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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

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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.

References

  1. Bravo-Cordero, J. J., Magalhaes, M. A. O., Eddy, R. J., Hodgson, L. & Condeelis, J. Functions of cofilin in cell locomotion and invasion. Nat. Rev. Mol. Cell Biol. 14, 405–415 (2013).

    CAS  PubMed  Google Scholar 

  2. Sidani, M. et al. Cofilin determines the migration behavior and turning frequency of metastatic cancer cells. J. Cell Biol. 179, 777–791 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Zoncu, R. et al. Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc. Natl Acad. Sci. USA 104, 3793–3798 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Oser, M. & Condeelis, J. The cofilin activity cycle in lamellipodia and invadopodia. J. Cell Biochem. 108, 1252–1262 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. DesMarais, V., Ghosh, M., Eddy, R. & Condeelis, J. Cofilin takes the lead. J. Cell Sci. 118, 19–26 (2005).

    CAS  PubMed  Google Scholar 

  6. Chen, Q. & Pollard, T. D. Actin filament severing by cofilin dismantles actin patches and produces mother filaments for new patches. Curr. Biol. 23, 1154–1162 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Yang, N. et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393, 809–812 (1998).

    CAS  PubMed  Google Scholar 

  8. Aizawa, H., Sutoh, K. & Yahara, I. Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in dictyostelium. J. Cell Biol. 132, 335–344 (1996).

    CAS  PubMed  Google Scholar 

  9. Aizawa, H. et al. Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nat. Neurosci. 4, 367–373 (2001).

    CAS  PubMed  Google Scholar 

  10. Ghosh, M. et al. Cofilin promotes actin polymerization and defines the direction of cell motility. Science 304, 743–746 (2004).

    CAS  PubMed  Google Scholar 

  11. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hughes, R. M. & Lawrence, D. S. Optogenetic engineering: light-directed cell motility. Angew. Chem. Int. Ed. Engl. 53, 10904–10907 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Courtemanche, N., Pollard, T. D. & Chen, Q. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nat. Cell Biol. 18, 676–683 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Dagliyan, O. et al. Engineering extrinsic disorder to control protein activity in living cells. Science 354, 1441–1444 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hayashi-Takagi, A. et al. Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature 525, 333–338 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–384 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Strickland, D., Moffat, K. & Sosnick, T. R. Light-activated DNA binding in a designed allosteric protein. Proc. Natl Acad. Sci. USA 105, 10709–10714 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Yi, J. J., Wang, H., Vilela, M., Danuser, G. & Hahn, K. M. Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides. ACS Synth. Biol. 3, 788–795 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Weitzman, M. & Hahn, K. M. Optogenetic approaches to cell migration and beyond. Curr. Opin. Cell Biol. 30, 112–120 (2014).

    CAS  PubMed  Google Scholar 

  21. Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).

    CAS  PubMed  Google Scholar 

  22. Zoltowski, B. D., Vaccaro, B. & Crane, B. R. Mechanism-based tuning of a LOV domain photoreceptor. Nat. Chem. Biol. 5, 827–834 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Kawano, F., Aono, Y., Suzuki, H. & Sato, M. Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins. PLoS One 8, e82693 (2013).

    PubMed  PubMed Central  Google Scholar 

  24. Wang, H. et al. LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat. Methods 13, 755–758 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang, H. & Hahn, K. M. LOVTRAP: a versatile method to control protein function with light. Curr. Protoc. Cell Biol. 73, 21.10.1–21.10.14 (2016).

    Google Scholar 

  26. Yu, Y. & Lutz, S. Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol. 29, 18–25 (2011).

    CAS  PubMed  Google Scholar 

  27. Zhou, X. X., Chung, H. K., Lam, A. J. & Lin, M. Z. Optical control of protein activity by fluorescent protein domains. Science 338, 810–814 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhou, X. X., Fan, L. Z., Li, P., Shen, K. & Lin, M. Z. Optical control of cell signaling by single-chain photoswitchable kinases. Science 355, 836–842 (2017).

    PubMed  PubMed Central  Google Scholar 

  29. Lai, F. P. L. et al. Arp2/3 complex interactions and actin network turnover in lamellipodia. EMBO J. 27, 982–992 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Pope, B. & Weeds, A. G. Binding of pig plasma gelsolin to F-actin and partial fractionation into calcium-dependent and calcium-independent forms. Eur. J. Biochem. 161, 85–93 (1986).

