RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence

1 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence Aida M. Lopez-Guerrero1,5, Noelia espinosa-Bermejo1,5, Irene Sanchez-Lopez1, Thomas Macartney2, Carlos pascual-caro1, Yolanda orantos-Aguilera1, Lola Rodriguez-Ruiz3, Ana B. perez-oliva 3, Victoriano Mulero3, Eulalia pozo-Guisado4* & Francisco Javier Martin-Romero 1* 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. ORAI1CTTN 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. To be efficient, cell migration requires tight spatiotemporal control of cellular signaling. Together with other molecules involved in the control of cell migration, calcium ion (Ca2+) has emerged as a key modulator of cytoskeleton reorganization in migrating cells1. Ca2+ signaling regulates cell migration in different aspects: the turnover of focal adhesions2,3, the contraction of actomyosin fibers (reviewed in4), plasma membrane ruffling5,6, and the formation of podosomes7. Indeed, in migrating cells a gradient is observed in the concentration of free cytosolic Ca2+ ([Ca2+]i) so that on the front side the [Ca2+]i is slightly lower than on the rear part of the cell8,9. This gradient of Ca2+ causes the Ca2+-dependent signaling to be polarized, as indeed is polarized the cytoskeleton in migrating cells. This difference in the [Ca2+]i is due to a differential subcellular localization of Ca2+ channels and pumps along the front-rear axis. For example, at the leading edge there is a greater concentration of PMCA (plasma membrane Ca2+-ATPase) and a higher density of SERCA (sarco(endo)plasmicreticulum Ca2+-ATPase), which facilitates a localized decrease of [Ca2+]i10. 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 retraction11. For these reasons, the subcellular localization of the different plasma membrane Ca2+ channels is critical 1Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain. 2MRCProtein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom. 3Department of Cell Biology and Histology, University of Murcia, IMIB-Arrixaca, Murcia, 30100, Spain. 4Department of Cell Biology, School of Medicine and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain. 5These authors contributed equally: Aida M. Lopez-Guerrero and Noelia Espinosa-Bermejo. *email: epozo@ unex.es; [email protected] open 2 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ when performing their function as cell migration regulators, either at the leading edge, the trailing edge, or modulating focal adhesion turnover. The influx of Ca2+ through store-operated Ca2+ channels (SOC channels) regulatesthe migration of both cancer and non-cancer cells12–17. 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 in18). Amongst these SOC channels, much attention has focused on ORAI1 since upregulation of its expression has been reported in a variety of cancer cell lines and primary tumors19,20, pointingto ORAI1 asbeing a potential target for cancer therapy. ORAI1 is involved in the epithelial to mesenchymal transition of breast cancer cells21, and the positive regulation of cell migration, but not in proliferation12. The closely related homolog ORAI3, however, is involved in the regulation of proliferation and cell death suppression of cancer cells19,22,23, but plays a minor role in cell migration. The control of the subcellular localization of ORAI1 is largely unknown, and its study should help explain the differential roles of ORAI homologs. In this regard, previous work has shown that ORAI1 presents a subcellular localization that overlaps with cortactin (CTTN) in HeLa cells, U2OS cells, and myoblasts5. CTTN is a well-known regulator of lamellipodia formation24, and therefore is a marker of the leading edge. The polarized localization of ORAI1 at the leading edge constitutes a good match with the polarized localization of phospho-STIM15, a pool of STIM1 that is phosphorylated by the ERK1/2 kinase. The function of STIM1 is to act as a Ca2+ sensor within the intraluminal side of the endoplasmic reticulum (ER) and to activate the opening of ORAI1 upon partial depletion of Ca2+ concentration within the ER25. In this way, i.e., being STIM1-dependent, ORAI1 can act as a SOC channel, although this channel can also be activated by Ca2+ store-independent mechanisms26,27. The polarized localization of STIM1 had been reported in a work that had shown an increasing gradient of diacylglycerol towards the front that promotes