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. Electronic supplementary materialThe online version of this article (10.1007/s00109-018-1677-y) contains supplementary material, which is available to authorized users.

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.

STIM1, the endoplasmic reticulum Ca sensor that modulates the activity of plasma membrane Ca channels, becomes phosphorylated at ERK1/2 target sites during Ca 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 Ca entry (SOCE) channel, leading to the activation of this Ca 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 Ca influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca 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 Ca, 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.

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 Ca 2+ 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 Ca 2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

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.