Regulation of membrane ruffling by polarized STIM1 and ORAI1in cortactin-rich domains

Aida M. López-Guerrero; Patricia Tomas-Martin; Carlos Pascual-Caro; Thomas Macartney; Alejandro Rojas-Fernandez; Graeme Ball; Dario R. Alessi; Eulalia Pozo-Guisado; Francisco Javier Martin-Romero

doi:10.1038/s41598-017-00331-4

Regulation of membrane ruffling by polarized STIM1 and ORAI1in cortactin-rich domains

Aida M. López-Guerrero, Patricia Tomas-Martin, Carlos Pascual-Caro, Thomas Macartney, Alejandro Rojas-Fernandez, Graeme Ball, Dario R. Alessi, Eulalia Pozo-Guisado, Francisco Javier Martin-Romero (Lead / Corresponding author)

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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.

Original language	English
Article number	383
Number of pages	15
Journal	Scientific Reports
Volume	7
DOIs	https://doi.org/10.1038/s41598-017-00331-4
Publication status	Published - 24 Mar 2017

Keywords

calcium
cortactin
cytoskeleton
membrane ruffling
migration
STIM1
ORAI1

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Final published version, 4.08 MBLicence: CC BY

Cite this

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title = "Regulation of membrane ruffling by polarized STIM1 and ORAI1in 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 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 tothe 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.",

keywords = "calcium, cortactin, cytoskeleton, membrane ruffling, migration, STIM1, ORAI1",

author = "{M. L{\'o}pez-Guerrero}, Aida and Patricia Tomas-Martin and Carlos Pascual-Caro and Thomas Macartney and Alejandro Rojas-Fernandez and Graeme Ball and Alessi, {Dario R.} and Eulalia Pozo-Guisado and Martin-Romero, {Francisco Javier}",

note = "DRA thanks the Medical Research Council (MC_UU_12016/2) for support. G.B. was supported by a “Next Generation Optical Microscopy” award from the Medical Research Council (MR/K015869/1). E.P.-G. was awarded with a short-term fellowship from the EMBO (ASTF-311-2014). ",

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doi = "10.1038/s41598-017-00331-4",

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AU - M. López-Guerrero, Aida

AU - Tomas-Martin, Patricia

AU - Pascual-Caro, Carlos

AU - Macartney, Thomas

AU - Rojas-Fernandez, Alejandro

AU - Ball, Graeme

AU - Alessi, Dario R.

AU - Pozo-Guisado, Eulalia

AU - Martin-Romero, Francisco Javier

N1 - DRA thanks the Medical Research Council (MC_UU_12016/2) for support. G.B. was supported by a “Next Generation Optical Microscopy” award from the Medical Research Council (MR/K015869/1). E.P.-G. was awarded with a short-term fellowship from the EMBO (ASTF-311-2014).

PY - 2017/3/24

Y1 - 2017/3/24

N2 - 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 tothe 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.

AB - 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 tothe 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.

KW - calcium

KW - cortactin

KW - cytoskeleton

KW - membrane ruffling

KW - migration

KW - STIM1

KW - ORAI1

U2 - 10.1038/s41598-017-00331-4

DO - 10.1038/s41598-017-00331-4

M3 - Article

C2 - 28341841

SN - 2045-2322

VL - 7

JO - Scientific Reports

JF - Scientific Reports

M1 - 383

ER -

Regulation of membrane ruffling by polarized STIM1 and ORAI1in cortactin-rich domains

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Keywords

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