We would like to thank the Neuroscience Imaging Service at Stanford University School of Medicine for providing equipment and space for this project. This research was supported by intramural funding of the Stanford Cancer Institute and the Gynecologic Oncology Division Stanford as well as GINOP-2.3.2-15-2016-00026, GINOP-2.3.3-15-2016-00030, NN129371, ANN135107 from the National Research, Development and Innovation Office, Hungary.

1. Plasmid construction
<ol>
	<li>For generating the eGFP-mCherry1 fusion probe, use an N1 mammalian cell expression vector (see Table of Materials) with mCherry110 inserted using the restriction sites AgeI and BsrGI.</li>
	<li>Use the following oligonucleotides to amplify eGFP11 without a stop codon as SalI-BamHI fragment: N-terminal primer 5&#39;-AAT TAA CAG TCG ACG ATG GTG AGC AAG GGC GAG G 3&#39; and C-terminal primer 5&#39;-AAT ATA TGG ATC CCG CTT GTA CAG CTC GTC CAT GC 3&#39;.</li>
	<li>Insert this SalI-BamHI fragment into the multiple cloning site of the N1 vector to introduce RNPPV linker (five amino-acid) linker between the green and red fluorescent protein. 
	NOTE: This linker yields a mean FRET efficiency for the GFP-Cherry donor-acceptor pair of about 0.25 -0.3 (Figure 1A). The choice of stiff12 and helical13 linkers of varying lengths to scale measured FRET efficiencies has been discussed elsewhere but is not required for our purpose of the fusion protein. Going forward for simplicity we will call the fluorescent proteins &#39;GFP&#39; and &#39;Cherry&#39;.</li>
</ol>
2. Cell culture and transfection
<ol>
	<li>Use any cell line, e.g., NRK cells, for FRET experiments in media, e.g., Dulbecco&#39;s modified Eagle&#39;s media (DMEM),&#160;without phenol red, to reduce background fluorescence. For the same reason, the usage of phenol red free trypsin is advised.</li>
	<li>Once cells are 80% confluent, detach cells with 1 mL of 0.05% trypsin-EDTA, count the number of cells in suspension using a Neubauer chamber and seed about 10,000 cells per well of an 8-well chambered cover glass; alternatively, from a confluent cell culture grown in T25 flask, use 1 drop of cell suspension from a 2 mL pipette or 3 drops from a 5-mL cell suspension from a confluent culture grown in a T 12.5-cell culture flask.</li>
	<li>Grow cells in 8-well chambers (0.8 cm2/ well) with #1.0 cover glass for fluorescence live-cell microscopy at standard cell culture conditions (37&#176;C and 5% CO2).</li>
	<li>24 h after plating transfect the cells using an appropriate commercially available transfection media (see Table of Materials), with GFP, Cherry, GFP/ Cherry mix (1:1 mix, i.e., 0.8 &#956;g and 0.8 &#956;g GFP and Cherry plasmid DNA), and the GFP-Cherry chimera.
	<ol>
		<li>For transfection, use 5 &#956;L of the transfection reagent in 45 &#956;L of DMEM and 1.6 &#956;g of plasmid DNA. Stir by gently flicking the microcentrifuge tube.</li>
		<li>After 15 min incubation of the mix at room temperature, add 1-2 &#956;L of the transfection reagent mixture to each well of the 8-well chamber slide. Return the chambered cover glass to the incubator.</li>
	</ol>
	</li>
	<li>Let 20 h after transfection elapse before live-cell imaging, to allow for proper fluorescent protein expression, folding and maturation, especially of the red fluorophore.</li>
</ol>
3. FRET Imaging
<ol>
	<li>Image transfected cells in a humidified and heated environmental chamber at 37 &#176;C. To buffer the cell media at physiological pH, use CO2 gas set to 5% flow, or add 20 mM HEPES to render the cell media CO2-independent.</li>
	<li>Use a confocal laser scanning microscope. Set the excitation and emission as follows to optimize the signal and minimize cross-talk.
	<ol>
		<li>Use the 488-nm line of the argon ion laser to excite GFP and the 561-nm diode pumped solid state laser (or 543-nm Helium Neon laser, depending upon available laser lines) to excite Cherry.</li>
		<li>Set the following in the software of a commercial confocal microscope. Set the Dichroic mirror to 488/ 561 by button click using the pull-down menu. Collect fluorescence using 488-nm laser light for excitation in channel 1 through an emission band of 505 - 530 nm (or 505 - 550 nm) and in channel 2 with a long pass filter &#62;585 nm and use the 561-nm laser light for excitation in channel 3 with a long pass filter &#62; 585 nm (type in wavelengths). Band pass filters e.g., 590 - 650 nm or similar can also be used which have the advantage of excluding Raman-scattering.</li>
	</ol>
	</li>
	<li>Excite with the two lasers sequentially and set the imaging mode to Switch after each line so that the excitation of the 512 x 512 pixels image alternates after each line (and not after each frame which would abrogate the ability to detect FRET due to diffusion of the labeled proteins while recording the images with different excitations; button click).</li>
