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 - 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.
UR - http://www.scopus.com/inward/record.url?scp=84981164236&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84981164236&partnerID=8YFLogxK
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 -