migration of endothelial cells10. To reach the leading edge, STIM1 requires the binding to microtubules through the microtubule plus-end binding protein EB110. However, the leading edge of migrating cells is devoid of microtubules, and the formation of filopodia and lamellipodia is driven by actin28, so an additional explanation for this re-localization was required. The explanation proposed was a mechanism of STIM1 regulation by phosphorylation29,30. The leading edge is enriched in receptor tyrosine kinases (RTKs)31, and many of these RTKs activate the ERK1/2 kinase pathway. ERK1/2 phosphorylates STIM1 at residues Ser575, Ser608, and Ser62129,32 which are close to the sequence that directly binds to EB1, leading to the dissociation of STIM1 from EB129, andenabling the dissociation of STIM1 from microtubules in the microtubule-free lamellipodia. However, the mechanisms that regulate the localization of ORAI1 at the leading edge remain far from clear. The small GTPase RAC1 regulates the localization of CTTNand the WAVE regulatory complex (WRC)33–35. RAC1 is a downstream effector of RTKs, and is highly enriched at the leading edge36, making this small GTPase a candidate for the regulation of the subcellular localization of ORAI1. We used an osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, toanalyze ORAI1 enrichment at the leading edge by means of immunofluorescence co-localization, and co-precipitation with cortactin, ARP2/3, and CYFIP1, which are at highly enriched levels in lamellipodia. We found that RAC1 potentiated ORAI1 translocation to the leading edge, and that this was sensitive to RAC1 inhibition, either by a chemical inhibitor or by a dominant negative mutation in RAC1. Our results demonstrate that RAC1 controls ORAI1 localization at the leading edge where this channel regulates lamellipodia formation, lamellipodial persistence, and cell directness, which are required for efficient cell migration and tumor cell invasiveness in vivo. Results ORAI1 potentiates lamellipodia formation and directional persistence. As noted above, 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. In those specific areas, ORAI1 and its regulator STIM1 are critical mediators for the Ca2+ entry required to sustain membrane ruffling. To further evaluate the role of ORAI1 at the leading edge, we analyzed different features of random cell migration on collagen-coated plates. Using U2OS cells edited by CRISPR/Cas to knock-out ORAI1 gene expression5, we observed that cell migration speed and total distance were greatly inhibited by the ORAI1 deficiency (Fig.1A, and Supplementary movieS1 and S2). More important was the fact that cell directness was significantly reduced (from 0.63 ± 0.03 to 0.43 ± 0.03) (Fig.1A). As a control, ORAI1-KO cells were transduced with retrovirus for the stable expression of ORAI1 (Supplementary Fig.S3), and this experiment proved that the over-expression of ORAI1 and the consequent rescue of ORAI1-dependent Ca2+ entry, reversed the inhibitory effect on cell directness observed in the ORAI1-KO cell line leading to the conclusion that the impairment of cell polarity was specifically due to the absence of ORAI1. The analysis of the leading edge length, i.e., the length of the sheet extending along the front of the moving cells, revealed that ORAI1 also plays a role in the formation or the stabilization of lamellipodia because ORAI1-deficient cells had smaller lamellipodiathan wild-type cells (Fig.1B and Supplementary Fig.S4). Indeed, the analysis of the cells shows the ORAI1-KO cells to be weakly polarized (Fig.1B and Supplementary Fig.S4). The combination of decreased polarity (or increased circularity) and shortened lamellipodia led us to analyze protrusion distance and lamellipodial persistence because a smaller lamellipodia could be the result of low levels of persistence or distance covered by the nascent lamellipodia. Our analysis confirmed that removal of ORAI1 triggered a significant reduction in both the extension of newly formed lamellipodiaand in lamellipodial persistence (Fig.1C), suggesting that there are defects in the formation of lamellipodia in the absence of ORAI1. Directional persistence and polarization are two key factors for successful cell migration and invasion in vivo. For this reason, we analyzed invasion rates in an in vivo 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 higher level of tumor cells in the larvae when wild-type U2OS cells