	<li>Set up a mini-time series of three images by button clicks to detect if significant photobleaching occurs, and potentially reduce the laser power. Photobleaching of less than 1% is optimal. High laser intensity can also lead to absorption saturation reducing the apparent FRET efficiency14. Laser power, up to 10-20 &#956;W measured at the objective lens are safe to use.</li>
	<li>First, image cells expressing the GFP-Cherry fusion construct. Set the parameters that define the time-integrated laser intensity per pixel in a confocal image, i.e., the pixel dwell time in microseconds, the acousto-optical tunable filter (AOTF) transmission in percent, and the zoom.
	<ol>
		<li>Image cells using a 63x oil objective and Zoom set to 3x. This provides sufficient magnification and resolution to image cells in its entirety. Aim for a pixel size of 70-80 nm.</li>
		<li>Set Pixel dwell time to 2-4 &#956;s and AOTF transmission for the 488-nm and 561-nm laser such that images have a good signal-to-noise ratio without bleaching and no pixels showing fluorescence intensity saturation. It is advantageous to adjust the laser power of 488 and 561 such that signal levels in channel 1 and channel 3 are similar.</li>
		<li>Set the Photomultiplier (averaging mode) gain to 600-800.</li>
	</ol>
	</li>
	<li>Image with these settings 15-20 cells expressing the GFP-Cherry fusion protein. 15-20 cells provide good statistics while keeping the total time of a FRET measurement session limited to a few hours to help ensure stability of the microscopic set-up.</li>
	<li>Image with the same settings cells expressing GFP, Cherry, GFP and Cherry and non-transfected cells. Search for expressing cells in the green channel or red channel, respectively.</li>
	<li>Then, image 15-20 cells co-expressing proteins of interest coupled to GFP and Cherry, respectively. Searching for expressing cells, avoid long exposure of the cells in order to not bleach the fluorescent proteins. Cherry has a lower photostability than GFP, and bleaching Cherry, the acceptor compromises FRET analysis. 
	NOTE: Absorption and emission spectra of GFP and Cherry is shown in Supplementary Figure 1. After measuring for 5-6 h, it is advisable to repeat imaging a few cells expressing the GFP-Cherry chimera at the end of the imaging session to document that the set-up remained stable and detected FRET efficiencies of the GFP-Cherry fusion protein did not significantly change during the course of an imaging session.</li>
</ol>
4. Image analysis for detecting absolute FRET efficiencies using donor quenching and sensitized emission
NOTE: Here, a practical step-by-step guide as to how to determine FRET efficiency with the use of the attached spreadsheet (Supplementary File 2) is provided. Theory and derivation of the presented equations can be found in detail in previous publications4,15,16,17. With the described settings, the following fluorescence intensities are collected.
<ol>
	<li>Measure the donor signal I1&#160;in channel 1, the donor channel, with 488-nm excitation and an emission band of 505-530 nm. 
	<img alt="Equation 2" src="/files/ftp_upload/62241/62241eq02.jpg" /> 
	where ID is the unquenched donor signal in channel 1 that would be measured in the absence of an acceptor, is the mean FRET efficiency, and B1 the average background signal in channel 1.</li>
	<li>Measure the acceptor signal I3 in channel 3, the acceptor channel, with 561-nm excitation and emission at &#62;585 nm. 
	<img alt="Equation 3" src="/files/ftp_upload/62241/62241eq03.jpg" /> 
	where IA is the acceptor signal and B3&#160;the background in channel 3.</li>
	<li>Measure the FRET signal in channel 2, the transfer channel, with 488-nm excitation and emission at &#62;585 nm. 
	<img alt="Equation 4" src="/files/ftp_upload/62241/62241eq04.jpg" /> 
	Where, the signal in channel 2 is a sum of four different components: (i) ID(1 - E)S1 is the spectral spill over from the quenched donor signal into the &#62;585 detection channel (with the cross-talk factor S1), (ii)&#160;IAS2 is the acceptor signal from the direct excitation by 488-nm light (with the cross-talk factor S2), (iii) IDE&#945; is the sensitized emission of the acceptor by FRET from the excited donor molecule (&#945; will be detailed further in 4.8. - 4.10.), and (iv) B2 is the background signal.</li>
	<li>Measure average background intensities in channels 1, 2, 3 in non-transfected or mock-transfected cells; either is fine with negligible difference. For all cell measurements, use the free-hand tool to delineate regions of interests and avoid perinuclear vesicles with increased autofluorescence. It is important to avoid significant autofluorescence from these perinuclear vesicles.
	<ol>
		<li>Enter the measurements into the columns X, Y, and Z of the provided spreadsheet. Average background intensities in the 3 channels are entered into A2, B2, and C2 of the excel spreadsheet (Supplementary File 2).</li>
	</ol>
	</li>
	<li>Measure average intensities in channels 1, 2, 3 of cells expressing GFP or Cherry alone, and enter the measurements into columns C, D, E, and N, O, P. Respective background intensities are subtracted (in F, G, H, and Q, R, S).</li>
