Format

Send to

Choose Destination
Nat Chem Biol. 2016 Oct;12(10):802-809. doi: 10.1038/nchembio.2145. Epub 2016 Aug 8.

FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.

Author information

1
Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine.
2
Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill.
3
Department of Cell Biology, The Scripps Research Institute.
4
Department of Immunology and Microbial Science, The Scripps Research Institute.
5
Department of Cell Biology, Harvard Medical School.
#
Contributed equally

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.

PMID:
27501396
PMCID:
PMC5030135
DOI:
10.1038/nchembio.2145
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center