Optogenetic Control of Nuclear Protein Import in Living Cells Using Light-Inducible Nuclear Localization Signals (LINuS)
Pierre Wehler
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Search for more papers by this authorDominik Niopek
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Department of Bioinformatics and Functional Genomics, Institute for Pharmacy and Biotechnology
Department of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany
Search for more papers by this authorRoland Eils
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Department of Bioinformatics and Functional Genomics, Institute for Pharmacy and Biotechnology
Department of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany
Search for more papers by this authorBarbara Di Ventura
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Search for more papers by this authorPierre Wehler
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Search for more papers by this authorDominik Niopek
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Department of Bioinformatics and Functional Genomics, Institute for Pharmacy and Biotechnology
Department of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany
Search for more papers by this authorRoland Eils
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Department of Bioinformatics and Functional Genomics, Institute for Pharmacy and Biotechnology
Department of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany
Search for more papers by this authorBarbara Di Ventura
Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany
Search for more papers by this authorAbstract
Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and time. The light-inducible nuclear localization signal (LINuS) was developed based on a natural plant photoreceptor that reversibly triggers the import of proteins of interest into the nucleus with blue light. Each LINuS is a small, genetically encoded domain that is fused to the protein of interest at the N or C terminus. These protocols describe how to carry out initial microscopy-based screening to assess which LINuS variant works best with a protein of interest. © 2016 by John Wiley & Sons, Inc.
Literature Cited
- Aravanis, A.M., Wang, L.-P., Zhang, F., Meltzer, L.A., Mogri, M.Z., Schneider, M.B., and Deisseroth, K. 2007. An optical neural interface: In vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural Eng. 4: S143-S156. doi: 10.1088/1741-2560/4/3/S02.
- Beyer, H.M., Juillot, S., Herbst, K., Samodelov, S.L., Müller, K., Schamel, W.W., Römer, W., Schäfer, E., Nagy, F., Strähle, U., Weber, W., and Zurbriggen, M.D. 2015. Red light-regulated reversible nuclear localization of proteins in mammalian cells and zebrafish. ACS Synth. Biol. 4: 951-958. doi: 10.1021/acssynbio.5b00004.
- Brameier, M., Krings, A., and MacCallum, R.M. 2007. NucPred—predicting nuclear localization of proteins. Bioinformatics 23: 1159-1160. doi: 10.1093/bioinformatics/btm066.
- Cautain, B., Hill, R., de Pedro, N., and Link, W. 2015. Components and regulation of nuclear transport processes. FEBS J. 282: 445-462. doi: 10.1111/febs.13163.
- Cokol, M., Nair, R., and Rost, B. 2000. Finding nuclear localization signals. EMBO Rep. 1: 411-415. doi: 10.1093/embo-reports/kvd092.
- Cosentino, C., Alberio, L., Gazzarrini, S., Aquila, M., Romano, E., Cermenati, S., Zuccolini, P., Petersen, J., Beltrame, M., Van Etten, J.L., Christie, J.M., Thiel, G., and Moroni, A. 2015. Engineering of a light-gated potassium channel. Science 348: 707-710. doi: 10.1126/science.aaa2787.
- Crefcoeur, R.P., Yin, R., Ulm, R., and Halazonetis, T.D. 2013. Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells. Nat. Commun. 4: 1779. doi: 10.1038/ncomms2800.
- Davidson, M.W. and Campbell, R.E. 2009. Engineered fluorescent proteins: Innovations and applications. Nat. Methods 6: 713-717. doi: 10.1038/nmeth1009-713.
- Diensthuber, R.P., Engelhard, C., Lemke, N., Gleichmann, T., Ohlendorf, R., Bittl, R., and Möglich, A. 2014. Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors. ACS Synth. Biol. 3: 811-819. doi: 10.1021/sb400205x.
- Engelke, H., Chou, C., Uprety, R., Jess, P., and Deiters, A. 2014. Control of protein function through optochemical translocation. ACS Synth. Biol. 3: 731-736. doi: 10.1021/sb400192a.
- Harper, S.M., Neil, L.C., and Gardner, K.H. 2003. Structural basis of a phototropin light switch. Science 301: 1541-1544. doi: 10.1126/science.1086810.
-
Idevall-Hagren, O. and De Camilli, P.
2014. Manipulation of plasma membrane phosphoinositides using photoinduced protein-protein interactions. In
Photoswitching Proteins, Methods and Protocols ( S. Cambridge, ed.), pp. 109-128. Springer, New York.
10.1007/978-1-4939-0470-9_8 Google Scholar
- Jacques, S.L. 2013. Optical properties of biological tissues: A review. Phys. Med. Biol. 58: R37. doi: 10.1088/0031-9155/58/11/R37.
- Kawano, F., Suzuki, H., Furuya, A., and Sato, M. 2015. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. 6: 6256. doi: 10.1038/ncomms7256.
- Kennedy, M.J., Hughes, R.M., Peteya, L.A., Schwartz, J.W., Ehlers, M.D., and Tucker, C.L. 2010. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7: 973-975. doi: 10.1038/nmeth.1524.
- Kosugi, S., Hasebe, M., Entani, T., Takayama, S., Tomita, M., and Yanagawa, H. 2008. Design of peptide inhibitors for the importin alpha/beta nuclear import pathway by activity-based profiling. Chem. Biol. 15: 940-949.
- Kosugi, S., Hasebe, M., Tomita, M., and Yanagawa, H. 2009a. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl. Acad. Sci. U.S.A. 106: 10171-10176. doi: 10.1073/pnas.0900604106.
