Source: Dunsing, V. et al. A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts. J. Vis. Exp. (2018).
This video demonstrates the use of fluorescence fluctuation spectroscopy to detect the interaction among cell surface proteins at cell-cell contacts. By expressing the transmembrane adhesion receptor of interest labeled with a fluorescent protein and mixing two different cell populations harboring two spectrally separated fluorescent labels, the trans-interaction between the receptors of two neighboring cells with different-colored fluorescence is assessed via cross-correlation in the fluctuations of fluorescence intensity.
1. Sample Preparation: Cell-Cell Mixing Assay
NOTE: The following protocol describes the mixing procedure for adherent cells. It may be modified for cells cultured in suspension.
2. Sample Preparation: Positive Control for Cross-Correlation Experiments and Homo-Dimer Construct for Brightness Analysis
3. Confocal Laser Scanning Microscopy: Setup and Focal Volume Calibration
NOTE: The following protocol is written for experiments performed with mEGFP/mEYFP and mCherry/mCardinal on the laser scanning confocal microscope used in this study. The optical setup, the software settings (laser lines, dichroic mirrors, filters), and the choice of calibration dyes may be modified for other FPs and microscope setups.
4. Scanning Fluorescence Cross-Correlation Spectroscopy: Acquisition
NOTE: The following protocol is written for experiments performed with mEGFP/mEYFP ('green') and mCherry/mCardinal ('red') on the laser scanning confocal microscope used in this study. The optical setup and the software settings (laser lines, dichroic mirrors, filters) may be different for other FPs or microscope setups.
Figure 1. Experimental workflow and schematic representation of scanning fluorescence cross-correlation spectroscopy and cross-correlation number and brightness analysis at cell-cell contacts. (A) Scheme of sample preparation: Two cell populations transfected with the protein of interest (e.g., APLP1) fused to two spectrally distinct fluorescent proteins (e.g., mEYFP and mCardinal) are mixed after transfection. Contacts of differently transfected cells are selected in the microscopy experiments. To avoid interference with extracellular binding domains, the fluorescent protein should be fused to the intracellular terminus of the protein of interest. (B) Scanning FCCS (sFCCS) measurements are performed perpendicular to the cell-cell contact in two spectral channels (channel 1, green, and channel 2, red). Scan lines (represented as kymographs) are aligned and membrane pixels summed. Then, ACFs and CCFs are calculated from the intensity traces Fi(t). ACFs are represented in red and green. CCF is represented in blue. (C) Cross-correlation N&B (ccN&B) acquisition results in a three-dimensional (x-y-time) image stack. An ROI is selected around the cell-cell contact. Then channel and cross-correlation brightness (ε1, ε2, and Bcc) values are calculated in each cell-cell contact pixel. The results are then visualized as histograms, pooling all selected pixels.
Figure 2. Scanning fluorescence cross-correlation spectroscopy control measurements. (A) Representative images of mixed HEK 293T cells expressing myr-palm-mEYFP/-mCardinal as a negative control for trans interactions. The yellow arrow indicates the sFCCS scan path. Scale bars are 5 µm. (B) Representative images of HEK 293T cells expressing myr-palm-mCardinal-mEYFP hetero-dimer (left: green channel, right: red channel) as positive cross-correlation control. The yellow arrow indicates the sFCCS scan path. Scale bars are 5 µm. (C) Representative CFs (green: ACF in the green channel (mEYFP), red: ACF in the red channel (mCardinal), blue: CCF) obtained in sFCCS measurements for negative control. Solid lines show fits of a two-dimensional diffusion model to the CFs. (D) Representative CFs (green: ACF in the green channel (mEYFP), red: ACF in the red channel (mCardinal), blue: CCF) were obtained in the sFCCS measurement of the positive control. Solid lines show fits of a two-dimensional diffusion model to the CFs.
The authors have nothing to disclose.
DMEM growth medium | PAN-Biotech | P04-01548 | |
DPBS w/o: Ca2+ and Mg2+ | PAN-Biotech | P04-36500 | |
DPBS w: Ca2+ and Mg2+ | PAN-Biotech | P04-35500 | |
Trypsin EDTA | PAN-Biotech | P10-023100 | |
TurboFect Transfection Reagent | Thermo Fisher Scientific | R0531 | |
HEK 293T cells | DSMZ | ACC 635 | |
Alexa Fluor 488 NHS Ester | Thermo Fisher Scientific | A20000 | |
Rhodamine B | Sigma-Aldrich | 83689-1G | |
Plasmid DNA | Addgene | NA | See Dunsing et. al., MBoC 2017, for a detailed description of all plasmids |
6-well plate | Starlab | CC7672-7506 | |
35-mm glass bottom dishes | CellVis | D35-14-1.5-N | |
Zeiss LSM780 confocal | Carl Zeiss | NA | |
Neubauer cell counting chamber | Marienfeld | 640110 |