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

Nature Chemical Biology volume 12, pages 802–809 (2016)Cite this article

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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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Figure 1: Design and validation of GDI.Cdc42 FLARE, a genetically encoded biosensor that reports localization of GDI–Cdc42 complexes.
Figure 2: GDI–Cdc42 complex localization in living cells.
Figure 3: Relationship between Cdc42 activation and GDI–Cdc42 localization monitored in the same cell.
Figure 4: Src-mediated phosphorylation of GDI at Y156 regulates the coordination of GDI–Cdc42 localization and Cdc42 activity in regions close to the edge.

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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).

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Author notes

  1. Louis Hodgson and Désirée Spiering: These authors contributed equally to this work.

Authors and 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.

Corresponding authors

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

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1 – 11. (PDF 4517 kb)

An example of imaging GDI–Cdc42 binding using the wild-type GDI.Cdc42 FLARE biosensor. (MOV 12693 kb)

An example of imaging GDI–Cdc42 binding using the wild-type GDI.Cdc42 FLARE biosensor (MOV 12440 kb)

A zoomed example of imaging GDI–Cdc42 binding using the wild-type GDI.Cdc42 FLARE biosensor. (MOV 10960 kb)

A zoomed example of imaging GDI–Cdc42 binding using the wild-type GDI.Cdc42 FLARE biosensor. (MOV 12561 kb)

Imaging Cdc42 activity (left) and GDI–Cdc42 binding (T35S mutant version; right) in the same MEF cell. (MOV 12797 kb)

41589_2016_BFnchembio2145_MOESM122_ESM.mov

A zoomed example of Cdc42 activity (left) and GDI–Cdc42 binding (T35S mutant version; right) in the same MEF cell. (MOV 12655 kb)

41589_2016_BFnchembio2145_MOESM123_ESM.mov

A zoomed example of Cdc42 activity (left) and GDI–Cdc42 binding (T35S mutant version; right) in the same MEF cell. (MOV 12117 kb)

41589_2016_BFnchembio2145_MOESM124_ESM.mov

A cell in which GDI has been knocked down and rescued with Y156F mutant GDI. Cdc42 activity is shown on the left, and GDI–Cdc42 binding is shown on the right (T35S mutant version). (MOV 18327 kb)

A zoomed example of a cell in which GDI has been knocked down and rescued with a Y156F GDI mutant. (MOV 10142 kb)

A cell in which GDI has been knocked down and rescued with a Y156F GDI mutant. (MOV 9125 kb)

A zoomed example of a cell in which GDI has been knocked down and rescued with a Y156F GDI mutant. (MOV 10596 kb)

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Hodgson, L., Spiering, D., Sabouri-Ghomi, M. et al. FRET binding antenna reports spatiotemporal dynamics of GDI–Cdc42 GTPase interactions. Nat Chem Biol 12, 802–809 (2016). https://doi.org/10.1038/nchembio.2145

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