Abstract
Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.
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Acknowledgements
We thank B. Glick (University of Chicago), S. Koide (University of Chicago) and F. Cross (Rockefeller University) for sharing plasmids, and members of the Glotzer, Weiss, Sosnick, Munro, Kovar and Glick laboratories for helpful discussions. This work was supported by research (GM088668, M.G. and T.R.S.) and training grants from the US National Institutes of Health, a grant from the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust (M.G., T.R.S. and E.L.W.), and by an American Cancer Society Postdoctoral Fellowship to D.S. (119248-PF-10-134-01-CCG).
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D.S. and M.G. conceived of the TULIPs strategy. D.S., Y.L., E.W., E.L.W. and M.G. designed experiments. D.S., Y.L., E.W. and C.M.H. performed experiments. D.S., Y.L., E.W., C.M.H., T.R.S., E.L.W. and M.G. analyzed data. J.Z., C.A. and T.R.S. provided new AsLOV2 mutations. D.S., E.L.W. and M.G. wrote the paper.
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A provisional patent application that includes portions of the research described in this manuscript has been filed by the University of Chicago Office of Technology and Intellectual Property.
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Supplementary Text and Figures
Supplementary Figures 1–11, Supplementary Tables 1–5 and Supplementary Notes 1–2 (PDF 3795 kb)
Supplementary Video 1
Repeated photoexcitation, mitochondrial recruitment of ePDZ-mCherry and dark-state recovery in a HeLa cell expressing Tom70-GFP-LOVpep (K–6R, T–2S) and ePDZb1-mCherry. mCherry fluorescence images were taken every 5 s. (MOV 2253 kb)
Supplementary Video 2
Photoexcitation, PM recruitment of ePDZ-mCherry and dark-state recovery in cells expressing Mid2-GFP-LOVpep (T406A, T407A). Left, ePDZb-mCherry. Right, ePDZb1-mCherry. Photoexcitation occurs between the first and second frames. mCherry fluorescence images were taken every 5 s. (MOV 106 kb)
Supplementary Video 3
Spot photoexcitation, PM recruitment of ePDZb1-mCherry and dark-state recovery in cells expressing Mid2GFP-LOVpep (V416I,T406A,T407A) and ePDZb1-mCherry. Left, spot recruitment of ePDZb1-mCherry only. Right, spot recruitment of ePDZb1-mCherry followed by global recruitment and cytoplasmic depletion. mCherry fluorescence images 1–17 were taken every 2 s, 18–30 every 10 s and 31–42 every 60 s. (MOV 144 kb)
Supplementary Video 4
Light-directed polarized growth in budding yeast. Cells were exposed to mating pheromone to induce cell cycle arrest, then stimulated with spot photoexcitation to recruit Cdc24-ePDZb1 to PM-tethered LOVpep. Red, actin labeled using Abp1-mCherry. Blue, phase contrast image processed using an edge detection filter. Frames were taken every 1 s. Spot photoexcitation occurs at the white crosshairs, only in the frames where they are visible. (MOV 1143 kb)
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Strickland, D., Lin, Y., Wagner, E. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9, 379–384 (2012). https://doi.org/10.1038/nmeth.1904
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DOI: https://doi.org/10.1038/nmeth.1904
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