Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains.
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.
Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Extremadura, Badajoz, 06006, Spain.
Aida M
AM
Lopez-Guerrero
Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Extremadura, Badajoz, 06006, Spain.
Patricia
P
Tomas-Martin
Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Extremadura, Badajoz, 06006, Spain.
Carlos
C
Pascual-Caro
MRC- Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.
Thomas
T
Macartney
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.|Center for Interdisciplinary Studies on the Nervous System (CISNe) and Institute of Medicine, Universidad Austral de Chile, Valdivia, Chile.
Alejandro
A
Rojas-Fernandez
Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.
Graeme
G
Ball
MRC- Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.
Dario R
0000-0002-2140-9185
DR
Alessi
Department of Cell Biology, School of Medicine, University of Extremadura, Badajoz, 06006, Spain.
Eulalia
E
Pozo-Guisado
Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Extremadura, Badajoz, 06006, Spain. fjmartin@unex.es.
Francisco Javier
0000-0001-6796-7396
FJ
Martin-Romero
D000818:Animals; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement; D051356:Cortactin / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D000071740:ORAI1 Protein / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1 / Q000378:metabolism*
D051356:Cortactin; C495869:Cttn protein, mouse; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C520430:Orai1 protein, mouse; C103530:STIM1 protein, human; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1