were injected 3 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 1. Genetic ablation of ORAI1 reduced motility, directness, lamellipodia formation, and invasion of U2OS cells. Panel A: Wild-type U2OS cells (black symbols) and ORAI1-KO cells (red symbols) were monitored for speed, accumulated distance, and directness in a 2D random motility assay (total assay time = 200 min). Representative traces of 19 cells/condition are shown in the left figure. Data from 3 independent assays/ condition (n = 44 WT cells; n = 78 KO cells) are shown as bar charts or dot plot. Panel B: The leading edge length was measured using bright-field images of wild-type cells and ORAI1-KO cells. Data from 3 independent experiments (>22 cells/condition) were measured. Bar = 20 μm. Circularity index was measured from 2 independent experiments (>20 cells/condition). Panel C: Protrusion distance and persistence were measured from 2D random motility assays. Using a 3-pixel width line drawn at the leading edge, protrusion distance and persistence were measured from the resulting kymograph (left panels). An example of a wild-type cell at two different times and the resulting kymograph are shown. Plotted data are individual data from 2 independent experiments. Panel D: The experimental design of xenotransplants of wild-type U2OS and ORAI1-deficient cells in casper zebrafish larvae is represented in the Supplementary Fig.S5. Imaging to analyze U2OS cells invasion was performed at 5 days post injection (dpi). Left panels: Representative images of wild-type and 4 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ (Fig.1D). The deficiency in ORAI1 significantly reduced the dissemination of osteosarcoma U2OS cells, a finding that we propose is 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 8–10 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 1–3 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 a 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 to co-precipitate with both endogenous CTTN 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. CTTN plays a key role in lamellipodia. It binds to the ARP2/3 complex in existing actin fibers (F-actin) to facilitate the formation of actin fiber branches. We therefore also monitored the co-precipitation of ORAI1 with the endogenous ARP2/3 complex (Fig.2C). This analysis confirmed the recruitment of ORAI1 to peripheral areas enriched in CTTN and ARP2/3, i.e., the translocation of ORAI1 to the leading edge where CTTN and ARP2/3 are cooperating to form lamellipodia. These results were confirmed by the use of epifluorescence microscopy to monitor ORAI1-GFPand mCherry-CTTN colocalization before and after the treatment with 50 ng/ml EGF (Fig.2D). Pearson correlation coefficient was calculated to quantify the degree of co-localization in both conditions. For this quantitation we analyzed the cell periphery that remained free of cell-cell contacts. The results indicated that there was a significant enrichment of ORAI1-CTTN co-localization at the cell periphery in response to the stimulation with EGF. RAC1 activity mediates the translocation of ORAI1 to the leading edge. An upstream regulator of CTTN, which is activated by EGF, is the member of the Rho family of GTPases RAC134. It is also known that RAC1 is enriched at the leading edge of migrating cells36 where it stimulates the recruitment of CTTN to branched actin networks33,34. In this regard, RAC1 also co-precipitates with the WAVE regulatory complex, aprotein complex that regulates lamellipodia formation (reviewed in38), although it is not part of thecore complex. To investigate the role of RAC1 in the control of ORAI1 subcellular localization, we first monitored RAC1 localization in cell lines expressing EGFP-RAC1 wild-type or EGFP-RAC1Q61L(a constitutively active form of RAC1) in FBS-containing assay medium. This mutation impairs GTPase activity which means that RAC1 is locked in the GTP-bound conformation39. Consistent with the role of RAC1 as an upstream regulator that triggers the translocation of CTTN to the cell periphery, we observed that the constitutively active mutant RAC1Q61L promoted a peripheral localization of CTTN (Fig.3A). The line scan of the fluorescence signal showed there to be significant enrichment of both RAC1 and CTTN at the cell periphery under each of the two conditions. We must emphasize, however, that, in contrast to what we observed with RAC1 wild-type, there was no polarization