Name: determination of tripartite interaction between two monomers of a mads-box transcription factor and a calcium sensor protein by bifc-fret-flim assay
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Description: Watch this Scientific Journal Video about Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay at JoVE.c

NB, GG, SB, KC sincerely thank the University Grants Commission (UGC), UGC-BSR, DBT-INSPIRE, and Council for Scientific and Industrial Research (CSIR) for their research fellowships. We thankfully acknowledge the Department of Biotechnology (DBT), Government of India, the Department of Science and Technology (DST-FIST), Government of India for financial support.

1. Cloning of genes in entry and destination vectors (Figure 2)
<ol>
	<li>Amplify the coding sequence (CDS) of the genes of interest (M and C genes in our case) by PCR and clone them in appropriate entry vectors (e.g., pENTR/D-TOPO vector; see Table 1 for vectors used in this experiment).</li>
	<li>Grow the clones on plates containing antibiotics. Validate clones that are selected on the antibiotics by restriction digestion and DN...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+highly+efficient+method+for+transient+gene+expression+in+rice+plants.">A Rapid and highly efficient method for transient gene expression in rice plants.</a> Frontiers in Plant Science. , 11 (2020).</li><li>Kudla, J., Bock, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+the+way+to+protein-protein+interactions:+Recommendations+on+best+practices+for+bimolecular+fluorescence+complementation+analyses.">Lighting the way to protein-protein interactions: Recommendations on best practices for bimolecular fluorescence complementation analyses.</a> Plant Cell. 28 (5), 1002-1008 (2016).</li><li>Shyu, Y. J., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+complementation:+an+emerging+tool+for+biological+research.">Fluorescence complementation: an emerging tool for biological research.</a> Trends in Biotechnology. 26 (11), 622-630 (2008).</li><li>Kodama, Y., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+bimolecular+fluorescence+complementation+assay+with+a+high+signal-to-noise+ratio.">An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.</a> BioTechniques. 49 (5), 793-803 (2010).</li><li>Kodama, Y., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC):+A+5-year+update+and+future+perspectives.">Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives.</a> BioTechniques. 53 (5), 285-298 (2012).</li><li>Piston, D. W., Kremers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET:+the+good,+the+bad+and+the+ugly.">Fluorescent protein FRET: the good, the bad and the ugly.</a> Trends in Biochemical Sciences. 32 (9), 407-414 (2007).</li><li>Chakrabarty, R., 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=pSITE+vectors+for+stable+integration+or+transient+expression+of+autofluorescent+protein+fusions+in+plants:+Probing+Nicotiana+benthamiana-+Virus+Interactions.">pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: Probing Nicotiana benthamiana- Virus Interactions.</a> Molecular Plant-Microbe Interactions. 20 (7), 740-750 (2007).</li><li>Walter, M., 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=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant Journal. 40 (3), 428-438 (2004).</li><li>Hellens, R., Mullineaux, P., Klee, H. <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+Agrobacterium+binary+Ti+vectors.">A guide to Agrobacterium binary Ti vectors.</a> Trends in Plant Science. 5 (10), 446-451 (2000).</li><li>Van Der Hoorn, R. A. L., Rivas, S., Wulff, B. B. H., Jones, J. D. G., Joosten, M. H. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+migration+in+gel+filtration+of+the+Cf-4+and+Cf-9+resistance+proteins+is+an+intrinsic+property+of+Cf+proteins+and+not+because+of+their+association+with+high-molecular-weight+proteins.">Rapid migration in gel filtration of the Cf-4 and Cf-9 resistance proteins is an intrinsic property of Cf proteins and not because of their association with high-molecular-weight proteins.</a> Plant Journal. 35 (3), 305-315 (2003).</li><li>Xie, X., 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=Engineering+SARS-CoV-2+using+a+reverse+genetic+system.">Engineering SARS-CoV-2 using a reverse genetic system.</a> Nature Protocols. 16, (2021).</li><li>Xu, J., 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=Optimized+plasmid+construction+strategy+for+Cas9.">Optimized plasmid construction strategy for Cas9.</a> Cellular Physiology and Biochemistry. 48, 131-137 (2018).</li><li>Mattanovich, D., 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=Efficient+transformation+of+Agrobacterium+spp.+by+electroporation.">Efficient transformation of Agrobacterium spp. by electroporation.</a> Nucleic Acids Research. 17 (16), 6747 (1989).</li><li>Rizzo, M. 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J., 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=SP8+FALCON:+a+novel+concept+in+fluorescence+lifetime+imaging+enabling+video-rate+confocal+FLIM.">SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM.</a> Nature Methods. 20, 2-4 (2019).</li><li>Postma, M., Goedhart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plotsofdata-a+web+app+for+visualizing+data+together+with+their+summaries.">Plotsofdata-a web app for visualizing data together with their summaries.</a> PLoS Biology. 17 (3), 1-8 (2019).</li><li>Galperin, E., Verkhusha, V. 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The present protocol demonstrates the use of BiFC-based FRET-FLIM assay to ascertain the formation of a ternary complex between two monomers of a MADS-box protein and a calcium sensor protein. The protocol is adapted from a report by Y. John Shyu et al. where they have developed a BiFC-based FRET method to visualize ternary complex formed between Fos-Jun heterodimers and NFAT or p65 using the sensitized emission method7. Earlier, a three-fluorophore FRET system was developed by Galperin and co-wor...

