane Ca(2+) channels upon depletion of Ca(2+) levels within the ER. During thapsigargin-triggered Ca(2+) store depletion, ERK1/2 phosphorylates STIM1 at Ser575, Ser608, and Ser621. This phosphorylation plays a role in the regulation of STIM1 dissociation from the microtubule plus-end binding protein EB1, an essential step for STIM1 activation by thapsigargin. However, little is known regarding the physiological role of this phosphorylation. Because IGF-1 triggers the activation of the RAF-MEK-ERK and the phosphoinositide pathways, the role of STIM1 phosphorylation in IGF-1 stimulation was studied. There was found to be phosphorylation of ERK1/2 in both the presence and the absence of extracellular Ca(2+), demonstrating that Ca(2+) influx is not essential for ERK1/2 activation. In parallel, IGF-1 triggered STIM1 phosphorylation at the aforementioned sites, an effect that was blocked by PD0325901, a MEK1/2 inhibitor used to block ERK1/2 activation. Also, STIM1-GFP was found in clusters upon IGF-1 stimulation, and STIM1-S575A/S608A/S621A-GFP strongly reduced this multimerization. Interestingly, phospho-STIM1 was mainly found in clusters when cells were treated with IGF-1, and IGF-1 triggered the dissociation of STIM1 from EB1, similarly to what has been observed for thapsigargin, suggesting that STIM1 mediates the IGF-1 signaling pathway. A study of IGF-1-stimulated NFAT translocation was therefore performed, finding that STIM1-S575A/S608A/S621A blocked this translocation, as did the fusion protein STIM1-EB1, confirming that both STIM1 phosphorylation and STIM1-EB1 dissociation are required for IGF-1-triggered Ca(2+)-dependent signaling, and demonstrating that STIM1 phosphorylation plays a role as a downstream effector of the RAF-MEK-ERK pathway and an upstream activator of Ca(2+) entry.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration.

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca²âº entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca²âº emptying triggered by the inhibition of the sarco(endo)plasmic Ca²âº -ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca²âº, indicating that both events are upstream steps for Ca²âºentry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca²âº channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.

Inhibition of STIM1 phosphorylation underlies resveratrol-induced inhibition of store-operated calcium entry.

Resveratrol, a natural phytoalexin that shows health-promoting benefits, is an inhibitor of store-operated calcium entry (SOCE). Knowledge of the molecular mechanism underlying this inhibition is required for the proper design of therapies that include resveratrol or related stilbenoids, but remains largely unknown. To unravel this mechanism, using HEK293 cells as a model, we found that resveratrol inhibited the ERK1/2 activation triggered by Ca²âº store depletion. As a consequence, resveratrol inhibited STIM1 phosphorylation at residues Ser575, Ser608, and Ser621. Because this phosphorylation regulates the dissociation of STIM1 from the microtubule plus-end binding protein EB1 under store depletion conditions, resveratrol inhibited STIM1-EB1 dissociation. This inhibition had downstream effects such as inhibition of STIM1 multimerization in response to store depletion, and a significant impairment in the binding of STIM1 to ORAI1. Although additional targets for resveratrol in the molecular mechanism that governs SOCE cannot be discarded, the present results demonstrate that ERK1/2 pathway is a major target for resveratrol, and that the impairment of its activation produces a significant inhibition of SOCE.

Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.

STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca(2+) concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.

The regulation of STIM1 by phosphorylation.

