Abstract
Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.
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References
Shcherbakova DM, Cox Cammer N, Huisman TM, Verkhusha VV, Hodgson L (2018) Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat Chem Biol 14(6):591–600. https://doi.org/10.1038/s41589-018-0044-1
Shcherbakova DM, Baloban M, Emelyanov AV, Brenowitz M, Guo P, Verkhusha VV (2016) Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nat Commun 7:12405. https://doi.org/10.1038/ncomms12405
Pertz O, Hodgson L, Klemke RL, Hahn KM (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440(7087):1069–1072
Hodgson L, Spiering D, Sabouri-Ghomi M, Dagliyan O, DerMardirossian C, Danuser G, Hahn KM (2016) FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions. Nat Chem Biol 12(10):802–809. https://doi.org/10.1038/nchembio.2145
Longo PA, Kavran JM, Kim MS, Leahy DJ (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol 529:227–240. https://doi.org/10.1016/B978-0-12-418687-3.00018-5
Hodgson L, Pertz O, Hahn KM (2008) Design and optimization of genetically encoded fluorescent biosensors: GTPase biosensors. Methods Cell Biol 85:63–81
Spiering D, Bravo-Cordero JJ, Moshfegh Y, Miskolci V, Hodgson L (2013) Quantitative ratiometric imaging of FRET-biosensors in living cells. Methods Cell Biol 114:593–609. https://doi.org/10.1016/B978-0-12-407761-4.00025-7
Bravo-Cordero JJ, Moshfegh Y, Condeelis J, Hodgson L (2013) Live cell imaging of RhoGTPase biosensors in tumor cells. Methods Mol Biol 1046:359–370. https://doi.org/10.1007/978-1-62703-538-5_22
Spiering D, Hodgson L (2012) Multiplex imaging of rho family GTPase activities in living cells. Methods Mol Biol 827:215–234. https://doi.org/10.1007/978-1-61779-442-1_15
Donnelly SK, Cabrera R, Mao SPH, Christin JR, Wu B, Guo W, Bravo-Cordero JJ, Condeelis JS, Segall JE, Hodgson L (2017) Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation. J Cell Biol 216(12):4331–4349. https://doi.org/10.1083/jcb.201704048
Miskolci V, Wu B, Moshfegh Y, Cox D, Hodgson L (2016) Optical tools to study the isoform-specific roles of small GTPases in immune cells. J Immunol 196(8):3479–3493. https://doi.org/10.4049/jimmunol.1501655
Moshfegh Y, Bravo-Cordero JJ, Miskolci V, Condeelis J, Hodgson L (2014) A trio-Rac1-Pak1 signalling axis drives invadopodia disassembly. Nat Cell Biol 16(6):574–586. https://doi.org/10.1038/ncb2972
Hanna S, Miskolci V, Cox D, Hodgson L (2014) A new genetically encoded single-chain biosensor for Cdc42 based on FRET, useful for live-cell imaging. PLoS One 9(5):e96469. https://doi.org/10.1371/journal.pone.0096469
Zawistowski J, Sabouri-Ghomi M, Danuser G, Hahn K, Hodgson L (2013) A RhoC biosensor reveals differences in the activation kinetics of RhoA and RhoC in migrating cells. Plos One 8(11):e79877
Hodgson L, Shen F, Hahn K (2010) Biosensors for characterizing the dynamics of rho family GTPases in living cells. Curr Protoc Cell Biol Chapter 14:Unit 14.11.1–Unit 14.1126
Shen F, Hodgson L, Rabinovich A, Pertz O, Hahn K, Price JH (2006) Functional proteometrics for cell migration. Cytometry A 69(7):563–572. https://doi.org/10.1002/cyto.a.20283
Hodgson L, Nalbant P, Shen F, Hahn K (2006) Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol 406:140–156
Kostenbader KD Jr, Cliver DO (1983) Membrane filter evaluations using poliovirus. J Virol Methods 7(5–6):253–257
Wallis C, Henderson M, Melnick JL (1972) Enterovirus concentration on cellulose membranes. Appl Microbiol 23(3):476–480
Beer C, Meyer A, Muller K, Wirth M (2003) The temperature stability of mouse retroviruses depends on the cholesterol levels of viral lipid shell and cellular plasma membrane. Virology 308(1):137–146
Wu B, Miskolci V, Sato H, Tutucci E, Kenworthy CA, Donnelly SK, Yoon YJ, Cox D, Singer RH, Hodgson L (2015) Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev 29(8):876–886. https://doi.org/10.1101/gad.259358.115
Shen F, Hodgson L, Hahn K (2006) Digital autofocus methods for automated microscopy. Methods Enzymol 414:620–632
Shen F, Hodgson L, Price JH, Hahn KM (2008) Digital differential interference contrast autofocus for high-resolution oil-immersion microscopy. Cytometry A 73(7):658–666
Crocker JC, Grier DG (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179:298–310
Del Pozo MA, Kiosses WB, Alderson NB, Meller N, Hahn KM, Schwartz MA (2002) Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat Cell Biol 4(3):232–239
Acknowledgments
This work was supported by National Institutes of Health grant GM136226 to LH. LH is an Irma T. Hirschl Career Scientist. RMB was supported by the Mamaroneck High School Original Science Research Program. MGW was supported by the Einstein-Montefiore Summer High School Research Program of the Albert Einstein College of Medicine, Graduate Division of Biomedical Sciences. NIR-Rac1 FRET biosensor was originally engineered, in part, by contributions from Tsipora M. Huisman, MD, and Natasha Cox Cammer [1]. Biosensor cDNA constructs and Matlab codes to enable processing of FRET data are available upon request.
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Bhalla, R.M., Hülsemann, M., Verkhusha, P.V., Walker, M.G., Shcherbakova, D.M., Hodgson, L. (2021). Multiplex Imaging of Rho GTPase Activities in Living Cells. In: Zamir, E. (eds) Multiplexed Imaging. Methods in Molecular Biology, vol 2350. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1593-5_4
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DOI: https://doi.org/10.1007/978-1-0716-1593-5_4
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