Protein-protein interactions (PPIs) form the basis of the proper functioning of the eukaryotic cells by regulating various metabolic and developmental processes. Some PPIs are stable, while others are transient in nature. The interactions may be categorized based on the number and type of members in the interaction such as dimeric, trimeric, tetrameric homomeric, and heteromeric1. The identification and characterization of protein interactions may lead to a better understanding of protein functions and regulatory networks.
Transcription factors are proteins that are involved in regulatory functions. They regulate the rate of transcription of their downstream genes by binding to the DNA. Sometimes oligomerization or formation of higher-order complexes by proteins is a prerequisite for carrying out their functions2. Plant MADS-box transcription factors are homeotic genes that regulate various processes such as floral transition, floral organ development, fertilization, seed development, senescence, and vegetative development. They are known to form higher-order complexes which bind to the DNA3,4. Studying PPI networks among transcription factors and their interactors provides insights into the complexity underlying transcriptional regulation.
Transient protein expression in Nicotiana benthamiana has been a popular approach to study protein localization or protein-protein interactions in vivo5. BiFC and FRET are methods for studying protein-protein interactions in vivo using fluorescent reporter systems6. A combination of these two techniques has been shown to reveal the interaction between three proteins7. FRET is measured using acceptor photobleaching, sensitized emission, and Fluorescence Lifetime Imaging (FLIM) technique. FLIM-based FRET assay has emerged as a tool that provides accurate quantification and spatiotemporal specificity to energy transfer measurements between two molecules based on their fluorescence lifetimes8. FLIM measures the time a fluorophore stays in an excited state before emitting a photon and is better than techniques that use intensity measurements alone9,10. Besides heterologous systems such as Nicotiana benthamiana and onion epidermal peels, more recent reports have demonstrated the use of Arabidopsis roots and young rice seedlings, etc., for in vivo analysis of protein-protein interactions under native conditions11,12.
Other than a suitable expression system, the selection of interacting partners for BiFC and FRET assays is also crucial for the success of this experiment. The PPI among partners used in BiFC configuration should be validated using appropriate controls prior to their use as a conjugated partner in the FRET experiment13. BiFC utilizes the structural complementation of N- and C-terminal parts of the fluorescent protein. A common limitation in most if not all fluorescent proteins used in BiFC assays has been the self-assembly between the two derivative nonfluorescent fragments, contributing to false-positive fluorescence and decreases the signal-to-noise (S/N) ratio14. Recent developments, including point mutations or position of splitting the fluorescent protein, have given rise to BiFC pairs with increased intensity, higher specificity, high S/N ratio15,16. These fluorescent proteins can also be used for carrying out BiFC depending on the suitability of the experiment.
Traditionally, CFP and YFP have been used as the donor and acceptor pair in FRET experiments17. However, YFP or m-Citrine were found to be better FRET donors (when used with RFP as the acceptor) because of the high quantum yield (QY) during the native expression of target proteins in the Arabidopsis root system. The selection of promoters (constitutive versus native/endogenous) and fluorophore also play a crucial role in designing a successful BiFC-FRET-FLIM experiment. It is essential to note that the efficiency of FRET donors and suitability of FRET pairs tend to change with the change in the promoter and the biological system being used for expression. The QY of the fluorophore, which relates to its brightness, depends on the pH, temperature, and the biological system in use. We suggest that these criteria be thoroughly considered before choosing the fluorophore pair for the FRET experiment. The biological system, promoters, and the proteins used for this protocol worked well with CFP-YFP fluorophores for the BiFC FRET-FLIM experiment.
