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Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors

Nature volume 447, pages 596–600 (2007)Cite this article

Abstract

Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases—the master regulators of the actin cytoskeleton1,2,3. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins4,5: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity2,3. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology.

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Figure 1: GEFs link diverse inputs to Rho GTPase modules that control cell morphology.
Figure 2: Modular recombination yields PKA-responsive synthetic GEFs.
Figure 3: Synthetic GEFs generate new PKA-dependent morphological changes in cells.
Figure 4: Two synthetic GEFs can be linked in series to form a higher order cascade.

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Acknowledgements

We thank J. C. Anderson, A. Arkin, H. Bourne, J. Dueber, G. Kapp, J. Kardon, M. Nyako, and members of the Lim and Bar-Sagi laboratories for assistance and discussion. This work was supported by grants from the NIH (D.B.-S. and W.A.L.), the Packard Foundation (W.A.L.), and the Rogers Family Foundation (W.A.L.). B.J.Y. was supported by a Post-Graduate Scholarship from NSERC and R.J.R. was supported by an NIH-NCI Cancer Biochemistry and Cell Biology training grant.

Author Contributions B.J.Y., R.J.R., D.B.-S. and W.A.L. conceived the experiments. B.J.Y. and A.D. designed and purified the constructs and performed the in vitro experiments. R.J.R. performed the in vivo experiments.

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Author notes

  1. Brian J. Yeh and Robert J. Rutigliano: These authors contributed equally to this work.

Authors and Affiliations

  1. Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, California 94158-2517, USA,

    Brian J. Yeh

  2. Department of Cellular and Molecular Pharmacology and the Cell Propulsion Lab, UCSF/UCB NIH Nanomedicine Development Center, University of California, San Francisco, San Francisco, California 94158-2517, USA,

    Brian J. Yeh, Anrica Deb & Wendell A. Lim

  3. Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA,

    Robert J. Rutigliano & Dafna Bar-Sagi

  4. Department of Biochemistry, New York University School of Medicine, New York, New York 10016, USA,

    Dafna Bar-Sagi

Authors
  1. Brian J. Yeh
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Correspondence to Dafna Bar-Sagi or Wendell A. Lim.

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Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Tables S1-S4, Supplementary Figures S1-S6 with Legends and additional references. (PDF 2394 kb)

Supplementary Video

This file contains Supplementary Video 1 which shows forskolin induced filopodia in cells injected with GEF1. A REF52 cell injected with GEF1 was visualized before (10 min) and during (60 min) forskolin treatment. Stimulation of filopodia can be observed within minutes of addition. (MOV 5239 kb)

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Yeh, B., Rutigliano, R., Deb, A. et al. Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors. Nature 447, 596–600 (2007). https://doi.org/10.1038/nature05851

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