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

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.