Calcium ion (Ca(2+)) concentration plays a key role in cell signaling in eukaryotic cells. At the cellular level, Ca(2+) directly participates in such diverse cellular events as adhesion and migration, differentiation, contraction, secretion, synaptic transmission, fertilization, and cell death. As a consequence of these diverse actions, the cytosolic concentration of free Ca(2+) is tightly regulated by the coordinated activity of Ca(2+) channels, Ca(2+) pumps, and Ca(2+)-binding proteins. Although many of these regulators have been studied in depth, other proteins have been described recently, and naturally far less is known about their contribution to cell physiology. Within this last group of proteins, STIM1 has emerged as a major contributor to Ca(2+) signaling by means of its activity as Ca(2+) channel regulator. STIM1 is a protein resident mainly, but not exclusively, in the endoplasmic reticulum (ER), and activates a set of plasma membrane Ca(2+) channels termed store-operated calcium channels (SOCs) when the concentration of free Ca(2+) within the ER drops transiently as a result of Ca(2+) release from this compartment. Knowledge regarding the molecular architecture of STIM1 has grown considerably during the last years, and several structural domains within STIM1 have been reported to be required for the specific molecular interactions with other important players in Ca(2+) signaling, such as Ca(2+) channels and microtubules. Within the modulators of STIM1, phosphorylation has been shown to both activate and inactivate STIM1-dependent Ca(2+) entry depending on the cell type, cell cycle phase, and the specific residue that becomes modified. Here we shall review current knowledge regarding the modulation of STIM1 by phosphorylation.

Intracellular cyclophilin A is an important Ca(2+) regulator in platelets and critically involved in arterial thrombus formation.

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.

Role of store-operated calcium entry during meiotic progression and fertilization of mammalian oocytes.

Calcium signaling is essential for many cellular events, including muscle contraction, secretion of hormones and neurotransmitters, and fertilization of oocytes. For the appropriate maturation and fertilization of mammalian oocytes, the influx of extracellular calcium through plasma membrane Ca(2+) channels is required. Although the molecular pathway of the Ca(2+) entry in other cell types has been reported, Ca(2+) channels involved in the regulation of Ca(2+) influx in oocytes have remained unknown for a long time. In this review, we summarize recent findings regarding the occurrence of store-operated calcium entry (SOCE) in mammalian oocytes and the expression and localization profiles of STIM1 and ORAI1, two important proteins that control SOCE. As we discuss here, STIM1, as an endoplasmic reticulum Ca(2+) sensor, and ORAI1, the major plasma Ca(2+) channel involved in SOCE, might help to explain the role of Ca(2+) entry in mammalian oocyte maturation and fertilization.

Calcium signaling in mouse oocyte maturation: the roles of STIM1, ORAI1 and SOCE.

Calcium handling is critical for the oocyte function, since the first steps of fertilization are dependent on the appropriate Ca(2+) mobilization to originate transient spikes of the cytosolic Ca(2+) concentration. It is well known that the Ca(2+) influx from the extracellular milieu is required to maintain this signaling in mammalian oocytes. However, the regulation of the Ca(2+) channels involved in this process is still unknown in oocytes. STIM1, a key regulator of store-operated Ca(2+) entry (SOCE), relocates in the mouse oocyte shortly after sperm stimulation, suggesting that SOCE is involved in the maintenance of cytosolic Ca(2+)-spiking in the fertilized oocyte. Here, we show that there is an up-regulation of the expression of STIM1 at the germinal vesicle breakdown stage, and this expression remains steady during following maturation stages. We found that oocytes express ORAI1, a store-operated Ca(2+) channel, and that ORAI1 expression level was stable during oocyte maturation. Immature oocytes showed no Ca(2+) entry and no increase in STIM1-ORAI1 colocalization in response to the store depletion induced by thapsigargin. On the contrary, in mature oocytes, STIM1-ORAI1 colocalization is enhanced 3-fold by depletion of Ca(2+) stores, enabling the activation of store-operated calcium channels and therefore Ca(2+) entry. Finally, the correlation between SOCE activation during the maturation of oocytes and STIM1-ORAI1 colocalization strongly suggests that ORAI1 is involved in the Ca(2+) entry pathway in the mature oocyte. SOCE up-regulation in the final stage of maturation is further evidence of a major role for SOCE in fully mature oocytes, and therefore in Ca(2+) signaling at fertilization.