Abstract
Optogenetic modules that use genetically encoded elements to control protein function in response to light allow for precise spatiotemporal modulation of signaling pathways. As one of optical approaches, LARIAT (Light-Activated Reversible Inhibition by Assembled Trap) is a unique light-inducible inhibition system that reversibly sequesters target proteins into clusters, generated by multimeric proteins and a blue light-induced heterodimerization module. Here we present a method based on LARIAT for optical inhibition of targets in living mammalian cells. In the protocol, we focus on the inhibition of proteins that modulate cytoskeleton and cell cycle, and describe how to transfect, conduct a photo-stimulation, and analyze the data.
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References
Doupe DP, Perrimon N (2014) Visualizing and manipulating temporal signaling dynamics with fluorescence-based tools. Sci Signal 7(319):1
Turgeon B, Meloche S (2009) Interpreting neonatal lethal phenotypes in mouse mutants: insights into gene function and human diseases. Physiol Rev 89(1):1–26
Stockwell BR (2004) Exploring biology with small organic molecules. Nature 432(7019):846–854
Zhou P (2005) Targeted protein degradation. Curr Opin Chem Biol 9(1):51–55
Banaszynski LA, Wandless TJ (2006) Conditional control of protein function. Chem Biol 13(1):11–21
Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn KM (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461(7260):104–108
Levskaya A, Weiner OD, Lim WA, Voigt CA (2009) Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461(7266):997–1001
Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, Tucker CL (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods 7(12):973–975
Bugaj LJ, Choksi AT, Mesuda CK, Kane RS, Schaffer DV (2013) Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 10(3):249–252
Lee S, Park H, Kyung T, Kim NY, Kim S, Kim J, Heo WD (2014) Reversible protein inactivation by optogenetic trapping in cells. Nat Methods 11(6):633–636
Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC, Leonhardt H (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3(11):887–889
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, Sixt M, Wedlich-Soldner R (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5(7):605–607
Shen K, Meyer T (1998) In vivo and in vitro characterization of the sequence requirement for oligomer formation of Ca2+/calmodulin-dependent protein kinase IIalpha. J Neurochem 70(1):96–104
Rosenberg OS, Deindl S, Sung RJ, Nairn AC, Kuriyan J (2005) Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme. Cell 123(5):849–860
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909
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Park, H., Lee, S., Heo, W.D. (2016). Protein Inactivation by Optogenetic Trapping in Living Cells. In: Kianianmomeni, A. (eds) Optogenetics. Methods in Molecular Biology, vol 1408. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3512-3_25
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DOI: https://doi.org/10.1007/978-1-4939-3512-3_25
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3512-3
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