- Kosugi, S., Hasebe, M., Matsumura, N., Takashima, H., Miyamoto-Sato, E., Tomita, M., and Yanagawa, H. 2009b. Six classes of nuclear localization signals specific to different binding grooves of importin alpha. J. Biol. Chem. 284: 478-485.
- Kosugi, S., Yanagawa, H., Terauchi, R., and Tabata, S. 2014. NESmapper: Accurate prediction of leucine-rich nuclear export signals using activity-based profiles. PLoS Comput. Biol. 10: e1003841. doi: 10.1371/journal.pcbi.1003841.
- Levskaya, A., Weiner, O.D., Lim, W.A., and Voigt, C.A. 2009. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461: 997-1001. doi: 10.1038/nature08446.
- Lungu, O.I., Hallett, R.A., Choi, E.J., Aiken, M.J., Hahn, K.M., and Kuhlman, B. 2012. Designing photoswitchable peptides using the AsLOV2 domain. Chem. Biol. 19: 507-517. doi: 10.1016/j.chembiol.2012.02.006.
- Macara, I.G. 2001. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65: 570-594. doi: 10.1128/MMBR.65.4.570-594.2001.
- Nakai, K. and Horten, P. 1999. PSORT: A program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. 24: 34-36. doi: 10.1016/S0968-0004(98)01336-X.
- Nihongaki, Y., Kawano, F., Nakajima, T., and Sato, M. 2015. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33: 755-760. doi: 10.1038/nbt.3245.
- Nikolaev, A.Y., Li, M., Puskas, N., Qin, J., and Gu, W. 2003. Parc: A cytoplasmic anchor for p53. Cell 112: 29-40. doi: 10.1016/S0092-8674(02)01255-2.
- Niopek, D., Benzinger, D., Roensch, J., Draebing, T., Wehler, P., Eils, R., and Di Ventura, B. 2014. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat. Commun. 5: 4404. doi: 10.1038/ncomms5404.
- Niopek, D., Wehler, P., Roensch, J., Eils, R., and Di Ventura, B. 2016. Optogenetic control of nuclear protein export. Nat. Commun. 7: 10624.
- Peter, E., Dick, B., and Baeurle, S.A. 2010. Mechanism of signal transduction of the LOV2-Jα photosensor from Avena sativa . Nat. Commun. 1: 122. doi: 10.1038/ncomms1121.
- Purvis, J.E. and Lahav, G. 2013. Encoding and decoding cellular information through signaling dynamics. Cell 152: 945-956. doi: 10.1016/j.cell.2013.02.005.
- Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22: 1567-1572. doi: 10.1038/nbt1037.
- Stevens, K.E. and Mann, R.S. 2007. A balance between two nuclear localization sequences and a nuclear export sequence governs extradenticle subcellular localization. Genetics 175: 1625-1636. doi: 10.1534/genetics.106.066449.
- Strickland, D., Yao, X., Gawlak, G., Rosen, M.K., Gardner, K.H., and Sosnick, T.R. 2010. Rationally improving LOV domain-based photoswitches. Nat. Methods 7: 623-626. doi: 10.1038/nmeth.1473.
- Strickland, D., Lin, Y., Wagner, E., Hope, C.M., Zayner, J., Antoniou, C., Sosnick, T.R., Weiss, E.L., and Glotzer, M. 2012. TULIPs: Tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9: 379-384. doi: 10.1038/nmeth.1904.
- Swartz, T.E., Corchnoy, S.B., Christie, J.M., Lewis, J.W., Szundi, I., Briggs, W.R., and Bogomolni, R.A. 2001. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J. Biol. Chem. 276: 36493-36500. doi: 10.1074/jbc.M103114200.
- Tolwinski, N.S. and Wieschaus, E. 2001. Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear anchor dTCF/Pan. Development 128: 2107-2117.
-
Tucker, C.L., Vrana, J.D., and Kennedy, M.J.
2014. Tools for controlling protein interactions using light. Curr
. Protoc. Cell Biol.
64: 17.16.1-17.16.20.
10.1002/0471143030.cb1716s64 Google Scholar
- Yang, X., Jost, A.P.-T., Weiner, O.D., and Tang, C. 2013. A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast. Mol. Biol. Cell 24: 2419-2430. doi: 10.1091/mbc.E13-03-0126.
- Yao, X., Rosen, M.K., and Gardner, K.H. 2008. Estimation of available free energy in a LOV2-Jα photoswitch. Nat. Chem. Biol. 4: 491-497. doi: 10.1038/nchembio.99.
- Yumerefendi, H., Dickinson, D.J., Wang, H., Zimmerman, S.P., Bear, J.E., Goldstein, B., Hahn, K., and Kuhlman, B. 2015. Control of protein activity and cell fate specification via light-mediated nuclear translocation. PloS One 10: e0128443. doi: 10.1371/journal.pone.0128443.
- Zayner, J.P., Antoniou, C., and Sosnick, T.R. 2012. The amino-terminal helix modulates light-activated conformational changes in AsLOV2. J. Mol. Biol. 419: 61-74. doi: 10.1016/j.jmb.2012.02.037.
Key Reference
- Niopek et al. (2014). See above.
Primary publication describing how LINuS was engineered and how nuclear protein import is quantitatively regulated by choosing appropriate illumination conditions, LINuS variants, and/or LOV2 mutants. This publication also showcases the utility of LINuS for cell biological applications by controlling entry into mitosis and gene expression with blue light.
