FIGURE SUMMARY
Title

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

Authors
Lopez-Guerrero, A.M., Espinosa-Bermejo, N., Sanchez-Lopez, I., Macartney, T., Pascual-Caro, C., Orantos-Aguilera, Y., Rodriguez-Ruiz, L., Perez-Oliva, A.B., Mulero, V., Pozo-Guisado, E., Martin-Romero, F.J.
Source
Full text @ Sci. Rep.

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 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.

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, total-PAK1, phospho-ERK1/2, and total-ERK1/2. Panel C: U2OS cells were transfected for the expression of ORAI1-GFP 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 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.

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 (wild-type), Flag-RAC1G12V, or Flag-RAC1T17N were transfected for the expression of mCherry-CTTN and ORAI1-GFP. 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.

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 wild-type RAC1), S9 (RAC1G12V), S10 (RAC1T17N). Bar = 10 μm.

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 GFP- signal. 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.

RAC1 co-precipitated with ORAI1 upon stimulation with EGF. Panel A: Cells were starved overnight in FBS-free cultured medium and then treated with 50 ng/ml EGF. At the indicated times, endogenous RAC1 activation (GTP-bound RAC1) was analyzed with a pull-down assay to evaluate the amount of RAC1 able to bind PAK1-PBD (see Methods section). Total RAC1 was analyzed as a loading control of the immunoblot. The blot is representative of 3 independent experiments and the quantification of active RAC1 was performed by analyzing the 3 independent experiments. Panel B: Cells were transfected for the expression of ORAI1-GFP, starved overnight with FBS-free medium and treated with 50 ng/ml EGF for 3 min. When required, cells were pre-incubated with 50 μM NSC 23766 for 8 h before the treatment with EGF. Then, ORAI1-GFP was pulled down with GFP beads, and the level of endogenous RAC1 co-precipitated was analyzed by immunoblot. The blot is representative of 3 independent experiments. Panel C: Cells were starved overnight in FBS-free cultured medium, treated with NSC 23766 when required, and then treated with 50 ng/ml EGF for 3 min. Inhibition of RAC1 by NSC 23766 was assessed with a pull-down assay to evaluate active RAC1, as in panel A. The blot is representative of 3 independent experiments. Quantification of RAC1 inhibition by NSC 23766 was performed by analyzing the 3 independent experiments. GTP-bound RAC1 levels were normalized with values obtained in the absence of stimulation (time = 0 min). Full-length blots are presented in Supplementary Fig. S13.

Inhibition of RAC1 impaired cell motility and reduced co-precipitation of ORAI1 with markers of the leading edge. Panel A: Tracking of U2OS cells (black symbols) and cells treated with NSC 23766 (green symbols) in a 2D random motility assay. Representative traces per condition are shown (20–21 per condition). The cells were monitored for speed, accumulated distance, and directness. The data in the panels are from 2 independent assays per condition and a minimum of 36 cells per condition. The leading edge length was measured using bright-field images of wild-type live cells growing in collagen-coated culture dishes. When required, cells were incubated with NSC 23766 for 8 h before the analysis. A minimum of 37 cells from 3 independent experiments were measured. Panel B: U2OS cells were transfected for the expression of ORAI1-GFP (or the empty vector) and mCherry-CTTN. Cells were starved overnight with FBS-free medium and treated with 50 ng/ml EGF for 3 min. Treatment with NSC 23766 was performed as indicated in the Fig. 6. ORA1-GFP was pulled down, and the co-precipitated mCherry-CTTN analyzed by immunoblot. 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. Whole cell lysates (WCL) were assessed for the level of mCherry-CTTN and ORAI1-GFP as the experimental control. Blots are representative of 3 independent experiments. Panel C: Cells were transfected for the expression of ORAI1-GFP and treated with EGF or NSC 23766 as indicated above. ORAI1-GFP was pulled down and the level of co-precipitated endogenous CYFIP1 and CTTN was analyzed by immunoblot. ORAI1-GFP pulled down was analyzed with an anti-GFP antibody. Blots are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S13.

Inhibition of RAC1 reduced surface ORAI1 and SOCE. Panel A: U2OS cells were transfected for the expression of ORAI1-GFP and cultured with FBS-containing DMEM. NSC 23766 was added to the culture medium 8 h before the biotinylation assay, when required. Cells were assessed for the level of ORAI1-GFP at the cell surface by a biotinylation assay of proteins at the plasma membrane in live intact cells and subsequent analysis of biotinylated ORAI1-GFP (top panel). Whole cell lysates were analyzed to assess the total amount of ORAI1-GFP (bottom panel). Quantification of results from 2 independent experiments is shown as a bar chart. Panel B: Wild-type U2OS cells or cells overexpressing Flag-RAC1T17N were assessed for the level of ORAI1-GFP at the plasma membrane, as in panel A. Quantification of results from 2 independent experiments is shown as bar chart. Full-length blots are presented in Supplementary Fig. S13. Panels C-D: Cells were loaded with fura-2-AM and treated with 1 μM thapsigargin in Ca2+-free Hank’s balanced salt solution. Then, 2 mM CaCl2 was added to this assay medium, and the increase rate of the ratio F340/F380 was measured during the first 90 sec after the addition of Ca2+. Results from 2 independent experiments are shown as a scatter plot.

Acknowledgments
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