Correlative Förster Resonance Electron Transfer‐Proximity Ligation Assay (FRET‐PLA) Technique for Studying Interactions Involving Membrane Proteins

Daniel Ivanusic1, Joachim Denner1, Norbert Bannert1

1 Robert Koch Institute, HIV and Other Retroviruses, Berlin
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.17
DOI:  10.1002/cpps.10
Online Posting Date:  August, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit provides a guide and detailed protocol for studying membrane protein‐protein interactions (PPI) using the acceptor‐sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET‐PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET‐PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.

Keywords: FRET; membrane proteins; NFRET; PLA; proximity ligation assay; protein‐protein interaction; sensitized emission

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Design of Plasmids Expressing Donor and Acceptor Proteins
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Design of Plasmids Expressing Donor and Acceptor Proteins

  Materials
  • HEK293T cells (ATCC)
  • DMEM (Gibco brand, Thermo Fisher Scientific)
  • FBS (Biochrom)
  • PBS without Ca2+/Mg2+ (Gibco brand, Thermo Fisher Scientific)
  • Purified DNA plasmid: pCMV‐CD63‐YFP, pCMV‐gp41‐CFP
  • Kanamycin (Sigma Aldrich)
  • Ampicillin (Carl Roth)
  • MetafectenePro (Biontex)
  • Maxi plasmid kit (Qiagen)
  • Paraformaldehyde (PFA; Carl Roth)
  • Trypsin/EDTA (Biochrom)
  • Saponin (Carl Roth)
  • p‐Phenylenediamine (Sigma Aldrich)
  • Glycerol (Carl Roth)
  • Nail polish (clear; Catrice cosmetics)
  • PLA kits and primary antibodies:
  • Duolink II in situ PLA Probe anti‐mouse PLUS (Eurogentec)
  • Duolink II in situ PLA Probe anti‐goat MINUS (Eurogentec)
  • Duolink II in situ PLA detection red reagents (Eurogentec)
  • Goat anti‐FLAG NB600‐344 (NovusBio)
  • Mouse anti‐V5 MCA1360 (Serotec)
  • DuolinkII antibody diluent buffer (Eurogentec)
  • Ligation mix (8 µl DuolinkII ligation buffer, 31 µl water, and 1 µl ligase)
  • Amplification mix (8 μl 5× Duolink amplification stock, 31.5 μl water, and 0.5 μl polymerase)
  • Wash buffer A (aqueous solution 0.01 M Tris, 0.15 M NaCl, and 0.05% Tween 20; adjust to pH 7.4; filter to avoid microbial contamination through a 0.2‐µm filter; and store at 4°C for up to 2 weeks)
  • Wash buffer B (aqueous solution 0.2 M Tris and 0.1 M NaCl; adjust to pH 7.5; filter to avoid microbial contamination through a 0.2‐µm filter; and store at 4°C for up to 2 weeks)
  • Wash buffer C (10 ml wash buffer B plus 990 ml water; filter to avoid microbial contamination through a 0.2‐µm filter; and store at 4°C for up to 2 weeks)
  • Mounting medium (glycerol containing 0.1% p‐phenylenediamine)
  • Blocking solution (lyophilized normal donkey serum [Jackson ImmunoResearch] reconstituted in water and diluted to 30% in PBS)
  • 6‐well plates (TPP)
  • Polysine microscope adhesion slides (Thermo Fisher Scientific)
  • Cover slip (Carl Roth)
  • PAP pen (Sigma Aldrich)
  • Zeiss 780 inverted confocal laser scanning microscope with implemented Zeiss Zen software ZEN2010 (ZEISS)
  • HeNelaser 594 nm, diode laser 405 nm, Argon multiline laser, 458 nm, 488 nm, 514 nm (ZEISS)
  • Plan‐Apochromat 63 ×/1.4 Oil DIC II Zeiss immersion oil (ZEISS)
  • Reaction tubes, 1.5 ml volume (Carl Roth)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Arhel, N. and Kirchhoff, F. 2010. Host proteins involved in HIV infection: New therapeutic targets. Biochim. Biophys. Acta 1802:313‐321. doi: 10.1016/j.bbadis.2009.12.003.
  Arinaminpathy, Y., Khurana, E., Engelman, D.M., and Gerstein, M.B. 2009. Computational analysis of membrane proteins: The largest class of drug targets. Drug Discov. Today 14:1130‐1135. doi: 10.1016/j.drudis.2009.08.006.
  Berggard, T., Linse, S., and James, P. 2007. Methods for the detection and analysis of protein‐protein interactions. Proteomics 7:2833‐2842. doi: 10.1002/pmic.200700131.
  Broussard, J.A., Rappaz, B., Webb, D.J., and Brown, C.M. 2013. Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nat. Protoc. 8:265‐281. doi: 10.1038/nprot.2012.147.
  Brzostowski, J.A., Meckel, T., Hong, J., Chen, A., and Jin, T. 2009. Imaging protein‐protein interactions by Förster resonance energy transfer (FRET) microscopy in live cells. Curr. Protoc. Protein Sci. 56:19.5.1‐19.5.12. doi: 10.1002/0471140864.ps1905s56.
  Elder, A.D., Domin, A., Kaminski Schierle, G.S., Lindon, C., Pines, J., Esposito, A., and Kaminski, C.F. 2009. A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer‐sensitized fluorescence emission. J. R. Soc. Interface 6:S59‐S81. doi: 10.1098/rsif.2008.0381.focus.