In the present study, we incorporate the feature of FLIM to visualize the interaction between three protein molecules using BiFC based FRET. In this technique, two proteins are tagged with split YFP protein and the third protein with CFP. Since we were interested in studying the interaction of a MADS-box protein (M) homodimer with a Calcium sensor protein (C), these proteins were tagged with fluorescent proteins in pSITE-1CA and pSITE-3CA vectors18. Two of the interacting partners, in this assay, were tagged with N- and C-terminal parts of the YFP in pSPYNE-35S and pSPYCE-35S vectors19, and their interaction results in the reconstitution of the functional YFP that acts as a FRET acceptor to the third interacting partner, which is tagged with CFP (acting as FRET donor) (Figure 1). In this particular case, the PPI between two M monomers and between M and C has been validated by performing BiFC in three different systems along with the yeast-two-hybrid system. These vectors were mobilized into Agrobacterium tumifaciens GV3101 strain by electroporation. The GV3101 strain has a disarmed Ti plasmid pMP90 (pTiC58DT-DNA) with gentamicin resistance20. A p19 Agrobacterium strain was added along with all infiltrations to prevent transgene silencing21. We recommend that the three proteins should also be used in opposite conformations to validate the tripartite interactions.
In this technique, we have employed FLIM, where first, the fluorescence lifetime of the donor (unquenched donor lifetime) is measured in the absence of an acceptor. After that, its lifetime is measured in the presence of the acceptor (quenched donor lifetime). This difference in donor fluorescence lifetimes is used to calculate FRET efficiency, which depends on the number of photons exhibiting a reduction in fluorescence lifetime. Mentioned below is a detailed protocol to determine the formation of a ternary complex between any three proteins by transiently expressing the fluorescent-tagged proteins in Nicotiana benthamiana and assaying their interaction by BiFC-FRET-FLIM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml Syringes without needles</td>
			<td>Dispovan</td>
			<td>&#160;-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway&#160; LR Clonase II Enzyme mix</td>
			<td>Thermo Fischer Scientific</td>
			<td>11791020</td>
			<td>The vectors used in the study are Gateway based</td>
		</tr>
		<tr>
			<td>Gentamycin Sulphate</td>
			<td>Himedia</td>
			<td>CMS461</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulphate</td>
			<td>Himedia</td>
			<td>MB105</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>K240020</td>
			<td>The vectors used in the study are Gateway based</td>
		</tr>
		<tr>
			<td>Phusion high fidelity Taq DNA polymerase</td>
			<td>Thermo Fischer Scientific</td>
			<td>F530-S</td>
			<td>Any High fidelity Polymerase can work</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Himedia</td>
			<td>CMS1889</td>
			<td></td>
		</tr>
		<tr>
			<td>SP8 FALCON Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>SP8 FALCON</td>
			<td>Any CLSM with FLIM capabilities can be used for this analysis</td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>Himedia</td>
			<td>TC034</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of tripartite interaction between two monomers of a mads-box transcription factor and a calcium sensor protein by bifc-fret-flim assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