	<li>In order to calculate E, the FRET efficiency, determine the cross-talk factors S1 and S2. The spectral cross-talk factor S1 is calculated from cells expressing only GFP 
	<img alt="Equation 6" src="/files/ftp_upload/62241/62241eq6v5.png" /> 
	in column I. Enter the mean value for S1 into cell D2 on the excel spreadsheet.</li>
	<li>Calculate the spectral cross-talk factor S2 from cells expressing only Cherry 
	<img alt="Equation 7" src="/files/ftp_upload/62241/62241eq07.jpg" /> 
	in column T. Enter the mean for S2 into cell E2 on the excel spreadsheet.</li>
	<li>Ensure that the &#945;&#160;factor relates the signal from any given number of excited GFP molecules in channel 1 to the signal of an equal number of excited Cherry molecules in channel 2, and is defined by 
	<img alt="Equation 8" src="/files/ftp_upload/62241/62241eq08.jpg" />&#160;&#160;&#160;<img alt="Equation 13" src="/files/ftp_upload/62241/62241eq13v5.png" /> 
	where QA and QD are the fluorescence quantum yields of Cherry and GFP; &#951;A and&#160;&#951;D the detection efficiencies of acceptor and donor fluorescence in channels 2 and 1, respectively. 
	NOTE: The &#945;&#160;factor could be determined from two samples expressing known absolute amounts of GFP and Cherry. It is, however, impossible to know the exact amount of GFP and Cherry expressed in a cell. Therefore, we calculated the factor by using cells that express the GFP-Cherry fusion protein. Here, while the absolute amount is still unknown, the ratio of donor and acceptor molecules is known to be one.</li>
	<li>Measure average intensities in channels 1, 2, 3 of cells expressing the GFP-Cherry fusion protein, and enter the measurements into the columns AE, AF, AG. Background intensities are subtracted (in AH, AI, AJ).</li>
	<li>Calculate the &#945;&#160;factor (AJ column) from the fluorescence intensities in channel 1, 2 and 3 of the GFP-Cherry fusion protein as follows: 
	<img alt="Equation 10" src="/files/ftp_upload/62241/62241eq10v6.png" /></li>
	<li>Background-corrected intensities measured in channel 1 (I1 - B1), 2 (I2 - B2) and 3 (I3 - B3), respectively, are measured using the GFP-Cherry chimera. The spectral cross talk factor S1 was determined using cells expressing GFP only (see 4.7.). &#949;D and &#949;A are the extinction coefficients of GFP, the donor, and Cherry, the acceptor, at 488 nm, and can be determined from the literature (&#949;GFP = 53,000 M-1cm-1)18 and the absorption curve of Cherry (&#949;Cherry &#8776; 5560 M-1cm1). The ratio&#160;&#160;<img alt="Equation 11" src="/files/ftp_upload/62241/62241eq11.jpg" />&#160;has been entered in cell G2 of the excel spreadsheet. Enter the mean value for the &#945; factor into J2.</li>
	<li>Use the determined &#945; factor for the calculation of the FRET efficiency, E, as follows (column AK): 
	<img alt="Equation 12" src="/files/ftp_upload/62241/62241eq12v6.png" /></li>
	<li>Alternatively, determine FRET efficiency, E, for the negative controls, i.e., the co-expression of GFP and Cherry and the expression of GFP alone by adding the measurements of channels 1, 2, and 3 in the excel sheet in column AD, AE, and AF under the GFP-Cherry fusion protein measurements. Determine FRET efficiencies between the GFP and Cherry labeled proteins of interest in the same way.</li>
	<li>Determine the unquenched donor intensity, ID as (I1 - B1)/(1 -&#160;E), and the acceptor intensity as IA = I3 - B3; these values are proportional to the expression levels of the tagged proteins.</li>
	<li>Determine the corrected acceptor-to-donor intensity ratio (Q) of the GFP-Cherry fusion protein as follows (column AL): 
	<img alt="Equation 13" src="/files/ftp_upload/62241/62241eq13v5.png" /></li>
	<li>For other co-transfected cells, calculate the acceptor-to-donor molecular ratio NA/ND as follows: 
	<img alt="Equation 14" src="/files/ftp_upload/62241/62241eq14.jpg" /> 
	NOTE: The rationale to determine the NA/ND&#160;ratio and plot the mean cellular FRET efficiency E versus NA/ND, is that one donor molecule can transfer energy to multiple acceptors while an acceptor molecule can only receive energy from one donor at a given time. Even if only one acceptor can interact with a donor because of the stoichiometry of the interaction, an increase of acceptor concentration is expected to increase the fraction of donors in complex with the acceptor because of the law of mass action. Thus, for a fixed (or narrow range of) donor expression, the FRET efficiency should rise with increasing NA/ND. When plotting the FRET efficiency E versus&#160;NA/ND for the co-expression of GFP and Cherry, i.e. the negative probe, however, an increase in NA/ND&#160;should not result in an increase in FRET efficiency (at least at sufficiently low acceptor concentrations where random FRET due to the vicinity of acceptor dyes to donor dyes does not occur).</li>
</ol>

