TY - JOUR
T1 - FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions
AU - Hodgson, Louis
AU - Spiering, Désirée
AU - Sabouri-Ghomi, Mohsen
AU - Dagliyan, Onur
AU - Der Mardirossian, Céline
AU - Danuser, Gaudenz
AU - MHahn, Klaus
N1 - Funding Information:
We thank the National Institute of General Medical Sciences (P01-GM103723 and T-R01 GM090317 to G.D. and K.M.H.; GM099837 to C.D.) and the National Cancer Institute (CA181838 to L.H.) for funding. O.D. is a Howard Hughes Medical Institute International Student Research Fellow. Mammalian expression cDNA constructs for Myc-WASP and HA-Tiam1 were gifts from D. Cox (Departments of Anatomy and Structural Biology and Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, USA). Myc-Intersectin1L was a gift from H. Bourne (Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA). Myc-mDia2 was from S. Narumiya (Department of Pharmacology, University of Kyoto, Kyoto, Japan). PAK1 was from Y. Wu (Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA).
Publisher Copyright:
© 2016 Nature America, Inc.
PY - 2016/10/1
Y1 - 2016/10/1
N2 - Guanine-nucleotide dissociation inhibitors (GDIs) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor, GDI.Cdc42 FLARE, in which Cdc42 is modified with a fluorescence resonance energy transfer (FRET) 'binding antenna' that selectively reports Cdc42 binding to endogenous GDIs. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed remarkably tight coordination of GTPase release and activation on a time scale of 10 s, suggesting that GDI-Cdc42 interactions are a critical component of the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.
AB - Guanine-nucleotide dissociation inhibitors (GDIs) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor, GDI.Cdc42 FLARE, in which Cdc42 is modified with a fluorescence resonance energy transfer (FRET) 'binding antenna' that selectively reports Cdc42 binding to endogenous GDIs. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed remarkably tight coordination of GTPase release and activation on a time scale of 10 s, suggesting that GDI-Cdc42 interactions are a critical component of the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.
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U2 - 10.1038/nchembio.2145
DO - 10.1038/nchembio.2145
M3 - Article
C2 - 27501396
AN - SCOPUS:84981164236
SN - 1552-4450
VL - 12
SP - 802
EP - 809
JO - Nature chemical biology
JF - Nature chemical biology
IS - 10
ER -