Abstract
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.
Highlights
To be efficient, cell migration requires tight spatiotemporal control of cellular signaling
We have recently reported that the plasma membrane Ca2+ channel ORAI1 is enriched at the leading edge of migrating cells, where it colocalizes with CTTN5
Using U2OS cells edited by CRISPR/Cas to knock-out ORAI1 gene expression[5], we observed that cell migration speed and total distance were greatly inhibited by the ORAI1 deficiency (Fig. 1A, and Supplementary movie S1 and S2)
Summary
Cell migration requires tight spatiotemporal control of cellular signaling. At the rear part of the migrating cell, the trailing edge, a wide range of Ca2+ channels increase [Ca2+]i and facilitate the activation of myosin light-chain kinase (MLCK) and Rho kinase-dependent processes which are necessary to stimulate actomyosin contraction and facilitate cell retraction[11] For these reasons, the subcellular localization of the different plasma membrane Ca2+ channels is critical. The influx of Ca2+ through store-operated Ca2+ channels (SOC channels) regulates the migration of both cancer and non-cancer cells[12,13,14,15,16,17] This Ca2+ influx pathway is controlled by a number of plasma membrane Ca2+ channels, including the ORAI protein family and some members of the TRPC protein family (reviewed in[18]). I.e., being STIM1-dependent, ORAI1 can act as a SOC channel, this channel can be activated by Ca2+ store-independent mechanisms[26,27]
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