Supplementary MaterialsSupplementary Information 41598_2020_63353_MOESM1_ESM. potentiated ORAI1 translocation towards 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 designed U2OS cells lacking ORAI1 expression that helped us to show the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness model using xenotransplants in zebrafish larvae. Casper zebrafish larvae were micro-injected with wild-type or ORAI1-KO U2OS cells, and 5 days post-injection the larvae were analyzed for cell dissemination by fluorescence microscopy (see experimental design in Supplementary Fig.?S5). The results showed a Rhosin higher level of tumor cells in the larvae when wild-type U2OS cells were injected (Fig.?1D). The deficiency in ORAI1 significantly reduced the dissemination of osteosarcoma U2OS cells, a finding that we propose is usually directly linked to the reduction in cell migration rate, in directional persistence, and in protrusion formation. EGF triggers the association between ORAI1 and CTTN Because EGF modulates cell migration and motility in epithelial cells and EGF receptors are enriched at the leading edge31, we investigated the binding of ORAI1 to CTTN in U2OS cells stimulated with EGF as an strategy to study the possible translocation or re-localization of ORAI1 to the leading edge in response to EGF. Cells were starved in FBS-free RPMI?1640 medium without phenol red for 8C10?h and then stimulated with 50?ng/ml EGF in the same medium. ORAI1-CTTN binding was monitored by ORAI1-GFP pulldown and subsequent analysis of co-precipitated mCherry-CTTN (Fig.?2A). The time?course of EGF stimulation was evaluated by monitoring the levels of (i) phospho-PAK1/2 (residues Thr423/Thr402), a well characterized serine-threonine kinase activated by the small GTPase RAC1 and a downstream mediator of EGFR, and (ii) phospho-ERK1/2, since the MAPK pathway becomes activated by EGF (Fig.?2B). The increase in PAK1/2 and ERK1/2 phosphorylation was observed after 1C3?min of stimulation with EGF. Within this time window, we analyzed the co-precipitation between ORAI1 and CTTN, observing greater binding after stimulation, and?this increase reached?statistical significance after 3?min of treatment with EGF (Fig.?2A). Because CTTN is usually a NKSF2 molecular marker of lamellipodia, this result suggests that EGF triggers the recruitment of ORAI1 to the leading edge. Also, when U2OS cells were stimulated with EGF under the above conditions, ORAI1-GFP was observed Rhosin to co-precipitate with both endogenous CTTN Rhosin and with endogenous CYFIP1 (cytosolic FMR-interacting protein 1) (Fig.?2C), also known as SRA-1 (specifically RAC1-associated protein 1)37, one of the subunits of the WRC, a molecular complex enriched at the leading edge. Open in a separate window Physique 2 EGF potentiated ORAI1 binding to CTTN, CYFIP1, and ARP2/3.?were subjected to electrophoresis on 10% acrylamide gels, blotted, and assessed for the level of mCherry-CTTN, ORAI1-GFP, phospho-PAK1/2, total-PAK1, phospho-ERK1/2, and total-ERK1/2. luciferase, as described previously44. Then, we measured the secreted luciferase activity?as a readout of the secretory pathway status, and we found that luciferase secretion was not inhibited by the overexpression of Flag-RAC1T17N (Fig.?5C) nor by the treatment of cells with NSC 23766, validating the use of this inhibitor in subsequent experiments. As a control of the experiment, we used brefeldin A, a well-known inhibitor of the ER-Golgi transport that inhibited the secretion of the luciferase. RAC1 inhibition reduced ORAI1 translocation and impaired cell migration To investigate further the role of RAC1 in the localization of ORAI1, FBS-starved cells were stimulated with EGF, and RAC1 activity in these experimental conditions was evaluated by a classical pull-down with GST-PAK1 protein-binding domain name (PBD) and the subsequent analysis of co-precipitated RAC1 (Fig.?6A). The results exhibited that RAC1 became activated within the first 30?sec-1?min of treatment with EGF, i.e., slightly earlier than the co-precipitation of ORAI1 with CTTN, ARP2/3, and CYFIP1 (see Fig.?2), in agreement with an upstream activation of RAC1 when compared with the effect observed in ORAI1-CTTN co-precipitation. Moreover, endogenous RAC1 co-precipitated with ORAI1-GFP in response to EGF (Fig.?6B), and the RAC1 inhibitor NSC 23766 inhibited the RAC1-ORAI1 co-precipitation observed upon stimulation with EGF. This result indicated that ORAI1-GFP binds to a molecular complex made up of active RAC1. The efficiency of NSC 23766, which prevents RAC1 activation by RAC-specific guanine nucleotide exchange factors45, as a RAC1 inhibitor was evaluated.