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

This method can help to understand key questions in the cell-to-cell interaction field such as which receptors mediate adhesion or recognition in cell-to-cell contacts and whether specific ligands trigger their interactions. The main advantage of this technique is that it allows one to investigate such interactions directly in living cells with no need for cell fixation or disruption. Though this method is used to investigate interactions between cultured cells, it can also be applied to other systems mono-membrane vesicles or even small model organisms.

Generally individuals new to this method should check that their sample is stable enough to allow acquisition over a few minutes by carefully inspecting cell movement and slow signal variations. First, seed the appropriate number of cells in at least four wells of a six-well plate a day before transfection. Culture the cells at 37 degrees Celsius 5%carbon dioxide in DMEM medium supplemented with 10%FBS and 1%L-glutamine.

To perform a basic experiment transfect plasmids for the protein of interest fused to a green or a yellow fluorescent protein and a red fluorescent protein in separate wells. After four hours remove the growth medium and wash each well gently with one milliliter of PBS supplemented with magnesium and calcium dropping PBS on the well edge to prevent detachment of the cells during washing. Then remove the PBS.

Add approximately 50 microliters of EDTA solution drop wise to each well to facilitate detachment of the cells. After incubating at 37 degrees Celsius for two minutes, slowly shake the six-well plate laterally to detach the cells. Next, add 950 microliters of growth medium to each well and resuspend the cells by pipetting a few times up and down thereby detaching all cells from the well bottom.

Ensure that cells are resuspended properly and detached from each other by visually checking for the absence of large cell aggregates after resuspension. Transfer the cell solution of one well to the corresponding well. Mix gently by pipetting a few times up and down.

Then seed the mixed cells on a 35-millimeter glass bottom dish and culture the seeded cells at 37 degrees Celsius in 5%carbon dioxide for one day. In the laser scanning confocal microscope software set up the optical path. To avoid spectral crosstalk select two separate tracks to excite and detect mEGFP or mEYFP and mCherry or mCardinal sequentially and select Switch tracks every Line.

For the detection use appropriate filters for both channels. Place the dish containing the mixed cells on the sample holder. After waiting 10 minutes to ensure temperature equilibration and to reduce focus drift, focus on the cells using the transmission light in the Locate menu.

Search for a pair of red and green cells in contact with each other. Next, select a scan path perpendicular to cell-to-cell contact using the Crop button. Zoom to achieve a pixel size of 50 to 200 nanometers and select Line in Scan mode.

Set frame size to 128 by one pixels. Set scan speed to the maximum allowed value. Then set cycles to 100, 000 to 500, 000.

Following this, choose the appropriate laser powers. Set the detectors to Photon Counting mode. Press Start Experiment to start the acquisition.

Export the raw data files to an RGB TIF image in raw data format. This file will contain a kymograph with the green and red channel data in the channel termed G and R of the image, respectively. Next, import the TIF file with the appropriate analysis software and proceed to perform the analysis.

Align the lines by performing a segment-wise time average with blocks of 500 to 1000 lines. Determine the membrane position in each block. Then shift all blocks to the same lateral position.

Sum up all aligned lines along the time axis and fit the average intensity profile using a Gaussian function. Define the pixels corresponding to the membrane as all pixels within plus or minus 2.5 sigma of the membrane position, and sum up the intensity of these pixels in each line obtaining a single fluorescent signal value for each time point. If photo bleaching is observed, apply a bleaching correction by fitting the membrane fluorescence time series with a double exponential function and applying the appropriate correction formula.

Calculate the auto and cross-correlation functions according to the appropriate equations. To improve the reliability of the analysis and avoid artifacts perform the calculations for 10 to 20 equal segments of the total measurement. Inspect the fluorescence time series and correlation functions in each segment and remove clearly distorted segments.

Finally, average all non-distorted segments. While analyzing your data, carefully inspect the intensity time series and correlation functions from each segment to avoid distortions, for instance, due to intracellular vesicles in the membrane periphery. An intensity image of HEK 293T cells expressing myristolyated-palmitoylated-mEYFP or mCardinal is shown here.

A relative cross-correlation close to zero was observed while the autocorrelation functions show characteristic decay times of 10 to 20 milliseconds for the diffusion of myristolyated-palmitoylated-mEYFP and mCardinal in the plasma membrane. The cross-correlation functions of the sFCCS measurements of HEK 293T cells expressing membrane-anchored heterodimer myristolyated-palmitoylated-mCardinal-mEYFP had positive amplitudes and showed similar decay times as the autocorrelation function. sFCCS measurements on cell-to-cell contacts between APLP1-mEYFP and APLP1-mCardinal-expressing cells resulted in a positive relative cross-correlation of 0.45.

The sFCCS correlation functions obtained from measurements across APLP1 clusters at cell-cell contacts showed strongly reduced dynamics as evident from large decay times in the oscillations at large lag times. Upon zinc ion addition, the relative cross-correlation increased to an average value of 0.8. Further the molecular brightness significantly increased from small oligomers to larger multimers consisting of 10 to 50 monomers on each cell.

Transient instabilities may result in a cross-correlation function having negative values or a high false positive cross-correlation. The corresponding correlation functions typically deviate strongly from those of the majority of segments. As a complementary approach to sFCCS cross-correlation number and brightness can be used to detect protein-protein interactions.

While attempting this procedure it's important to remember that mixing of transfected cells is a critical step. Gently mix the cells and optimize transfection efficiencies to increase the chance of finding red and green cells in contact. After its development this technique has been used by researchers in the field of immunology to explore the interactions of signaling molecules in immunological synapses.