in the CTTN localization when RAC1 was permanently active (RAC1Q61L). This finding can beeasily explained by the fact that RAC1Q61L activity is independent of polarized receptor tyrosine kinases and is able to trigger lamellipodia formation over the entire cell periphery. We then monitored ORAI1 and CTTN localization in cell lines stably expressing (i) Flag-RAC1 wild-type, (ii) Flag-RAC1G12V (another constitutively active form of RAC1 that impairs the GTPase activity leading to the locking of RAC1 in the GTP-bound conformation), and (iii) Flag-RAC1T17N (a dominant negative mutant of RAC1) (Fig.3B). As well as the Q61L, all these mutant forms of RAC1 have been widely used in other cell types to study the role of RAC1 inmembrane ruffling, lamellipodia formation, and cell invasion40–43. In Flag-RAC1 (wt)-overexpressing cells, ORAI1-GFP was found polarized in restricted areas of CTTN. However, the active form of RAC1G12V triggered significant re-localization of ORAI1 to the entire cell periphery, with an identical localization profile to that found for CTTN in these conditions. Similar localization of ORAI1 was found when the cells were transfected for the expression of mCherry-RAC1G12Vand ORAI1-GFP (Supplementary Fig.S6). In contrast, the dominant negative mutant of RAC1, RAC1T17N, reduced the translocation of CTTN to the periphery in the same way as it inhibited the peripheral localization of ORAI1. To quantify the extension of ORAI1 enrichment at CTTN-areas, we assessed the level of cortical ORAI1-GFP/ total ORAI1-GFP in individual cells and the ratio cortical CTTN/total CTTN. The analysis revealed that ORAI1-KO U2OS cells dissemination in zebrafish at 5 dpi. Magnification bar: 500 µm. Right panel: Percentage of invaded larvae of both genotypes with different invasion levels. Data shown in bar chart are from 2 independent experiments (n = 119 larvae injected with WT cells, n = 87 larvae injected with ORAI1-KO cells). **p < 0.01 according to Chi-square tests. 5 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 2. EGF potentiated ORAI1 binding to CTTN, CYFIP1, and ARP2/3.Panel A: U2OS cells transfected for the expression of ORAI1-GFP (or the empty vector, i.e., GFP only) and mCherry-CTTN were starved overnight with FBS-free medium and treated with 50 ng/ml EGF for the times indicated in the figure. Images are representative of 3 independent experiments. From total lysates, ORAI1-GFP was pulled down and the co-precipitated mCherry-CTTN analyzed by immunoblot. In co-IP assays, proteins were separated using 6.5% acrylamide gels. Total ORAI1-GFP pulled down was assessed with an anti-GFP antibody. The quantification of ORAI1-CTTN co-precipitation was evaluated with an anti-CTTN antibody from 3 independent experiments (scatter plot). Panel B: Whole cell lysates (WCL) from panel A were subjected to electrophoresis on 10% acrylamide gels, blotted, and assessed for the level of mCherry-CTTN, ORAI1-GFP, phospho-PAK1/2, totalPAK1, phospho-ERK1/2, and total-ERK1/2. Panel C: U2OS cells were transfected for the expression of ORAI1GFP or the empty vector. Cells were starved overnight with FBS-free medium and treated with 50 ng/ml EGF for 3 min. ORAI1-GFP was pulled down, and the co-precipitated endogenous CYFIP1, CTTN, and ARP2/3 were analyzed by immunoblot. Total ORAI1-GFP pulled down was assessed with an anti-GFP antibody. Blots 6 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ RAC1G12V triggered a significant increase of cortical ORAI1 and CTTN, whereas RAC1T17N reduced the level of both proteins at the cell cortex. The relative level of cortical ORAI1/CTTN did not change between these experimental conditions (Supplementary Fig.S7). These results suggest that both proteins have a similar behavior when RAC1 is modulated by the mutations used in the study. In addition to the localization of ORAI1, we studied the ruffling of the leading edge in response to the activation or inhibition of RAC1. Cells stably expressing Flag-RAC1 wild-type, Flag-RAC1G12V, and Flag-RAC1T17N were transfected for the transient expression of ORAI1-GFP and mCherry-CTTN. Cells growing in FBS-containing medium were visualized under epifluorescence microscopy and the cortical ruffling was evaluated as described previously by our group5. Supplementary MoviesS8, S9, and S10 show time-lapse sequences with the dynamics of GFP/Cherry in Flag-RAC1 wild-type, Flag-RAC1G12V, and Flag-RAC1T17N, respectively. Figure4 