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LOVTRAP: an optogenetic system for photoinduced protein dissociation

Nature Methods volume 13, pages 755758 (2016) | Download Citation

Abstract

LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.

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Acknowledgements

This work was funded by NIH grants R01-GM090317 (K.M.H. and G.D.), P01-GM103723 (K.M.H. and G.D.), R01-DA036877 (K.M.H. and B.K.), and R01-CA157738 (R.L.) and by the Max Planck Society, German Research Foundation DFG, FOR 1279 (I.S.). H.W. is a recipient of an Arthritis Foundation Postdoctoral Fellowship. We thank E. Hartmann for crystallizing Zdk–LOV complexes, R. Littlefield (UNC Chapel Hill Department of Biochemistry and Biophysics) for providing LOV2 peptides, and E. Trudeau for cloning help. Diffraction data were collected at the Swiss Light Source, beamline X10SA, of the Paul Scherrer Institute, Villigen, Switzerland. We thank the Heidelberg data collection team and the PXII staff for their support in setting up the beamline and C. Roome for expert support of the crystallographic software. The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.

Author information

Author notes

    • Andreas Winkler

    Present address: Institute of Biochemistry, Graz University of Technology, Graz, Austria.

    • Hui Wang
    •  & Marco Vilela

    These authors contributed equally to this work.

Affiliations

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

    • Hui Wang
    •  & Klaus M Hahn
  2. Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, Texas, USA.

    • Marco Vilela
    •  & Gaudenz Danuser
  3. Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg, Germany.

    • Andreas Winkler
    • , Miroslaw Tarnawski
    •  & Ilme Schlichting
  4. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Hayretin Yumerefendi
    •  & Brian Kuhlman
  5. Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Rihe Liu
  6. Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Rihe Liu
  7. Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Klaus M Hahn

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Contributions

H.W. performed the screening experiments, engineered proteins, and performed imaging studies. M.V. performed analysis of oscillating signaling behavior. A.W., M.T., and I.S. carried out crystallography. H.W., H.Y., and B.K. purified proteins for crystallography. R.L., G.D., and K.M.H. directed the work and carried out final edits for the paper, which was written using contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rihe Liu or Gaudenz Danuser or Klaus M Hahn.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1–6 and Supplementary Note

Zip files

  1. 1.

    Supplementary Software

    Spectral analysis code

Videos

  1. 1.

    The crystal structure of LOV2 bound to Zdk1.

    Crystal structure of the LOV2-Zdk1 complex. Zdk1: magenta; LOV2 core domain: green; LOV2 Jα helix: blue; FMN cofactor: cyan.

  2. 2.

    Release of mCherry from mitochondria using blue light.

    HeLa cells were imaged without blue light for 5 min, then irradiated with blue light for 5 min (a blue dot appears), then again imaged without blue light for 6.5 min. Before irradiation, mCherry is localized on mitochondria. Blue light causes release of mCherry, leading to diffuse distribution in the cytosol. When the blue light is turned off, mCherry returns to the mitochondria.

  3. 3.

    Dark mutant of LOV2 abolishes the release of mCherry from mitochondria.

    HeLa cells were imaged for 5 min, then irradiated with blue light for 5 min (a blue dot appears) and then imaged without blue light for an additional 6.5 min. The dark mutant of LOV2 (LOVSD) showed only mitochondrial localization even after prolonged irradiation.

  4. 4.

    Release of mCherry from plasma membrane using blue light.

    HEK-293 cells were irradiated with 5 second pulses of blue light (a blue dot appears) every 100 seconds. mCherry was released from the plasma membrane during irradiation and returned to the plasma membrane when the blue light was turned off.

  5. 5.

    Release of Vav2 from mitochondria induced reversible increases in the velocity of cell edge oscillations.

    HeLa cells were imaged for 30 min, then irradiated with blue light for 30 min (a green dot appears), and then imaged again after blue light irradiation for 30 min. Images are scaled to optimize visualization of the edge.

  6. 6.

    Release of Rac1 Q61L from mitochondria induced reversible increases in the extent and velocity of cell edge ruffling.

    HeLa cells were imaged without blue light illumination for 30 min, then irradiated with blue light for 30 min (a blue dot appears), and then imaged for 30 min without blue light.

  7. 7.

    Release of RhoA Q63L from mitochondria induced contraction.

    HeLa cells were imaged without blue light for 30 min, then irradiated with blue light for 30 min (a blue dot appears) and then imaged without irradiation for 30 min.

  8. 8.

    Tracking cell edges.

    Upper left, a HeLa cell with the cell edge highlighted before, during and after optogenetically stimulated VAV2 release. Upper right, traces of the cell edge overlaid at 10 sec intervals before optogenetic stimulation; Lower left, traces overlaid during VAV2 release; Lower right, traces overlaid after blue light irradiation had been halted; Warmer colors indicate later time points. Scale bar: 10 μm.

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DOI

https://doi.org/10.1038/nmeth.3926

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