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FRET binding antenna reports spatiotemporal dynamics of GDI–Cdc42 GTPase interactions

Nature Chemical Biology volume 12, pages 802809 (2016) | Download Citation

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.

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Acknowledgements

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).

Author information

Author notes

    • Louis Hodgson
    •  & Désirée Spiering

    These authors contributed equally to this work.

Affiliations

  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA.

    • Louis Hodgson
    •  & Désirée Spiering
  2. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.

    • Louis Hodgson
    •  & Désirée Spiering
  3. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Louis Hodgson
    • , Onur Dagliyan
    •  & Klaus M Hahn
  4. Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Louis Hodgson
    • , Onur Dagliyan
    •  & Klaus M Hahn
  5. Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.

    • Mohsen Sabouri-Ghomi
    •  & Gaudenz Danuser
  6. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.

    • Céline DerMardirossian
  7. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Gaudenz Danuser

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Contributions

L.H. and K.M.H. conceived the biosensor; L.H. optimized and built the biosensor; L.H., D.S. and G.D. designed and interpreted correlation experiments; L.H. and D.S. performed biological experiments; M.S.-G. and L.H. performed the computational analysis; O.D. and K.M.H. performed structural analysis and interpreted studies to examine the antennae mechanism; C.D. subcloned the shRNA expression constructs and gave critical feedback; and L.H., G.D. and K.M.H. wrote the manuscript with input from all other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Louis Hodgson or Gaudenz Danuser or Klaus M Hahn.

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DOI

https://doi.org/10.1038/nchembio.2145