<ol>
	<li>F&#246;rster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zwischenmolekulare+Energiewanderung+und+Fluoreszenz.">Zwischenmolekulare Energiewanderung und Fluoreszenz.</a> Annal der Physik. 437, 55-75 (1948).</li><li>Jovin, T. M., Arndt-Jovin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescence+digital+imaging+microscopy.">Luminescence digital imaging microscopy.</a> Annual Review of Biophysics and Biophysical Chemistry. 18, 271-308 (1989).</li><li>Mekler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photochemical+technique+to+enhance+sensitivity+of+detection+of+fluorescence+resonance+energy+transfer.">A photochemical technique to enhance sensitivity of detection of fluorescence resonance energy transfer.</a> Photochemistry and Photobiology. 59, 615-620 (1994).</li><li>Vamosi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+of+the+c-Fos/c-Jun+complex+in+vivo:+A+combined+FRET,+FCCS,+and+MD-modeling+study.">Conformation of the c-Fos/c-Jun complex in vivo: A combined FRET, FCCS, and MD-modeling study.</a> Biophysical Journal. 94 (7), 2859-2868 (2008).</li><li>Renz, M., Daniels, B. R., Vamosi, G., Arias, I. M., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+asialoglycoprotein+receptor+deciphered+by+ensemble+FRET+imaging+and+single-molecule+counting+PALM+imaging.">Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.</a> Proceedings of the National Academy of Science U. S. A. 109 (44), 2989-2997 (2012).</li><li>van Rheenen, J., Langeslag, M., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+confocal+acquisition+to+optimize+imaging+of+fluorescence+resonance+energy+transfer+by+sensitized+emission.">Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission.</a> Biophysical Journal. 86 (4), 2517-2529 (2004).</li><li>Muller, S. M., Galliardt, H., Schneider, J., Barisas, B. G., Seidel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Forster+resonance+energy+transfer+by+monitoring+sensitized+emission+in+living+plant+cells.">Quantification of Forster resonance energy transfer by monitoring sensitized emission in living plant cells.</a> Frontiers in Plant Sciences. 4, 413 (2013).</li><li>Gates, E. M., LaCroix, A. S., Rothenberg, K. E., Hoffman, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+quality,+reproducibility,+and+usability+of+FRET-based+tension+sensors.">Improving quality, reproducibility, and usability of FRET-based tension sensors.</a> Cytometry Part A. 95 (2), 201-213 (2019).</li><li>Menaesse, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+instrument+calibration+for+wide-field+fluorescence+resonance+energy+transfer+(FRET)+measured+by+the+sensitized+emission+method.">Simplified instrument calibration for wide-field fluorescence resonance energy transfer (FRET) measured by the sensitized emission method.</a> Cytometry Part A. , 24194 (2020).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22 (12), 1567-1572 (2004).</li><li>Cormack, B. P., Valdivia, R. H., Falkow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-optimized+mutants+of+the+green+fluorescent+protein+(GFP).">FACS-optimized mutants of the green fluorescent protein (GFP).</a> Gene. 173, 33-38 (1996).</li><li>Nagy, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+calibration+method+for+flow+cytometric+fluorescence+resonance+energy+transfer+measurements+between+visible+fluorescent+proteins.">Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.</a> Cytometry Part A. 67 (2), 86-96 (2005).</li><li>Arai, R., Ueda, H., Kitayama, A., Kamiya, N., Nagamune, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+the+linkers+which+effectively+separate+domains+of+a+bifunctional+fusion+protein.">Design of the linkers which effectively separate domains of a bifunctional fusion protein.</a> Protein Engineering. 14 (8), 529-532 (2001).</li><li>Szendi-Szatmari, T., Szabo, A., Szollosi, J., Nagy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+the+detrimental+effects+of+saturation+phenomena+in+FRET+microscopy.">Reducing the detrimental effects of saturation phenomena in FRET microscopy.</a> Analytical Chemistry. 91 (9), 6378-6382 (2019).</li><li>Tron, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+fluorescence+resonance+energy+transfer+on+cell+surfaces.+Quantitative+evaluation+of+the+transfer+efficiency+on+a+cell-by-cell+basis.">Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis.</a> Biophysical Journal. 45 (5), 939-946 (1984).</li><li>Sebestyen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+wavelength+fluorophores+and+cell-by-cell+correction+for+autofluorescence+significantly+improves+the+accuracy+of+flow+cytometric+energy+transfer+measurements+on+a+dual-laser+benchtop+flow+cytometer.">Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer.</a> Cytometry. 48 (3), 124-135 (2002).</li><li>Szaloki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+FRET+analysis+of+protein-protein+interactions+by+slide-based+imaging+laser+scanning+cytometry.">High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry.</a> Cytometry Part A. 83 (9), 818-829 (2013).</li><li>McRae, S. R., Brown, C. L., Bushell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+EGFP,+EYFP,+and+ECFP+with+high+yield+and+purity.">Rapid purification of EGFP, EYFP, and ECFP with high yield and purity.</a> Protein Expression and Purification. 41 (1), 121-127 (2005).</li><li>Kremers, G. J., Goedhart, J., van Munster, E. B., Gadella, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyan+and+yellow+super+fluorescent+proteins+with+improved+brightness,+protein+folding,+and+FRET+Forster+radius.">Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius.</a> Biochemistry. 45 (21), 6570-6580 (2006).</li><li>Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., Chu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Fluorescent+Protein+FRET+Pairs.">A Guide to Fluorescent Protein FRET Pairs.</a> Sensors (Basel). 16 (9), (2016).</li><li>Scott, B. L., Hoppe, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+fluorescent+protein+trios+for+3-Way+FRET+imaging+of+protein+interactions+in+living+cells.">Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.</a> Science Reports. 5, 10270 (2015).</li><li>Chen, Y. C., Spring, B. Q., Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+comes+of+age+how+to+do+it+and+how+to+interpret+it.">Fluorescence lifetime imaging comes of age how to do it and how to interpret it.</a> Methods Molecular Biology. 875, 1-22 (2012).</li><li>King, C., Sarabipour, S., Byrne, P., Leahy, D. J., Hristova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FRET+signatures+of+noninteracting+proteins+in+membranes:+Simulations+and+experiments.">The FRET signatures of noninteracting proteins in membranes: Simulations and experiments.</a> Biophysical Journal. 106 (6), 1309-1317 (2014).</li></ol>

