Abstract
Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.
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Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141(7):1117–1134. https://doi.org/10.1016/j.cell.2010.06.011
Neben CL, Lo M, Jura N, Klein OD (2019) Feedback regulation of RTK signaling in development. Dev Biol 447(1):71–89. https://doi.org/10.1016/j.ydbio.2017.10.017
Simi A, Ibáñez CF (2010) Assembly and activation of neurotrophic factor receptor complexes. Dev Neurobiol 70(5):323–331. https://doi.org/10.1002/dneu.20773
Casaletto JB, McClatchey AI (2012) Spatial regulation of receptor tyrosine kinases in development and cancer. Nat Rev Cancer 12(6):387–400. https://doi.org/10.1038/nrc3277
Robertson SC, Tynan JA, Donoghue DJ (2000) RTK mutations and human syndromes: when good receptors turn bad. Trends Genet 16(6):265–271. https://doi.org/10.1016/S0168-9525(00)02021-7
Goglia AG, Toettcher JE (2019) A bright future: optogenetics to dissect the spatiotemporal control of cell behavior. Curr Opin Chem Biol 48:106–113. https://doi.org/10.1016/j.cbpa.2018.11.010
Guglielmi G, Falk HJ, De Renzis S (2016) Optogenetic control of protein function: from intracellular processes to tissue morphogenesis. Trends Cell Biol 26(11):864–874. https://doi.org/10.1016/j.tcb.2016.09.006
Grusch M, Schelch K, Riedler R, Reichhart E, Differ C, Berger W, Ingles-Prieto A, Janovjak H (2014) Spatio-temporally precise activation of engineered receptor tyrosine kinases by light. EMBO J 33(15):1713–1726. https://doi.org/10.15252/embj.201387695
Kim N, Kim Jin M, Lee M, Kim Cha Y, Chang K-Y, Heo Won D (2014) Spatiotemporal control of fibroblast growth factor receptor signals by blue light. Chem Biol 21(7):903–912. https://doi.org/10.1016/j.chembiol.2014.05.013
Chang KY, Woo D, Jung H, Lee S, Kim S, Won J, Kyung T, Park H, Kim N, Yang HW, Park JY, Hwang EM, Kim D, Heo WD (2014) Light-inducible receptor tyrosine kinases that regulate neurotrophin signalling. Nat Commun 5:4057. https://doi.org/10.1038/ncomms5057
Bugaj LJ, Spelke DP, Mesuda CK, Varedi M, Kane RS, Schaffer DV (2015) Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering. Nat Commun 6:6898. https://doi.org/10.1038/ncomms7898
Duan L, Hope JM, Guo S, Ong Q, François A, Kaplan L, Scherrer G, Cui B (2018) Optical activation of TrkA signaling. ACS Synth Biol 7(7):1685–1693. https://doi.org/10.1021/acssynbio.8b00126
Kainrath S, Stadler M, Reichhart E, Distel M, Janovjak H (2017) Green-light-induced inactivation of receptor signaling using cobalamin-binding domains. Angew Chem Int Ed 56(16):4608–4611. https://doi.org/10.1002/anie.201611998
Reichhart E, Ingles-Prieto A, Tichy A-M, McKenzie C, Janovjak H (2016) A phytochrome sensory domain permits receptor activation by red light. Angew Chem Int Ed 55(21):6339–6342. https://doi.org/10.1002/anie.201601736
Li Y, Lee M, Kim N, Wu G, Deng D, Kim JM, Liu X, Heo WD, Zi Z (2018) Spatiotemporal control of TGF-β signaling with light. ACS Synth Biol 7(2):443–451. https://doi.org/10.1021/acssynbio.7b00225
Ramachandran A, Vizán P, Das D, Chakravarty P, Vogt J, Rogers KW, Müller P, Hinck AP, Sapkota GP, Hill CS (2018) TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition. ELife 7:e31756. https://doi.org/10.7554/eLife.31756
Sako K, Pradhan SJ, Barone V, Inglés-Prieto Á, Müller P, Ruprecht V, Čapek D, Galande S, Janovjak H, Heisenberg C-P (2016) Optogenetic control of nodal signaling reveals a temporal pattern of nodal signaling regulating cell fate specification during gastrulation. Cell Rep 16(3):866–877. https://doi.org/10.1016/j.celrep.2016.06.036
Muthuswamy SK, Gilman M, Brugge JS (1999) Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers. Mol Cell Biol 19(10):6845–6857. https://doi.org/10.1128/MCB.19.10.6845
Polyethylenimine (PEI), linear (1 mg/mL) (2008). Cold Spring Harb Protoc 2008 (3):pdb.rec11323. doi:https://doi.org/10.1101/pdb.rec11323
Baker JM, Boyce FM (2014) High-throughput functional screening using a homemade dual-glow luciferase assay. J Vis Exp 88. https://doi.org/10.3791/50282
Watanabe N, Mitchison TJ (2002) Single-molecule speckle analysis of actin filament turnover in lamellipodia. Science 295(5557):1083–1086. https://doi.org/10.1126/science.1067470
Ingles-Prieto A, Reichhart E, Muellner MK, Nowak M, Nijman SM, Grusch M, Janovjak H (2015) Light-assisted small-molecule screening against protein kinases. Nat Chem Biol 11(12):952–954. https://doi.org/10.1038/nchembio.1933
Bae JH, Boggon TJ, Tome F, Mandiyan V, Lax I, Schlessinger J (2010) Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells. Proc Natl Acad Sci U S A 107(7):2866–2871. https://doi.org/10.1073/pnas.0914157107
Takahashi F, Yamagata D, Ishikawa M, Fukamatsu Y, Ogura Y, Kasahara M, Kiyosue T, Kikuyama M, Wada M, Kataoka H (2007) AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles. Proc Natl Acad Sci U S A 104(49):19625–19630. https://doi.org/10.1073/pnas.0707692104
Strauss HM, Schmieder P, Hughes J (2005) Light-dependent dimerisation in the N-terminal sensory module of cyanobacterial phytochrome 1. FEBS Lett 579(18):3970–3974. https://doi.org/10.1016/j.febslet.2005.06.025
Jost M, Fernandez-Zapata J, Polanco MC, Ortiz-Guerrero JM, Chen PY, Kang G, Padmanabhan S, Elias-Arnanz M, Drennan CL (2015) Structural basis for gene regulation by a B12-dependent photoreceptor. Nature 526(7574):536–541. https://doi.org/10.1038/nature14950
Welm BE, Freeman KW, Chen M, Contreras A, Spencer DM, Rosen JM (2002) Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland. J Cell Biol 157(4):703–714. https://doi.org/10.1083/jcb.200107119
Mohammadi M, Dikic I, Sorokin A, Burgess WH, Jaye M, Schlessinger J (1996) Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol Cell Biol 16(3):977–989
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Kainrath, S., Janovjak, H. (2020). Design and Application of Light-Regulated Receptor Tyrosine Kinases. In: Niopek, D. (eds) Photoswitching Proteins . Methods in Molecular Biology, vol 2173. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0755-8_16
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DOI: https://doi.org/10.1007/978-1-0716-0755-8_16
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