Abstract
Store-operated calcium entry (SOCE) provided through channels formed by ORAI proteins is a major regulator of several cellular processes. In immune cells, it controls fundamental processes such as proliferation, cell adhesion, and migration, while in cancer, SOCE and ORAI1 gene expression are dysregulated and lead to abnormal migration and/or cell proliferation. In the present study, we used the CRISPR/Cas9 technique to delete the ORAI1 gene and to identify its role in proliferative and migrative properties of the model cell line HEK-293. We showed that ORAI1 deletion greatly reduced SOCE. Thereby, we found that this decrease and the absence of ORAI1 protein did not affect HEK-293 proliferation. In addition, we determined that ORAI1 suppression did not affect adhesive properties but had a limited impact on HEK-293 migration. Overall, we showed that ORAI1 and SOCE are largely dispensable for cellular proliferation, migration, and cellular adhesion of HEK-293 cells. Thus, despite its importance in providing Ca2+ entry in non-excitable cells, our results indicate that the lack of SOCE does not deeply impact HEK-293 cells. This finding suggests the existence of compensatory mechanism enabling the maintenance of their physiological function.
Highlights
Cells were maintained in high-glucose, GlutaMAXTM Dulbecco’s modified Eagle medium (DMEM; Thermo Fischer Scientific, Illkirch, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Dominique Dutscher, Brumath, France) and cultured in 75 cm2 flasks kept at 37 ◦ C in a humidified incubator containing 5% CO2
Our results indicate that ORAI1 KO increases the collective migration speed but does not affect single-cell migration (Transwell® )
We have demonstrated that ORAI1 is responsible for the majority of store-operated calcium entry (SOCE) (~80%) and that proliferation, adhesion and migration rates are barely affected by ORAI1 deletion
Summary
Calcium (Ca2+ ) is a major intracellular second messenger controlling several fundamental cellular processes [1]. Ca2+ signals are finely regulated both spatially and temporally due to a variety of specialized Ca2+ channels, exchangers, and transporters [2]. This fine regulation results in a tight control of crucial cellular functions such as proliferation and migration [3]. It is widely accepted that one of the main Ca2+ entry pathways in non-excitable cells is represented by store-operated calcium entry (SOCE) [4]. SOCE is defined by Ca2+ entry across the cell plasma membrane (PM) consecutive to Ca2+ depletion from the endoplasmic reticulum (ER) [5]. The molecular components of SOCE are represented by the ER-resident protein family stromal interaction molecule (STIM), including
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