The authors have nothing to disclose.

The presented protocol details the use of the genetically coupled one-to-one fluorescent protein calibration probe for quantifying FRET using the detection of sensitized emission of the acceptor and quenching of the donor molecule by confocal microscopy. This method can be applied to assess protein interactions in the physiological context of the living cell in different subcellular compartments. Spatial resolution can be further improved by applying the presented algorithm to calculate FRET efficiencies in each pixel of an image (pixel-by-pixel FRET). Intensity-based determination of absolute FRET efficiencies requires the determination of the cross-talk, quantified here with the S factors, and the detection efficiency of donor and acceptor molecules by the given microscopic set-up, quantified by the &#945;&#160;factor. Here, a protocol is provided that permits quantification of both cross-talk and detection efficiency. Direct coupling of the genetic information of fluorescent protein guarantees equimolar expression in live cells and thereby makes the determination of the &#945; factor possible. Knowledge of the &#945; factor in turn is a prerequisite for the quantification of the FRET efficiency in live cells. Critical steps in the provided protocol are the proper cloning of the directly coupled fluorescent protein chimera, sufficient time after transfection to allow for fluorescent protein maturation, use of fluorescent proteins in a similar cellular microenvironment, e.g., fluorescent proteins in cytoplasm and on cytoplasmic side of plasma membrane, and a stable microscope set-up.
Experimental set-up and algorithm presented here are designed for the GFP-Cherry FRET pair. We provide in the Supplementary File 1 the algorithm for different versions of cyan (ECFP, CyPet, mTFP1, Cerulean, mTurquoise2) and yellow fluorescent proteins (EYFP, Citrine, Venus, SYFP2, YPet). The advantage of these fluorescent protein pairs is a greater spectral overlap between the emission spectrum of the cyan and the absorption spectrum of the yellow protein (as compared to GFP-Cherry) resulting in somewhat higher R0 values and larger FRET efficiencies. However, for the same reason, the number of non-negligible crosstalk factors and their magnitudes are also larger (see Supplementary Text).
Limitations of quantitative microscopic ratiometric FRET in live cells need to be considered and is discussed. Prerequisite of using FRET for detecting molecular interactions is the application of fluorescent tags. Our protocol uses genetically encoded fluorescent proteins. Since these fluorescent proteins may be comparable in size (27 kDa) to the tagged protein of interest, they may change localization and function of the protein of interest. Therefore, both localization and functionality of any tagged protein of interest should be tested and compared with those of the endogenous unlabeled protein. Another critical point to keep in mind is the endogenous unlabeled pool of the protein of interest. The interactions of the labeled with unlabeled endogenous proteins will decrease the FRET signal. Ideally, all proteins of interest are labeled. This can be achieved by using cells which do not have the protein of interest endogenously (such as in the example given in Figure 2C and 2E), using cells derived from knock-in mice, using CRISPR modified cells, etc. Even with the use of pixel-by-pixel FRET, the signal of donor and acceptor molecules will be averaged over a diffraction-limited spot, the resolution limit of a confocal microscope. Therefore, it is impossible to resolve different donor populations within a diffraction-limited spot. There might be donors without an acceptor, or donors with multiple acceptors contributing to the average FRET signal. Dissecting different molecular subpopulations by FRET requires fluorescence lifetime imaging22. Another problem, especially at high expression levels is the so-called random FRET between fluorophores in close proximity without underlying interaction of the proteins of interest23. This random FRET can be significant in the plasma membrane given the 2-D confinement of the membrane proteins compared to freely diffusive cytoplasmic proteins. Therefore, control experiments should always be done such as deleting domains that mediate the presumed interaction between molecules and test for reduction in the detected FRET signal.
The uncertainty of exact stoichiometries of interacting proteins in live cells limits the use of FRET as a spectroscopic ruler to assess molecular distances between proteins in live cells. Even in the case of a strict one-to-one interaction, (i) the flexibility of the linker attaching the fluorescent protein to the protein of interest as well as (ii) the lack of knowledge of the relative orientation of the dipole moments of the dyes (determining the so-called &#954;2 factor) may confound exact distance measurements. Studies on acceptor fusion constructs with stiff linkers of different lengths separating the two fluorophores and displaying different FRET efficiencies have been reported elsewhere12. Conversely, assessing stoichiometries with FRET measurements in live cells is complex, however possible using the presented ratiometric FRET quantification. The inclined reader may be referred to previous work detailing a feasible approach5.
In summary, the quantitative FRET approach presented here permits the detection of (i) protein interactions in the physiological context of the living cell, (ii) changes in protein interactions over time and (iii) differences in interactions in different subcellular compartments down to the pixel-by-pixel level of a confocal image, and (iv) the dependence of the detected FRET signal upon the molecular acceptor-to-donor ratio expressed in a live cell.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62241">Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission</a>. The Authors section was updated from:
Gy&#246;rgy V&#225;mosi1 
Sarah Miller2 
Molika Sinha2 
Gabor Mocs&#225;r1 
Malte Renz2 
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen 
2Gynecologic Oncology Division, Stanford University School of Medicine
to:
Gy&#246;rgy V&#225;mosi1 
Sarah Miller2 
Molika Sinha2 
Maria Kristha Fernandez2 
Gabor Mocs&#225;r1 
Malte Renz2 
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen 
2Gynecologic Oncology Division, Stanford University School of Medicine

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62241">Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission</a>. The Authors section was updated.

