Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains
Abstract Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understand...
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Nature Portfolio
2017-03-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-00331-4 |
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| author | Aida M. Lopez-Guerrero Patricia Tomas-Martin Carlos Pascual-Caro Thomas Macartney Alejandro Rojas-Fernandez Graeme Ball Dario R. Alessi Eulalia Pozo-Guisado Francisco Javier Martin-Romero |
| author_facet | Aida M. Lopez-Guerrero Patricia Tomas-Martin Carlos Pascual-Caro Thomas Macartney Alejandro Rojas-Fernandez Graeme Ball Dario R. Alessi Eulalia Pozo-Guisado Francisco Javier Martin-Romero |
| author_sort | Aida M. Lopez-Guerrero |
| collection | DOAJ |
| description | Abstract Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immunoprecipitated with endogenous CTTN. This latter result, in addition to the KO cells’ phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling by the Ca2+-channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling. |
| first_indexed | 2024-12-20T21:05:20Z |
| format | Article |
| id | doaj.art-def4895300394d3fa46a7edb9c8abe1b |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-20T21:05:20Z |
| publishDate | 2017-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-def4895300394d3fa46a7edb9c8abe1b2022-12-21T19:26:37ZengNature PortfolioScientific Reports2045-23222017-03-017111510.1038/s41598-017-00331-4Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domainsAida M. Lopez-Guerrero0Patricia Tomas-Martin1Carlos Pascual-Caro2Thomas Macartney3Alejandro Rojas-Fernandez4Graeme Ball5Dario R. Alessi6Eulalia Pozo-Guisado7Francisco Javier Martin-Romero8Department of Biochemistry and Molecular Biology, School of Life Sciences, University of ExtremaduraDepartment of Biochemistry and Molecular Biology, School of Life Sciences, University of ExtremaduraDepartment of Biochemistry and Molecular Biology, School of Life Sciences, University of ExtremaduraMRC- Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeCentre for Gene Regulation and Expression, School of Life Sciences, University of DundeeDundee Imaging Facility, School of Life Sciences, University of DundeeMRC- Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeDepartment of Cell Biology, School of Medicine, University of ExtremaduraDepartment of Biochemistry and Molecular Biology, School of Life Sciences, University of ExtremaduraAbstract Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immunoprecipitated with endogenous CTTN. This latter result, in addition to the KO cells’ phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling by the Ca2+-channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling.https://doi.org/10.1038/s41598-017-00331-4 |
| spellingShingle | Aida M. Lopez-Guerrero Patricia Tomas-Martin Carlos Pascual-Caro Thomas Macartney Alejandro Rojas-Fernandez Graeme Ball Dario R. Alessi Eulalia Pozo-Guisado Francisco Javier Martin-Romero Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains Scientific Reports |
| title | Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains |
| title_full | Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains |
| title_fullStr | Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains |
| title_full_unstemmed | Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains |
| title_short | Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains |
| title_sort | regulation of membrane ruffling by polarized stim1 and orai1 in cortactin rich domains |
| url | https://doi.org/10.1038/s41598-017-00331-4 |
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