This protocol represents an optimized method to study in vivo tripartite protein-protein interactions in plants. The basic principle of the protocol is to combine two fluorescence-tagged protein-interaction techniques, i.e., BiFC and FRET, to create an assay to measure ternary complex formation between three protein partners. Here, we have used FLIM to measure the fluorescence lifetime of the FRET donor partner in the presence and absence of the FRET acceptor. A reduction in the fluorescence lifetime of the dono...

Watch this Scientific Journal Video about Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay at JoVE.c

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

The study of protein-protein interactions provides an understanding of the regulation of many biological processes. Here we demonstrate a procedure described initial by Y John shoe and co. Workers in 2007, where the authors have used BiFC in combination with FRET to validate the tripartite interaction between three protein molecules.However, we have included fluorescence lifetime measurements in this procedure to substantiate FRET measurements. To demonstrate a tripartite interaction, we have chosen a protein, which is known to homodimerize. Furthermore, it has also been validated in-house to interact with a calcium sensor of protein referred to as C protein in this protocol.The MNC proteins interact in the cytoplasm. In this technique, two proteins are tagged with split YFP proteins. Their interaction results in the restitution of functional YFP that act as a FRET acceptor to the third interacting partner, which is tagged with CFP acting as a FRET donor.Start with the amplification of the coding sequences of the genes of interest that are M and C genes in our case by PCR and clone them in appropriate entry vectors. Validate clones that are selected on the antibiotics containing plates by restriction digest and DNA sequencing. Mobilize the reconstituted CDS'from entry clones to the destination vectors.All the vectors used in this experiment are listed in table one. Finally, transform agrobacterium GV3101 cells with the destination vectors by electroporation. In here we grow Nicotiana Benthamiana plants still four to six leave stage in controlled conditions in a grow chamber.Prepare the soil mix by mixing soilrite, coco peat and compost in a ratio of 2:1:1. Spread a one-inch thick layer of this mix in a plastic tray to make the soil bed and saturate it with a little water. Sprinkle about 200 seeds on top of the soil and transfer it to a bigger tray that about one centimeter of standing water.Cover this tray with plastic wrap to create a moisture chamber. Transfer this tray to a grow chamber set at 23 degrees centigrade with 16-hours light and eight-hours dark cycle. After two weeks transfer the young seedlings to small three to four inch pots containing water saturated soil mix.Place these pots in a plastic trays and transfer them to grow chamber for four more weeks. For agroinfiltration, the bacterial strains need to be freshly sub-cultured and mixed along with P19 strain of agrobacterium in appropriate ratios. Start this procedure by inoculating GV3101 agrobacterium strains containing BiFC and FRET constructs from streak plates in 10 ml.to the broth containing appropriate antibiotics. Alongside, also initiate a culture of P19 strain of agrobacteria, which is added to prevent transgene silencing. Cover the flask with aluminum foil and keep them in an incubator shaker, set it 28 degrees centigrade at 170 RPM for 16 hours.After the overnight growth transfer 1 ml. of these cultures to a disposable cubit to measure the optical density at 600 nanometer by using a spectrophotometer. Mix the cultures of appropriate BiFC and FRET partner containing strains so that the final OD is 0.5 And that of P19 is 0.3 in a total volume of 2 ml.To achieve these ratios we follow the formula shown on the screen for these calculations. Centrifuge the mixed agrobacterium cultures at 3000G for five minutes at room temperature and carefully discard the supernatant. Suspend the pellets in a 2 ml.infiltration buffer. You may have to use a vortex mixer to re-suspend the cells in a homogenous cell suspension completely. After this incubate the tubes at room temperature in the dark for three hours.Meanwhile, label each plant pot for the construct mixture it's going to be infiltrated with. Fill a 1 ml. needleless syringe with the agrobacterial mix.Gently but firmly press the syringe tip to the abaxial side of a fully expanded leaf while supporting the leaf from the other side. Gently push the plunger till the solution fills up in the leaf area equivalent of two to three times as that of the syringe tip. Infiltrate the bacterial mix at up to four spots on one leaf and repeat this procedure for three to four leaves for one kind of infiltration.Also, include at least two plants per construct for infiltration. Remember to wipe your hands with 70%alcohol or change your gloves to prevent any cross contamination. Transfer all the pots to a tray and incubate in the growth chamber at the same growth condition.Check a small part of the agroinfiltrated leaf using a fluorescence microscope. When the fluorescence from both YFP and CFP are detectable in cells proceed to the confocal microscope for BiFC FRET-FLIM assay. In our case, this analysis was carried out three days after agroinfiltration.Now that the plants are ready to be visualized under a microscope cut square leaf samples from five to 8 mm. away from the syringe tip wound and mount them on distilled water. Place a clean cover slip over it and seal.Visualize