depicts a single frame from the time-lapse experiments. The analysis of the fluorescence at the cell cortex shows the spiking of the fluorescence for both tags, which was strongly inhibited in Flag-RAC1T17N-expressing cells. In addition to the translocation of ORAI1 to lamellipodia, also striking was the alteration of the intracellular/peripheral ratio of ORAI1 stimulated by the activation of RAC1. As observed in cells expressing RAC1T17N, the inhibition of RAC1 activity triggered accumulation of ORAI1 in intracellular vesicles compared with RAC1 (wt)-expressing cells (Fig.3B). We have investigated the intracellular trapped ORAI1 using markers for ER-Golgi transport (Sec13a), cis-Golgi (GM130), trans-Golgi (TGN46), endosomes (EEA1), and lysosomes (LAMP1), and found that these vesicles were GM130 positive (Fig.5). Therefore, an important conclusion from this experiment is the partial inhibition of ORAI1 translocation to the plasma membrane due to RAC1 inactivation. Because NSC23766 is a widely used inhibitor of RAC1 we also investigated whether the treatment of cells with NSC23766 led to ORAI1 accumulation into cis-Golgi vesicles. In this latter case, ORAI1 was found much more diffusively distributed throughout the cytoplasm, without evident accumulation at Golgi (Fig.5B), suggesting that the pharmacological inhibition of RAC1 did not impaired the traffic between ER and Golgi. To investigate whether RAC1 inhibition was impairing the entire secretory pathway we generated a stable cell line expressing the Gaussia 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 Gaussia 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 (PBD) and the subsequent analysis of co-precipitated RAC1 (Fig.6A). The results demonstrated 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 containing active RAC1. The efficiency of NSC 23766, which prevents RAC1 activation by RAC-specific guanine nucleotide exchange factors45, as a RAC1 inhibitor was evaluated by directly assessing the level of active RAC1 by pull-down with GST-PBD and analyzing the co-precipitated RAC1 (Fig.6C). Because surface ORAI1 was concentrated at the leading edge, where it colocalized with leading edge markers, we analyzed the effect of RAC1 inhibition on lamellipodia formation and cell directness. As shown in Fig.7A, the treatment of U2OS cells with NSC 23766 reduced cell speed, total distance migrated, cell directness, and the lamellipodia length, similarly to what had been observed in ORAI1-deficient cells (Fig.1), confirming that RAC1 activity and ORAI1 are essential for the efficient development of the lamellipodia. As a consequence of the RAC1 inhibition by NSC 23766, the level of ORAI1-CTTN interaction was reduced. This interaction was monitored by pulling down ORAI1-GFP from cells expressing Cherry-CTTN (Fig.7B), as well as the co-precipitation with endogenous CTTN (Fig.7C). While EGF stimulated this co-precipitation, the pretreatment of cells with NSC 23766 reduced ORAI1-CTTN co-precipitation, a result that confirmed the role of RAC1 in the translocation of ORAI1 to lamellipodia. As stated above, CYFIP1 is one of the subunits of the WRC. Because CYFIP1 interacts with the active GTP-bound form of RAC1, we also monitored the co-precipitation of ORAI1 with CYFIP1 in response to EGF. As shown in Fig.7C, ORAI1-CYFIP1 co-precipitation was sensitive to RAC1 inhibition, a result that strongly supported a role for RAC1 in the recruitment of ORAI1 in CTTNand CYFIP1-rich areas, i.e, the lamellipodia. In coherence with these results, the treatment of cells with NSC 23766 reduced the amount of ORAI1 at the cell surface (Fig.8A) as assessed with a biotinylation assay of surface proteins. A similar result was found when are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig.S13. Panel D:U2OS cells transfected for the expression of ORAI1-GFP and mCherry-CTTN were starved overnight with FBS-free medium and treated with 50 ng/ml EGF for 3 min (+EGF). Controls without the addition of EGF (-EGF) were processed in parallel. Fixed cells were analyzed under epifluorescence microscopy to evaluate the Pearson correlation coefficient of GFP and mCherry in the cell periphery only. Circular ROIs of 1.1–1.18 μm2 were set over the cell periphery that remained free of cell-cell contacts. Pearson correlation coefficient values within the range 0–1 are plotted in the bottom left panel. Right panel shows the histogram of the frequency distribution of Pearson coefficient values. 