Erratum: Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Microscopy-based analysis of F&#246;rster Resonance Energy Transfer (FRET) permits assessment of interactions between proteins in live cells. It provides spatial and temporal information, including information on where in the cell and in which subcellular compartment the interaction takes place and if this interaction changes over time.
Theodor F&#246;rster laid the theoretical foundation of FRET in 19481. FRET is a radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance of the molecules and the relative orientation of their transition dipoles as well as the overlap between the donor emission and acceptor absorption spectra. The rate of energy transfer is inversely proportional to the sixth power of the donor-acceptor distance. Thus, FRET can be used to measure molecular proximity in the range of 1-10 nm.
FRET competes with other de-excitation processes of the donor molecule and results in the so-called donor-quenching and sensitized emission of the acceptor. Donor-quenching is a reduction of the number of emitted donor photons, while sensitized emission is an increase in emitted acceptor photons. Many microscopic FRET analyses use fluorescence intensity measurements, including acceptor photobleaching2, donor photobleaching2, or FRET-sensitized photobleaching of the acceptor3.
Here, a step-by-step experimental protocol and mathematical algorithm are presented to quantify FRET using donor quenching and acceptor sensitized emission4,5, a method often referred to as ratiometric FRET. Many protocols on how to approximate sensitized emission have been published, few have quantified the absolute FRET efficiency6,7,8,9. The quantification of FRET efficiencies in the living cell requires determining (i) the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins and, also (ii) the detection efficiency of the microscopic setup. While crosstalk can be assessed by imaging cells expressing only one of the fluorophores, the assessment of the relative detection efficiency of the donor and acceptor fluorescence is more complicated. It requires the knowledge of at least the ratio of the number of donor and acceptor molecules giving rise to the measured signals. The number of fluorophores expressed in live cells varies, however, from cell to cell and is unknown. The so-called &#945;&#160;factor characterizes the relative signal strengths from a single excited donor and acceptor molecule. Knowledge of the factor is a prerequisite for quantitative ratiometric FRET measurements in samples with variable acceptor-to-donor molecule ratios as encountered during live-cell imaging with fluorescent proteins. Using a 1-to-1 donor-acceptor fusion protein as a calibration probe permits the determination of the &#945;&#160;factor and also serves as a positive control. This genetically coupled probe is expressed by cells in unknown total amounts but in a fixed and known relative amount of one-to-one. The following protocol lays out how to construct the 1-to-1 probe and how to use it for quantification of FRET efficiency. A spreadsheet that includes all formulae can be found in the supplement and can be used by the readers to enter their own measurements in the respective columns as outlined below.
While the protocol uses the GFP-Cherry donor/ acceptor pair, the presented approach can be performed with any other FRET pair. The Supplementary File 1 provides details on cyan-yellow pairs.

<table><tbody><tr><td>0.5% Trypsin-EDTA without phenol red (10x)</td><td>Thermo Fisher Scientific</td><td>15400054</td><td></td></tr><tr><td>Clontech mCherry N1 vector</td><td>Addgene</td><td>3553</td><td></td></tr><tr><td>DMEM without phenol red</td><td>Thermo Fisher Scientific</td><td>11054020</td><td></td></tr><tr><td>Fugene 6</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>LabTek 8-well chambers #1.0</td><td>Thermo Fisher Scientific</td><td>12565470</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Thermo Fisher Scientific</td><td>25030081</td><td></td></tr></tbody></table>

assessing protein interactions in live-cells with fret-sensitized emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