these samples in confocal laser scanning microscope. In this procedure, the basis of determining and quantifying interaction between two proteins is the reduction in fluorescence lifetime of the FRET donor partner upon its interaction with the acceptor, which is used to calculate the efficiency of FRET. The complexity in the case of tripartite interaction further increases because FRET acceptor in this case is not a single molecule, but a split YFP BiFC pair, which should first get reconstituted in vivo to become a functional FRET acceptor fluorophore.To carry out FRET-FLIM, we have to determine the donor molecules fluorescence lifetime, first alone and then in presence of a FRET partner. Open the FLIM application in the confocal laser scanning microscope, start the console and use the pattern recognition photon counting to measure fluorescence lifetime. Select the standard all photon counting measurement mode.We will take two types of agroinfiltrated plants. One that has the CCFP gene and the other that has CCFP along with MYFP because the interaction of M protein has already been validated with the C protein using BiFC and Y2H, we expect good FRET efficiency with this interacting pair. Now we first scan the CCFP agroinfiltrated leaf and look for a cell showing good CFP fluorescence.The microscope settings are shown on the screen. The sample is illuminated at sufficient laser power to achieve approximately 0.1 photons per pulse. For samples with variable fluorescence intensity, we will capture 50 frames to collect adequate photons required for the lifetime measurement.As CFP is known to exhibit two fluorescence lifetimes due to its confirmational adaptation, we feed the data using an exponential reconvolution model while keeping the value of N equal to two. At these settings, the two lifetimes are visible at one and 3.2 nanoseconds. We will use the higher lifetime for all subsequent calculations.We are now set to measure FRET between CCFP and MYFP. For this let's use the leaf sample from the agroinfiltrated plant with CCFP and MYFP. We look for a cell that expresses both CCFP and MYFP and confirm their respective emission patterns by exciting them using full 40 nanometer pulse laser and 514 nanometer white light laser.After that we switch to the FLIM console to measure the lifetime of CFP using the same settings that we used earlier for measuring the lifetime of CCFP. But this time the cell is also expressing MYFP that can potentially interact with CCFP and cause a reduction in the lifetime of CCFP. As we've read the graph obtained using the N exponential reconvolution model with N equal to two, there is in fact a decrease in the CFP lifetime from 3.2 to 2.6 nanoseconds.We now start the FRET console in the software and calculate the FRET efficiency by manually entering unquestionable lifetime in the equation provided in the software and the observed FRET efficiency is 56%Now let's move to the final step, that is to visualize the interaction between three partners. For this, we take the leaf sample from a plant that was co-infiltrated with CCFP and MYFPN with MYFPC. We scan the leaf plant for a cell which shows both CFP and reconstituted YFP fluorescence emanating from interaction between two M proteins.We use the same laser and emission wavelength as used earlier. Subsequently, we switch off the 514 nanometer laser and move to the FLIM console. If the MYFP dimer interacts with CCFP.We should see a reduction in the lifetime of CCFP as observed during its interaction with MYFP. However, if the CCFP fails to interact with the MYFP dimer, its fluorescence lifetime should remain seen at 3.2 nanoseconds. Using the similar settings as mentioned above, we measure the CFP lifetime in the presence of reconstituted YFP.We fit the graph using the N exponential reconvolution model with an equal to two and move to the FRET console. And yes there is a decline in the CFP lifetime from 3.2 to 2.3 nanoseconds. We calculate the FRET efficiency as described above.The calculated FRET efficiency is 55%Here we have used FLIM to measure the fluorescence lifetime of the FRET donor partner in the presence and absence of the FRET acceptor. A reduction in the fluorescence lifetime of the donor was expected if there were a positive interaction between the two proteins. In case of positive control, that is CCFP and MYFP.We observe a reduction in the fluorescence lifetime of the donor from 3.3 to 2.5 nanoseconds and the FRET efficiency was 56%A similar exercise with reconstituted YFP resulted from the interaction between two amonomers and C proteins also resulted in a positive FRET interaction leading to the reduction in fluorescence lifetime of the donor to 2.6 nanoseconds. The calculated FRET efficiency was about 55%in this case. The reduction in the fluorescence lifetime of the FRET donor provides a strong evidence for a tripartite interaction between these proteins.The protocol described here is a powerful tool to determine tripartite protein-protein interactions in living cells. This procedure can also help determine the intracellular localization of the resultant complexes, which is important in deciphering the function and physiological significance of any protein complex. In conclusion, BiFC based FRET-FLIM assay provides an opportunity to study and characterize important aspects of tripartite protein interactions, which can be helpful in protein-protein interaction studies in both animal and plant systems.

determination-tripartite-interaction-between-two-monomers-mads-box

Research

JoVE Journal

Biology

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...