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Optogenetic control of cofilin and αTAT in living cells using Z-lock

Nature Chemical Biology volume 15, pages 1183–1190 (2019)Cite this article

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An Author Correction to this article was published on 21 July 2020

This article has been updated

Abstract

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

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Fig. 1: Design of Z-lock cofilin.
Fig. 2: Optimization of designs based on Zdk1 and Zdk2.
Fig. 3: Effect of Z-lock cofilin activation on leading edge protrusions and invadopodium formation in tumor cells.
Fig. 4: Z-lock αTAT.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

Code availability

Code is available from the authors upon request or at http://www.hahnlab.com.

Change history

  • 21 July 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank the NIH for supporting this work (grant no. GM122596 to K.M.H., grant nos. CA150344 and CA216248 to J.S.C.). O.J.S. is a recipient of a Ruth L. Kirschstein National Research Service Award (no. 1F31CA192739). We thank M. Azoitei for assisting with Rosetta modeling, T. Watanabe for assistance with localized photoactivation experiments and C. Onyeji for help with cloning and biochemical assays. N.P. was supported by grant PF-16-18-01-CSM from the American Cancer Society.

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Author notes

  1. These authors contributed equally: Orrin J. Stone, Neha Pankow, Bei Liu.

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA

    Orrin J. Stone, Neha Pankow, Bei Liu, Hui Wang, Andrew T. Putz & Klaus M. Hahn

  2. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY, USA

    Ved P. Sharma, Robert J. Eddy & John S. Condeelis

  3. Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, New York, NY, USA

    Ved P. Sharma & John S. Condeelis

  4. Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA

    Frank D. Teets & Brian Kuhlman

  5. Integrated Imaging Program, Albert Einstein College of Medicine, New York, NY, USA

    John S. Condeelis

  6. Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Klaus M. Hahn

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Contributions

J.S.C. and K.M.H. conceived of the project. O.J.S., B.L. and N.P. carried out the experiments, with assistance and advice from V.P.S. and R.J.E. in live cell imaging and assay of effects on cofilin. K.M.H. and H.W. conceived of Z-lock, and J.S.C. initiated the focus on cofilin. K.M.H. and J.S.C. supervised the project. O.J.S., F.D.T. and B.K. performed Rosetta modeling. A.T.P. purified proteins. O.J.S. and K.M.H. wrote the manuscript with input from all authors.

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Correspondence to John S. Condeelis or Klaus M. Hahn.

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Supplementary information

Supplementary Information

Supplementary Tables 1–2 and Supplementary Figures 1–13.

Reporting Summary

Supplementary Video 1

Original images of blots and stain-free gels used for Fig. 4b.

Supplementary Video 2

Photoactivation of Z-lock cofilin leads to protrusion.

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Stone, O.J., Pankow, N., Liu, B. et al. Optogenetic control of cofilin and αTAT in living cells using Z-lock. Nat Chem Biol 15, 1183–1190 (2019). https://doi.org/10.1038/s41589-019-0405-4

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