  Förster, T. 1948. Zwischenmolekulare energiewanderung und fluoreszenz [Intermolecular energy migration and fluorescence]. Ann. Phys. 437:55‐75. doi: 10.1002/andp.19484370105.
  Frankel, A.D. and Young, J.A. 1998. HIV‐1: Fifteen proteins and an RNA. Annu. Rev. Biochem. 67:1‐25. doi: 10.1146/annurev.biochem.67.1.1.
  Fruh, K., Finlay, B., and McFadden, G. 2010. On the road to systems biology of host‐pathogen interactions. Future Microbiol. 5:131‐133. doi: 10.2217/fmb.09.130.
  Gajadhar, A. and Guha, A. 2010. A proximity ligation assay using transiently transfected, epitope‐tagged proteins: Application for in situ detection of dimerized receptor tyrosine kinases. BioTechniques 48:145‐152. doi: 10.2144/000113354.
  Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702‐2713. doi: 10.1016/S0006‐3495(98)77976‐7.
  Gustafsdottir, S.M., Schallmeiner, E., Fredriksson, S., Gullberg, M., Soderberg, O., Jarvius, M., Jarvius, J., Howell, M., and Landegren, U. 2005. Proximity ligation assays for sensitive and specific protein analyses. Anal. Biochem. 345:2‐9. doi: 10.1016/j.ab.2005.01.018.
  Ivanusic, D., Eschricht, M., and Denner, J. 2014. Investigation of membrane protein‐protein interactions using correlative FRET‐PLA. BioTechniques 57:188‐198. doi: 10.2144/000114215.
  Jarvius, M., Paulsson, J., Weibrecht, I., Leuchowius, K.J., Andersson, A.C., Wahlby, C., Gullberg, M., Botling, J., Sjoblom, T., Markova, B., Ostman, A., Landegren, U., and Soderberg, O. 2007. In situ detection of phosphorylated platelet‐derived growth factor receptor βusing a generalized proximity ligation method. Mol. Cell Proteomics 6:1500‐1509. doi: 10.1074/mcp.M700166‐MCP200.
  Klasener, K., Maity, P.C., Hobeika, E., Yang, J., and Reth, M. 2014. B cell activation involves nanoscale receptor reorganizations and inside‐out signaling by Syk. Elife 3:e02069. doi: 10.7554/eLife.02069.
  Kremers, G.J., Goedhart, J., van Munster, E.B., and Gadella, T.W. Jr. 2006. Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry 45:6570‐6580. doi: 10.1021/bi0516273.
  Maity, P.C., Yang, J., Klaesener, K., and Reth, M. 2015. The nanoscale organization of the B lymphocyte membrane. Biochim. Biophys. Acta 1853:830‐840. doi: 10.1016/j.bbamcr.2014.11.010.
  Müller, S.M., Galliardt, H., Schneider, J., Barisas, B.G., and Seidel, T. 2013. Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Front. Plant Sci. 4:413. doi: 10.3389/fpls.2013.00413.
  Piston, D.W. and Kremers, G.J. 2007. Fluorescent protein FRET: The good, the bad and the ugly. Trends Biochem. Sci. 32:407‐414 doi: 10.1016/j.tibs.2007.08.003.
  Sekar, R.B. and Periasamy, A. 2003. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160:629‐633. doi: 10.1083/jcb.200210140.
  Selvin, P.R. 2000. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7:730‐734. doi:10.1038/78948.
  Söderberg, O., Gullberg, M., Jarvius, M., Ridderstrale, K., Leuchowius, K.J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L.G., and Landegren, U. 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3:995‐1000. doi: 10.1038/nmeth947.
  Söderberg, O., Leuchowius, K.J., Kamali‐Moghaddam, M., Jarvius, M., Gustafsdottir, S., Schallmeiner, E., Gullberg, M., Jarvius, J., and Landegren, U. 2007. Proximity ligation: A specific and versatile tool for the proteomic era. In Genetic Engineering: Principles and Methods, Vol. 28 (J.K. Setlow, ed.) pp. 85‐93. Springer Science+Business Media, New York.
  Stryer, L. 1978. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47:819‐846. doi: 10.1146/annurev.bi.47.070178.004131.
  Szöllősi, J., Damjanovich, S., Nagy, P., Vereb, G., and Mátyus, L. 2006. Principles of resonance energy transfer. Curr. Protoc. Cytom. 38:1.12.1‐1.12.16. doi: 10.1002/0471142956.cy0112s38.
  Wallin, E. and von Heijne, G. 1998. Genome‐wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 7:1029‐1038. doi: 10.1002/pro.5560070420.
  Weibrecht, I., Leuchowius, K.J., Clausson, C.M., Conze, T., Jarvius, M., Howell, W.M., Kamali‐Moghaddam, M., and Söderberg, O. 2010. Proximity ligation assays: A recent addition to the proteomics toolbox. Exp. Rev. Proteomics 7:401‐409. doi: 10.1586/epr.10.10.
  Xia, Z. and Liu, Y. 2001. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81:2395‐2402. doi: 10.1016/S0006‐3495(01)75886‐9.
  Youvan, D.C., Silva, C.M., Bylina, E.J., Coleman, W.J., Dilworth, M.R., and Yang, M.M. 1997. Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads. Biotechnol. Alia 3:1‐18.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library