7 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ the dominant negative mutant RAC1T17N was overexpressed (Fig.8B) as a strategy to inhibit RAC1. As a control of the specificity of the biotinylation assay for plasma membrane proteins, the absence of biotinylated p38 (cytosolic marker) and the presence of biotinylated Na+/K+-ATPase (plasma membrane marker) were confirmed by Figure 3. Activation of RAC1 triggered the translocation of ORAI1 to CTTN-containing cell periphery. Panel A: Cells growing onto collagen-coated coverslips in FBS-containing DMEM were transfected for the expression of mCherry-CTTN and EGFP-RAC1 (wt) or EGFP-RAC1Q61L (active RAC1). At 36 h after transfection, cells were fixed and visualized under wide-field epifluorescence microscopy. Cherry and GFP channels were recorded sequentially using independent filter blocks. The two images were merged, and the fluorescence of both channels was measured over the arrow depicted in the image. Fluorescence values, plotted on right panels, were quantified with the NIS-Elements AR software. Images are representative of a minimum of 32 cells per condition from 3 independent experiments. Bar = 10 μm. Panel B: Cells stably expressing Flag-RAC1 (wildtype), Flag-RAC1G12V, or Flag-RAC1T17N were transfected for the expression of mCherry-CTTN and ORAI1GFP. As in panel A, cells were analyzed under epifluorescence microscopy, and Cherry and GFP channels were recorded. Images are representative of >26 cells per condition from 3 independent experiments. Bar = 10 μm. 8 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ immunoblot (Supplementary Fig.S11). Moreover, the inhibition of RAC1 led to a reduced speed of Ca2+ entry in fura 2-loaded cells treated with thapsigargin to deplete intracellular stores (Fig.8C,D), a result that fits well with the reduced amount ofsurface ORAI1 when RAC1 is inhibited. Discussion In this study, we have investigated the molecular mechanisms that control ORAI1 localization in migrating cells using the osteosarcoma cell line U2OS, a cellular model with a high invasion and migration potential46. We had previously shown that ORAI1 is enriched in CTTN-rich areas5, but the regulation of this localization remained unexplained. The results revealed that, upon stimulation with EGF, there is greater co-precipitation of ORAI1 withCTTN, ARP2/3, and CYFIP1, all of which are well-known regulators of cortical cytoskeleton at the leading edge. Active RAC1 (i.e., GTP-bound RAC1) recruits CTTN to the leading edge to help, together with ARP2/3, in the formation of lamellipodia, and CYFIP1 (also known as specifically RAC1-activated protein, or SRA1) is a Figure 4. RAC1 regulated the dynamics of peripheral ORAI1-CTTN and ruffling. U2OS cells stably expressing Flag-RAC1 (wild-type), Flag-RAC1G12V or Flag-RAC1T17N, were transfected for the transient expression of ORAI1-GFP and mCherry-CTTN. Fluorescence was monitored in live cells in Leibovitz’s L-15 medium supplemented with 10% FBS. The ROI indicated by the arrow was selected to assess the variation of fluorescence intensity (right panels). Emission of fluorescence of ORAI1-GFP (green line) and mCherry-CTTN (red line) was recorded every 3 sec for 4 min. Full time-lapse sequences are shown as Supplementary MoviesS8 (for wildtype RAC1), S9 (RAC1G12V), S10 (RAC1T17N). Bar = 10 μm. 9 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 5. RAC1T17N blocked the transport of ORAI1 to the cell surface. Panel A: U2OS cells stably expressing Flag-RAC1T17N were transfected for the transient expression of ORAI1-GFP. Cells were fixed 24 h after transfection, and used for the immunolocalization of GM130, TGN46, EEA1, LAMP1, and Sec13a. Secondary antibodies were labelled with Alexa Fluor 594. Images are representative of 2 independent experiments (>20 cells per condition). Fluorescence of both channels was measured over the arrow depicted in the image, which was placed over the intracellularly trapped GFPsignal. Panel B: U2OS cells were treated with 50 μM NSC 23766 for 8 h, fixed, and the immunolocalization of GM130 was performed as in panel A. Images are representative of 26 cells from 2 independent experiments. Bar = 10 μm. Panel C:Left: Cells were cultured on 96-well plates in DMEM + 10% FBS medium. The secreted and the intracellular luciferase activity were measured after 28 h of culture. The treatment with 50 μM NSC 23766 was performed during the last 8 h of culture. The overexpression of Flag-RAC1T17N was triggered with doxycycline during the last 22 h of culture. Data from n = 15 wells and 3 independent experiments are shown as bar chart. Middle: Intracellular Gluc-YFP protein was evaluated from cell lysates by immunoblot using an anti-GFP antibody. Right: Cells were treated with 5 μg/ml brefeldin A for 2–4 h to assess the inhibition of the secretory pathway, as a control of the experiment. 