Abstract
Real-time noninvasive fluorescence-based protein assays enable a direct access to study interactions in their natural environment and hence overcome the limitations of other methods that rely on invasive cell disruption techniques. The determination of Förster resonance energy transfer (FRET) by means of fluorescence lifetime imaging microscopy (FLIM) is currently the most advanced method to observe protein–protein interactions at nanometer resolution inside single living cells and in real-time. In the FRET-FLIM approach, the information gained using steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction. The combination of confocal laser scanning microscopy with the sensitive advanced technique of time-correlated single photon counting allows the mapping of the spatial distribution of fluorescence lifetimes inside living cells on a pixel-by-pixel basis that is the same as the fluorescence image. Moreover, the use of multiphoton excitation particularly for plant cells provides further advantages such as reduced phototoxicity and photobleaching. In this protocol, we briefly describe the instrumentation and experimental design to study protein interactions within the plant endomembrane system, with a focus on the imaging of plant cells expressing fluorescent proteins and acquisition and analysis of fluorescence lifetime resolved data.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Förster T (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann Phys 437:55–75
Becker W (2012) Fluorescence lifetime imaging-techniques and applications. J Microsc 247:119–136
Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R (2005) Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc Natl Acad Sci U S A 102:3135–3140
Stubbs CD, Botchway SW, Slater SJ, Parker AW (2005) The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells. BMC Cell Biol 6:22
Adjobo-Hermans MJ, Goedhart J, Gadella TW (2006) Plant G protein heterotrimers require dual lipidation motifs of Galpha and Ggamma and do not dissociate upon activation. J Cell Sci 119:5087–5097
Aker J, Hesselink R, Engel R, Karlova R, Borst JW, Visser AJ, de Vries SC (2007) In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol 145:339–350
Osterrieder A, Carvalho CM, Latijnhouwers M, Johansen JN, Stubbs C, Botchway S, Hawes C (2009) Fluorescence lifetime imaging of interactions between Golgi tethering factors and small GTPases in plants. Traffic 10:1034–1046
Sparkes I, Tolley N, Aller I, Svozil J, Osterrieder A, Botchway S, Mueller C, Frigerio L, Hawes C (2010) Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties. Plant Cell 22:1333–1343
Crosby KC, Pietraszewska-Bogiel A, Gadella TW, Winkel BS (2011) Förster resonance energy transfer demonstrates a flavonoid metabolon in living plant cells that displays competitive interactions between enzymes. FEBS Lett 585:2193–2198
Berendzen KW, Böhmer M, Wallmeroth N, Peter S, Vesi M, Zhou Y, Tiesler FK, Schleifenbaum F, Harter K (2012) Screening for in planta protein–protein interactions combining bimolecular fluorescence complementation with flow cytometry. Plant Methods 8(1):25
Yadav RB, Burgos P, Parker AW, Iadevaia V, Proud CG, Allen RA, O‘Connell JP, Jeshtadi A, Stubbs CD, Botchway SW (2013) mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging. BMC Cell Biol 14:3
Schoberer J, Liebminger E, Botchway SW, Strasser R, Hawes C (2013) Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta. Plant Physiol 161:1737–1754
Runions J, Hawes C, Kurup S (2007) Fluorescent protein fusions for protein localization in plants. Methods Mol Biol 390:239–255
Schoberer J, Vavra U, Stadlmann J, Hawes C, Mach L, Steinkellner H, Strasser R (2009) Arginine/lysine residues in the cytoplasmic tail promote ER export of plant glycosylation enzymes. Traffic 10:101–115
Sparkes I, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025
Brandizzi F, Snapp E, Roberts A, Lippincott-Schwartz J, Hawes C (2002) Membrane protein transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching. Plant Cell 14:1293–1309
Sun Y, Day RN, Periasamy A (2011) Investigating protein–protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc 6:1324–1340
Becker W, Su B, Bergmann A (2009) Fast-acquisition multispectral FLIM by parallel TCSPC. Proceedings of the SPIE 7183, multiphoton microscopy in the biomedical sciences IX, 718305
Weber G, Teale FWJ (1957) Determination of the absolute quantum yield of fluorescent solutions. Trans Faraday Soc 53:646–655
Acknowledgements
This work was funded by the Austrian Science Fund (FWF): J2981-B20 (to J.S.), and Oxford Brookes University. Access to the Central Laser Facility, Rutherford Appleton Laboratory, was funded by a Science and Technology Facilities Council Program Access grant. We thank Chris Hawes for carefully reading the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Schoberer, J., Botchway, S.W. (2014). Investigating Protein–Protein Interactions in the Plant Endomembrane System Using Multiphoton-Induced FRET-FLIM. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 1209. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1420-3_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1420-3_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1419-7
Online ISBN: 978-1-4939-1420-3
eBook Packages: Springer Protocols