title: FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.
abstract: 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.
authors: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine.|Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill.
authors: Louis
authors: L
authors: Hodgson
authors: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine.
authors: Désirée
authors: D
authors: Spiering
authors: Department of Cell Biology, The Scripps Research Institute.
authors: Mohsen
authors: M
authors: Sabouri-Ghomi
authors: Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill.
authors: Onur
authors: O
authors: Dagliyan
authors: Department of Immunology and Microbial Science, The Scripps Research Institute.
authors: Céline
authors: C
authors: DerMardirossian
authors: Department of Cell Biology, The Scripps Research Institute.|Department of Cell Biology, Harvard Medical School.
authors: Gaudenz
authors: G
authors: Danuser
authors: Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill.
authors: Klaus M
authors: KM
authors: Hahn
mesh_terms: D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D062211:Spatio-Temporal Analysis; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020730:Guanine Nucleotide Dissociation Inhibitors; D020764:cdc42 GTP-Binding Protein