16 Scientific RepoRtS | (2020) 10:6580 | https://doi.org/10.1038/s41598-020-63353-5 www.nature.com/scientificreports www.nature.com/scientificreports/ were resuspended in 67% PBS + 5% FBS. Two hundred cells per embryo were then injected in the yolk sac of transparent roya9/a9; nacrew2/w2 (casper) zebrafish larvae65 of 48 h post-fertilization (hpf) and after 5 days at 35 °C the larvae were analyzed for human U2OS cells dissemination by fluorescence microscopy (see experimental design in Supplementary Fig.S5). U2OS cell invasion score was calculated as the percentage of zebrafish U2OS cell-invaded larvae over the total number of larvae analyzed taking into account also the number of tumor foci per larvae. Three tumor foci were established to score a larva as positive for invasion. Furthermore, larvae positive for invasion were also distinguished in three groups considering the number of positive foci per larvae: 3–5 foci per larvae, 5–15 foci per larvae and >15 foci per larvae. Images were acquired using a Leica MZ16F fluorescence stereo microscope and processed using ImageJ software (http://rsb.info.nih.gov/ij/). Image analysis. Persistence and protrusion distance were calculated as described elsewhere66. Basically, cells were grown on collagen-coated culture dishes. After a minimum of 4 h after plating, cells were selected when they showed free migration without cell-cell contacts for 2 h. Kymographs were prepared by plotting a 3 pixel-width line to evaluate lamellipodial persistence and protrusion. Circularity was evaluated by measuring the perimeter and total area of individual cells. Circularity was defined as 4 x pi x area/perimeter. Evaluation of Pearson correlation coefficient (Fig.2D) was performed by designing circular ROIs of 1.1–1.18 μm2 over the cellular periphery. The number of ROIs per cell was 70–90 in order to evaluate all the perimeter of the cell that remained free of cell-cell contacts. Normalization of cortical ORAI1/CTTN over total ORAI1/CTTN was performed by defining a single ROI for every cell that enclosed the cortical region, excluding cell-cell contact sites. An additional ROI surrounding the entire cell was designed to evaluate total fluorescence. Fluorescence intensity for GFPand mCherrychannels was recorded in both ROIs, and data of cortical ORAI1 (or CTTN)/total ORAI1 (or CTTN) was calculated and plotted (see Supplementary Fig.S7). 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Cortactin promotes cell motility by enhancing lamellipodial persistence. Curr. Biol. 15, 1276–1285 (2005). Acknowledgements This work was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (grants BFU2017-82716-P to FJMR, and BIO2017-84702-R to VM), and Junta de Extremadura (grants GR18084 and IB16088 to FJMR). All grants were co-financed by FEDER funds. ALG, NEB, and CPC were supported by predoctoral fellowships from the Spanish Ministerio de Economia y Competitividad (BES-2012-052061), Junta de Extremadura (PD18040), and Ministerio de Educación, Cultura y Deporte (FPU13/03430), respectively. None of these funding agencies was involved in the design of the study, completion of the research, or manuscript preparation. Author contributions A.M.L.G., N.E.B. and E.P.G. designed and performed experiments; I.S.L. and C.P.C. contributed to the generation of cell lines and data analysis; Y.O.A. performed the analysis of Gaussia luciferase secretion; T.M. designed the strategy for CRISPR/Cas-mediated gene editing; L.R.R., A.B.P.O. and V.M. designed, performed, and analyzed cell invasion experiments; E.P.G. and F.J.M.R. conceived the project and designed experiments; F.J.M.R. wrote the manuscript with inputs from all authors. All authors reviewed and approved the final manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-63353-5. Correspondence and requests for materials should be addressed to E.P.-G. or F.J.M.-R. Reprints and permissions information is available at www.nature.com/reprints. 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