Figure 1&#160;shows the images obtained in the donor channel, channel 1 (488, 505-530 nm), the transfer channel, channel 2 (488, &#62;585 nm), and the acceptor channel, channel 3 (561, &#62;585 nm), respectively. Representative images of cells expressing GFP only, Cherry only, co-expressing GFP and Cherry, and expressing the GFP-Cherry fusion protein. The mean cellular FRET efficiencies calculated in NRK cells expressing GFP-Cherry fusion protein (positive control, Figure 2A) and those co-expressing GFP-Cherry (negative control, Figure 2B) are plotted versus the acceptor-to-donor ratio intensity ratio (Q) or molecular ratio&#160;NA/ND in each cell. Figure 2C illustrates an example on how to outline a region of interest and avoid perinuclear vesicles with high autofluorescence.
The presented algorithm can be used to quantify FRET efficiency in any region of interest including the quantification in every pixel of the image in the transfer channel. Figure 2D shows normalized pixel-by-pixel FRET images of cells expressing the GFP-Cherry fusion protein, co-expressing GFP and Cherry as negative control, and expressing receptor subunits of the Ashwell-Morell receptor. The rat variant of this receptor, the rat hepatic lectin (RHL1 and RHL2), is a two-subunit receptor system that is known to hetero-oligomerize. All FRET efficiencies were normalized to that of the GFP-Cherry fusion protein. We labeled RHL1 and 2 with GFP and Cherry on the cytoplasmic side of the plasma membrane. The image shows distinct FRET values at the plasma membrane compared to intracellular vesicles. Deleting the stalk domain of RHL1 (GFP-RHL1&#916;stalk) which is thought to mediate the tight interaction between the two subunits decreases the detected FRET efficiencies. In Figure 2E, mean cellular FRET efficiencies of cells expressing GFP-RHL1 and Cherry-RHL2, and GFP-RHL1&#916;stalk and Cherry-RHL2 are plotted versus the acceptor-to-donor&#160;molecular ratio (NA/ND). For further FRET analyses of this receptor system the reader may refer to a previous publication5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62241/62241fig1v2.jpg" /> 
Figure 1: Representative images of cells expressing fluorescent proteins. Cells expressing GFP only (A), Cherry only (B), GFP and Cherry co-expression (C), and GFP-Cherry fusion protein (D). Images in channel 1 (488, 505-530 nm), channel 2 (488, &#62;585 nm), and channel 3 (561, &#62;585 nm), respectively. Images were obtained with 63x oil objective and zoom set to 3x. Scale bar: 10 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62241/62241fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62241/62241fig2v2.jpg" /> 
Figure 2: Quantification of FRET images. Mean cellular FRET efficiencies for cells expressing the GFP-Cherry fusion protein plotted versus the acceptor-to-donor intensity ratio (Q) (A) and versus the acceptor-to-donor molecular ratio (NA/ND) for cells co-expressing GFP and Cherry (B). Open circle thick line represents single cell expressing GFP-Cherry fusion protein. Open circle thin line represents a cell co-expressing GFP and Cherry.&#160;Note that at higher&#160;NA/ND ratios the fraction of the useful signal, the sensitized emission (IDE&#945;) in the transfer channel becomes smaller and smaller relative to the direct excitation of the acceptor (IAS2). This results in a larger error in the determination of E.&#160;Pixel-by-pixel FRET images calculated using the presented algorithm (C). This panel illustrates a cell co-expressing GFP and Cherry, the negative control, in the donor channel, the transfer channel, and the acceptor channel. It also shows a possible outline of a region of interest avoiding perinuclear vesicles with high autofluorescence which may negatively impact the precision of the FRET calculation. (D) All FRET efficiencies were normalized to that of the GFP-Cherry fusion protein. From left to right, GFP-Cherry fusion protein (positive control), GFP Cherry co-expression (negative control), GFP-RHL1 and Cherry-RHL2, and GFP-RHL1&#916;stalk and Cherry-RHL2. Scale bar: 10 &#956;m. Color-coded scale bar: normalized mean FRET efficiency (normalized to the mean value of the positive control, the GFP-Cherry fusion protein).&#160;(E) This panel shows mean cellular FRET efficiencies for cells expressing the GFP-RHL1 and Cherry-RHL2 as well as GFP-RHL1&#916;stalk and Cherry-RHL2 plotted versus the acceptor-to-donor&#160;molecular ratio (NA/ND). Closed grey circle represents a single cell expressing GFP-RHL1 and Cherry-RHL2. Closed black circle represents a cell expressing GFP-RHL1&#916;stalk and Cherry-RHL2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62241/62241fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Algorithm for other donor-acceptor pairs. Algorithm for other donor-acceptor pairs such as different versions of cyan (ECFP, CyPet, mTFP1, Cerulean, mTurquoise2) and yellow (EYFP, Citrine, Venus, SYFP2, YPet) fluorescent proteins. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62241/Vamosi et al. Supplemental Text.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Spreadsheet with the presented FRET algorithm and use of the GFP-Cherry fusion protein to quantify FRET by sensitized emission of the acceptor and donor-quenching. Cells A2, B2, C2: Mean background signals (B1, B2, B3) in channels 1, 2, and 3, respectively. Cells D2 and E2: Mean values for cross-talk factors S1, and S2. Cell G2: Value for extinction coefficient ratio <img alt="Equation 11" src="/files/ftp_upload/62241/62241eq11.jpg" />. Cell I1, and I2: Extinction coefficients of GFP and Cherry at 488-nm laser light. Cell J2: Mean value for factor. Column C (C5 and up): measured fluorescence intensity of a cell expressing GFP in channel 1. Column D (D5 and up): measured fluorescence intensity of a cell expressing GFP in channel 2. Column E (E5 and up): measured fluorescence intensity of a cell expressing GFP in channel 3. Columns F, G, and H (F5, G5, H5 and up, respectively): measured fluorescence intensities subtracted by mean background intensities in all 3 channels. Column I (I5 and up): Calculated cross-talk factor S1. Column M (M5 and up): measured fluorescence intensity of a cell expressing Cherry in channel 1. Column N (N5 and up): measured fluorescence intensity of a cell expressing Cherry in channel 2. Column O (O5 and up): measured fluorescence intensity of a cell expressing Cherry in channel 3. Columns P, Q, and R (P5, Q5, R5 and up, respectively): measured fluorescence intensities subtracted by mean background intensities in all 3 channels. Column S (S5 and up): Calculated cross-talk factor S2. Column W (W5 and up): measured fluorescence intensity of a non- or mock-transfected cell in channel 1. Column X (X5 and up): measured fluorescence intensity of a non- or mock-transfected cell in channel 2. Column Y (Y5 and up): measured fluorescence intensity of a non- or mock-transfected cell in channel 3.Column AD (AD5 and up): measured fluorescence intensity of a cell expressing the GFP-Cherry fusion protein (or co-expressing GFP and Cherry, or any protein pair of interest) in channel 1. Column AE (AE5 and up): measured fluorescence intensity of a cell expressing the GFP-Cherry fusion protein (or co-expressing GFP and Cherry, or any protein pair of interest) in channel 2. Column AF (AF5 and up): measured fluorescence intensity of a cell expressing the GFP-Cherry fusion protein (or co-expressing GFP and Cherry, or any protein pair of interest) in channel 3. Columns AG, AH, and AI (AG5, AH5, AI5 and up, respectively): measured fluorescence intensities subtracted by mean background intensities in all 3 channels. Column AJ (AJ5 and up): Calculated &#945;&#160;factor. Column AK (AK 5 and up): Calculated mean FRET efficiency E. Column AL (AL5 and up): calculated corrected acceptor-to-donor intensity ratio (Q).&#160;Examples for calculated parameters from a FRET experiment as expressed as mean and standard deviation: S1 = 0.2232&#160;&#177; 0.0060. S2&#160;= 0.2039 &#177; 0.0074. &#945; = 1.9463&#160;&#177; 0.1409. E = 0.2713&#160;&#177; 0.0220. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62241/Supplementary File 2.xls" target="_blank">Please click here to download this File.</a>
Supplementary Figure 1: GFP and Cherry absorption and emission spectra. Normalized absorption and fluorescence emission spectra of eGFP and mCherry. The excitation laser lines (488 and 543 nm) and filter transmissions used for the donor channel (ch1: 505-530 nm) and the transfer/acceptor channels (ch2 &#38; 3: &#62;585 nm) in the confocal microscope are marked by shading. For excitation of the acceptor, 561 or 590-nm laser lines can also be used. Source fluorescent protein data base (fpbase.org). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62241/Vamosi et al. suppl. Figure 1.eps" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission at JoVE.com

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

assessing-protein-interactions-live-cells-with-fret-sensitized

Nanyang Technological University

Research

JoVE Journal

Biology

3.1K Views.  University of Debrecen. Dit protocol beschrijft een stap-voor-stap experimenteel proces en wiskundig algoritme om FRET te kwantificeren met behulp van donorblussing en acceptor gesensibiliseerde emissie. De kwantificering van FRET-efficiënties in de levende cel vereist het bepalen van de overspraak van de fluorescerende eiwitten en de relatieve emissie- en detectie-efficiëntie van de fluoroforen in de microscopische opstelling. Kruisverwijzing kan worden beoordeeld door cellen af te beelden die slechts één fluor...

