研究目的
To measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts, using fluorescence fluctuation spectroscopy techniques.
研究成果
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. It provides molecular specificity and information by detection of fluorescent proteins genetically fused to the protein of interest, with no requirement for the fluorescent labels to be localized on the extracellular side.
研究不足
The stability of the system needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes. The presence of diffusive dynamics is an essential condition for the method to work. The technique may be pushed to its limits by very slow protein complexes.
1：Experimental Design and Method Selection
The assay combines fluorescence cross-correlation approaches (scanning fluorescence cross-correlation spectroscopy (sFCCS), cross-correlation number and brightness (ccN&B)) and mixing of cells expressing a fusion construct of the protein of interest, e.g., an adhesion receptor. The investigated receptors in the two interacting cells are labeled with two spectrally separated fluorescent proteins (FPs), from the intracellular side.
2：Sample Selection and Data Sources
Adherent cells (e.g., HEK 293T cells) are transfected with plasmids for the protein of interest fused to a 'green' or 'red' fluorescent protein. Cells are then mixed and seeded on glass bottom dishes for microscopy experiments.
3：List of Experimental Equipment and Materials
Confocal laser scanning microscope, fluorescent dye solutions, 35-mm glass bottom dishes, water immersion objective with NA 1.2, photon counting or analog detectors.
4：Experimental Procedures and Operational Workflow
The procedure involves cell mixing, transfection, seeding on glass bottom dishes, and fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. Detailed steps include instrument calibration, data acquisition, and analysis.
5：Data Analysis Methods
Data analysis involves alignment algorithms correcting for lateral cell movement, determination of the position with maximum fluorescence signal, and calculation of auto- and cross-correlation functions (ACFs/CCFs) of the fluorescence signals. A two-dimensional diffusion model is fitted to all correlation functions.
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