“…In this particular experiment, to promote cell migration, C2C12 cells were stimulated with FBS, and HeLa and U2OS cells were stimulated with epidermal growth factor, EGF (50 ng/ml). Because the phosphorylation of STIM1 at ERK1/2 target sites has been shown to be essential for the dissociation of STIM1 from EB1 and for the activation of SOC channels 9, 21–23, 28 , this polarized distribution of phospho-STIM1 suggests a role for Ca 2+ influx through STIM1-dependent pathways in the remodelling of the cortical cytoskeleton required for lamellipodia and filopodia formation.Figure 2Immunolocalization of phospho-STIM1 and cortactin (CTTN). C2C12, HeLa, and U2OS cells were cultured for a minimum of 48 h as indicated in Materials and Methods.…”
Section: Resultsmentioning
confidence: 99%
“…This mutant failed to polarize in migrating cells, although it is known to activate overall SOC influx to a similar degree as wild-type STIM1 13, 21 , indicating that the transport to the leading edge is microtubule dependent event. In this regard, in previous work, our group has found that STIM1 becomes phosphorylated at three ERK1/2 target sites, Ser575, Ser608, and Ser621, upon store depletion conditions triggered by thapsigargin, EGF, or IGF-1 21–23 . Once wild-type STIM1 is phosphorylated by ERK1/2, it dissociates from EB1, whilst a non-phosphorylatable mutant (STIM1 S575A/S608A/S621A ) remains bound to EB1 even after store depletion 21, 24 .…”
Section: Introductionmentioning
confidence: 81%
“…Phosphorylation of STIM1 at ERK1/2 sites is attained during stimulation of cells with activators of the MAPK (mitogen-activated protein kinases) pathway such as EGF 9 or IGF-1 23 , and we have reported that this phosphorylation facilitates the dissociation of STIM1 from microtubules (i.e., EB1). Because of the receptor tyrosine kinase (RTK) enrichment at the front of migrating cells, a model is plausible in which activation of RTKs by ligands could lead to partial depletion of ER Ca 2+ levels because of the activation of the phosphoinositide pathway 9, 23 .…”
Section: Discussionmentioning
confidence: 99%
“…Because of the receptor tyrosine kinase (RTK) enrichment at the front of migrating cells, a model is plausible in which activation of RTKs by ligands could lead to partial depletion of ER Ca 2+ levels because of the activation of the phosphoinositide pathway 9, 23 . This partial and local emptying of the ER, together with the ERK1/2-dependent phosphorylation of STIM1, would increase local and transient Ca 2+ pulses.…”
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.
“…In this particular experiment, to promote cell migration, C2C12 cells were stimulated with FBS, and HeLa and U2OS cells were stimulated with epidermal growth factor, EGF (50 ng/ml). Because the phosphorylation of STIM1 at ERK1/2 target sites has been shown to be essential for the dissociation of STIM1 from EB1 and for the activation of SOC channels 9, 21–23, 28 , this polarized distribution of phospho-STIM1 suggests a role for Ca 2+ influx through STIM1-dependent pathways in the remodelling of the cortical cytoskeleton required for lamellipodia and filopodia formation.Figure 2Immunolocalization of phospho-STIM1 and cortactin (CTTN). C2C12, HeLa, and U2OS cells were cultured for a minimum of 48 h as indicated in Materials and Methods.…”
Section: Resultsmentioning
confidence: 99%
“…This mutant failed to polarize in migrating cells, although it is known to activate overall SOC influx to a similar degree as wild-type STIM1 13, 21 , indicating that the transport to the leading edge is microtubule dependent event. In this regard, in previous work, our group has found that STIM1 becomes phosphorylated at three ERK1/2 target sites, Ser575, Ser608, and Ser621, upon store depletion conditions triggered by thapsigargin, EGF, or IGF-1 21–23 . Once wild-type STIM1 is phosphorylated by ERK1/2, it dissociates from EB1, whilst a non-phosphorylatable mutant (STIM1 S575A/S608A/S621A ) remains bound to EB1 even after store depletion 21, 24 .…”
Section: Introductionmentioning
confidence: 81%
“…Phosphorylation of STIM1 at ERK1/2 sites is attained during stimulation of cells with activators of the MAPK (mitogen-activated protein kinases) pathway such as EGF 9 or IGF-1 23 , and we have reported that this phosphorylation facilitates the dissociation of STIM1 from microtubules (i.e., EB1). Because of the receptor tyrosine kinase (RTK) enrichment at the front of migrating cells, a model is plausible in which activation of RTKs by ligands could lead to partial depletion of ER Ca 2+ levels because of the activation of the phosphoinositide pathway 9, 23 .…”
Section: Discussionmentioning
confidence: 99%
“…Because of the receptor tyrosine kinase (RTK) enrichment at the front of migrating cells, a model is plausible in which activation of RTKs by ligands could lead to partial depletion of ER Ca 2+ levels because of the activation of the phosphoinositide pathway 9, 23 . This partial and local emptying of the ER, together with the ERK1/2-dependent phosphorylation of STIM1, would increase local and transient Ca 2+ pulses.…”
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.
“…In contrast, Ser/Glu mutations enhanced SOCE, whereas facilitated the clustering of STIM1 in response to store depletion, as well as the binding to ORAI1. Later, we reported that IGF-1 [48] and EGF [49] also trigger Ca 2+ release from the ER and phosphorylation of STIM1, an efect that has been proven to be essential for the dissociation from EB1 and inally for the activation of SOCE (see Figure 1).…”
Cell motility is a complex cellular event that involves reorganization of cytoskeleton. This reorganization encompasses the transient polarization of the cell to facilitate the plasma membrane ruling, a rearrangement of cortical actin cytoskeleton required for the development of cellular protrusions. It is known that extracellular Ca 2+ inlux is essential for cell migration and for the positive-feedback cycle that maintains leading-edge structures and ruling activity. The aim of this review is to summarize our knowledge regarding the Ca 2+ -dependent signaling pathways, Ca 2+ transporters and sensors involved in cell migration. Also, we show here reported evidences that support for a crosstalk between Ca 2+ transport and the reorganization of the cytoskeleton required for cell migration. In this regard, we will analyze the role of store-operated Ca 2+ entry (SOCE) as a modulator of cytoskeleton and cell migration, but also the modulation of this Ca 2+ entry pathway by microtubules and the actin cytoskeleton. As a main conclusion, this review will show that data reported in the last years support a role for SOCE in shaping cytoskeleton, but at the same time, SOCE is strongly dependent on cytoskeletal proteins, in an interesting interplay between cytoskeleton and Ca 2+ dynamics.
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.
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