Video: Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Biochemistry

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Bioengineering

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within &#60;10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone ...

F&#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred&#8212;and consequently the overall emission of sample&#8212;depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry techniques to obtain detailed information of biomolecular structures and interactions from this &#8220;spectroscopic ruler.&#8221;
This video covers the principles and concepts of FRET analysis. The procedure focuses on preparing samples for FRET and ways to present and interpret data. Finally, the applications include monitoring conformational and cellular processes by labeling parts of a cell or protein, monitoring enzyme reactions that alter protein structures, and using FRET to monitor aggregation of monomers expressed by cells.
<div class="chapter_text" id="script_0">
F&#246;rster Resonance Energy Transfer, or FRET, is a non-radiative transfer of energy between light-emitting molecules, and is often used to investigate close-range biochemical interactions. FRET only occurs when fluorescent molecules are spaced within 10 nm of each other. FRET analysis can be combined with other techniques to obtain detailed structural information. This video will introduce the underlying principles of FRET, summarize a protocol and data presentation, and discuss some biochemical applications.
</div>
<div class="chapter_text" id="script_1">
A photoluminescent molecule such as a fluorophore is excited by absorbing electromagnetic radiation at a wavelength in its absorption spectrum. As it relaxes, it emits light at a wavelength within its emission spectrum. For more information about fluorescence, see JoVE&#39;s video on fluorescence microscopy. Different fluorophores absorb and emit light at different wavelengths, which frequently overlap. If the emission spectrum of a fluorophore overlaps significantly with the absorption spectrum of another fluorophore, the &#8220;donor&#8221; will release a virtual photon, which is absorbed by the &#8220;acceptor&#8221;. When an excited donor is within 10 nm of an acceptor, energy is transferred from donor to acceptor by dipole-dipole interactions. The release of energy by emission of light from the donor correspondingly decreases. Meanwhile, the excited acceptor emits light at its emission wavelength. The FRET response is evaluated in terms of efficiency, or the percentage of energy released from the donor by FRET rather than by fluorescence or other radiative processes. The efficiency depends strongly on the distance between the donor and acceptor, which allows FRET to act as a &#39;molecular&#39; or &#39;spectroscopic&#39; ruler.
In biochemistry, FRET is often used qualitatively to observe conformational changes in molecules by monitoring fluorophores as they move in and out of FRET range of each other. Similarly, cellular functions can be studied with molecules containing a FRET pair. If the labeled molecule is cleaved by enzyme activity, FRET stops and the observed fluorescence wavelength changes.
Now that you understand the principles behind FRET, let&#39;s look at an overview of a protocol and a few ways to present and interpret the data.
</div>
<div class="chapter_text" id="script_2">
Prior to the experiment, the biomolecules of interest, typically DNA or proteins, are engineered with fluorescent tags, using molecular biology techniques.&#160;Common ways to introduce the modified genetic material into the cells include transfection and electroporation.
Then, the cells are prepared for FRET visualization on a fluorescence microscope.&#160;For instance, the molecules may be immobilized on a slide for single-molecule FRET, or samples are loaded into wells for high-throughput screening.
Then, the excitation lasers, microscope, and associated equipment are prepared. (A) FRET experiments often involve powerful lasers; (B) so appropriate PPE and safety procedures should be used.&#160;The sample is then placed in the instrument and illuminated with the excitation laser.
</div>
<div class="chapter_text" id="script_3">
For experiments monitoring cell behavior, color images showing differences or changes in emission intensity are used. Donor and acceptor emission intensities are plotted together to track FRET response over time.
FRET data can also be fitted to various functions for more complex analyses. Depending on the experiment, data may be presented in multiple ways to best represent the results, making FRET a flexible experimental tool.
</div>
<div class="chapter_text" id="script_4">
Now that you&#39;re familiar with the basics of running and analyzing a FRET experiment, let&#39;s look at some applications of FRET in biochemistry research.
FRET can be used to study conformational changes or cellular processes by labeling parts of the protein or cell predicted to move within 10 nm of each other with a FRET pair. For example, protein sensors are prepared by labeling receptors with a pair of fluorophores. The FRET response is monitored live by confocal microscopy. Variation of emission wavelength and intensity indicate conformational changes.
FRET can also be used by preparing molecules with an active FRET pair and observing changes in the response. When the substrate is cleaved, FRET is disrupted, causing an increase in donor emission and a decrease in acceptor emission. The emissions are analyzed to determine contributions by donor, acceptor, and FRET. Once the direct emission factors are calculated for the cyan and yellow fluorescent proteins, the concentration and kinetic parameters of the substrate can be determined.
Cells designed to express monomers containing either of a FRET pair function as &#39;sensors&#39; for interactions between those monomers. If aggregation of those monomers is induced, a FRET response is observed. This can be used to investigate protein aggregation triggered by &#39;seeding&#39; of misfolded proteins. Here, cells were transduced with aggregates of the protein of interest, incubated, and analyzed with flow cytometry.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on F&#246;rster Resonance Energy Transfer, or FRET. This video contained the underlying principles of FRET, preparation and analysis of a FRET experiment, and a few biochemical applications.
Thanks for watching!
</div>

F&amp;#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video