    CAS  PubMed  Google Scholar 

  31. Huang, P.-S. et al. RosettaRemodel: a generalized framework for flexible backbone protein design. PLoS One 6, e24109 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kellogg, E. H., Leaver-Fay, A. & Baker, D. Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Proteins 79, 830–838 (2011).

    CAS  PubMed  Google Scholar 

  33. Chan, A. Y., Bailly, M., Zebda, N., Segall, J. E. & Condeelis, J. S. Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion. J. Cell Biol. 148, 531–542 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Neri, A., Welch, D., Kawaguchi, T. & Nicolson, G. Development and biologic properties of malignant cell sublines and clones of a spontaneously metastasizing rat mammary adenocarcinoma. J. Natl. Cancer Inst. 68, 507–517 (1982).

    CAS  PubMed  Google Scholar 

  35. Chan, A. Y. et al. EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells. J. Cell Sci. 111(Pt 2), 199–211 (1998).

    CAS  PubMed  Google Scholar 

  36. Eddy, R. J., Weidmann, M. D., Sharma, V. P. & Condeelis, J. S. Tumor cell invadopodia: invasive protrusions that orchestrate metastasis. Trends Cell Biol. 27, 595–607 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yamaguchi, H. et al. Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J. Cell Biol. 168, 441–452 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Oser, M. et al. Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation. J. Cell Biol. 186, 571–587 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Beaty, B. T. et al. β1 integrin regulates Arg to promote invadopodial maturation and matrix degradation. Mol. Biol. Cell 24, 1661–75, S1 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Haggarty, S. J., Koeller, K. M., Wong, J. C., Grozinger, C. M. & Schreiber, S. L. Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl Acad. Sci. USA 100, 4389–4394 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458 (2002).

    CAS  PubMed  Google Scholar 

  42. North, B. J., Marshall, B. L., Borra, M. T., Denu, J. M. & Verdin, E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol. Cell 11, 437–444 (2003).

    CAS  PubMed  Google Scholar 

  43. Miotto, B. & Struhl, K. HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by geminin. Mol. Cell 37, 57–66 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Borra, M. T., Langer, M. R., Slama, J. T. & Denu, J. M. Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases. Biochemistry 43, 9877–9887 (2004).

    CAS  PubMed  Google Scholar 

  45. Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B. & Nachury, M. V. The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc. Natl Acad. Sci. USA 107, 21517–21522 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Friedmann, D. R., Aguilar, A., Fan, J., Nachury, M. V. & Marmorstein, R. Structure of the α-tubulin acetyltransferase, αTAT1, and implications for tubulin-specific acetylation. Proc. Natl Acad. Sci. USA 109, 19655–19660 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Szyk, A. et al. Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase. Cell 157, 1405–1415 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Meth. Enzymol. 487, 545–574 (2011).

    CAS  Google Scholar 

  49. Kuhlman, B., Jacobs, T. & Linskey, T. Computational design of protein linkers. Methods Mol. Biol. 1414, 341–351 (2016).

    CAS  PubMed  Google Scholar 

  50. Suhre, K. & Sanejouand, Y.-H. ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res. 32, W610–W614 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Li, T. et al. Incorporation of DDR2 clusters into collagen matrix via integrin-dependent posterior remnant tethering. Int. J. Biol. Sci. 14, 654–666 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Sharma, V. P. et al. Tks5 and SHIP2 regulate invadopodium maturation, but not initiation, in breast carcinoma cells. Curr. Biol. 23, 2079–2089 (2013).

    CAS  PubMed  Google Scholar 

  53. Leibovitz, A. The growth and maintenance of tissue–cell cultures in free gas exchange with the atmosphere. Am. J. Epidemiol. 78, 173–180 (1963).

    CAS  Google Scholar 

  54. Sharma, V. P., Entenberg, D. & Condeelis, J. High-resolution live-cell imaging and time-lapse microscopy of invadopodium dynamics and tracking analysis. Methods Mol. Biol. 1046, 343–357 (2013).

    PubMed  PubMed Central  Google Scholar 

  55. Hodgson, L., Shen, F. & Hahn, K. Biosensors for characterizing the dynamics of rho family GTPases in living cells. Curr. Protoc. Cell Biol. Chapter 14, Unit 14.11.1–14.11.26 (2010).

    Google Scholar 

  56. Otsu, N. A threshold selection method from gray-level histograms. IEEE Trans. Syst. Man. Cybern. 9, 62–66 (1979).

    Google Scholar 

  57. Klejnot, M. et al. Analysis of the human cofilin 1 structure reveals conformational changes required for actin binding. Acta Crystallogr. D 69, 1780–1788 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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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.

Author information

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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