Hodgson, LouisSpiering, DésiréeSabouri-Ghomi, MohsenDagliyan, OnurDerMardirossian, CélineDanuser, GaudenzHahn, Klaus M.2017-03-282016Hodgson, Louis, Désirée Spiering, Mohsen Sabouri-Ghomi, Onur Dagliyan, Céline DerMardirossian, Gaudenz Danuser, and Klaus M. Hahn. 2016. “FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.” Nature chemical biology 12 (10): 802-809. doi:10.1038/nchembio.2145. http://dx.doi.org/10.1038/nchembio.2145.http://nrs.harvard.edu/urn-3:HUL.InstRepos:31731651Guanine-nucleotide dissociation inhibitors (GDI) 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 design (GDI.Cdc42 FLARE), in which Cdc42 was modified with a FRET ‘binding antenna’ that selectively reported Cdc42 binding to endogenous GDI. 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 a remarkably tight coordination of GTPase release and activation on a time scale of 10 seconds, suggesting that GDI-Cdc42 interactions are a critical component in the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.en-USJournal Article2017-03-2810.1038/nchembio.2145