Published: December 1st, 2018
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 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.
Many biological processes occur at the sites of cell-cell interactions, e.g., cell-cell adhesion1,2,3, cell-cell fusion4 and cellular recognition5. Such events are particularly important during the development of multicellular organisms and for cell-cell communication, e.g., during immune responses. These processes are typically mediated by proteins that are localized at the surface, i.e., at the plasma membrane (PM) of neighboring cells and undergo specific interactions at the cell-cell contact that a....
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.
A first test for the protein-protein interaction assay, i.e., mixing of cells expressing spectrally distinct fluorescent proteins followed by sFCCS/ccN&B measurements (Figure 1), should be performed on proteins that are not expected to interact at the cell-cell contact (i.e., a negative control). Therefore, HEK 293T cells expressing myristoylated-palmitoylated-mEYFP (myr-palm-mEYFP) or -mCardinal were mixed and sFCCS was performed across.......
The experimental procedure described here allows the investigation of protein-protein trans interactions at cell-cell contacts, employing fluorescence fluctuation spectroscopy techniques, namely sFCCS and ccN&B. These methods involve a statistical analysis of fluorescence fluctuations emitted by two spectrally separated FPs fused to the protein(s) of interest at a contact of two neighboring cells, each expressing one or the other fusion protein. The presence of trans complexes is quantified by probi.......
|DMEM growth medium
|DPBS w/o: Ca2+ and Mg2+
|DPBS w: Ca2+ and Mg2+
|TurboFect Transfection Reagent
|Thermo Fisher Scientific
|HEK 293T cells
|Alexa Fluor 488 NHS Ester
|Thermo Fisher Scientific
|See reference 12 (Dunsing et. al., MBoC 2017),for a detailed description of all plasmids
|35-mm glass bottom dishes
|Zeiss LSM780 confocal
|MATLAB software package
|Neubauer cell counting chamber
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