Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts
DEŞİFRE METNİ
Cell-cell interactions — mediated by proteins at neighboring cell surfaces — can be studied in live cells using a fluorescence fluctuation spectroscopy assay.
First, take separate cell cultures expressing an adhesion receptor — a transmembrane protein. The receptor in the cells from the two populations is labeled with spectrally-separated fluorescent proteins. These fluorescent labels are present on the receptor's cytoplasmic region, preventing interference with the cell-cell interaction.
Discard the medium and add trypsin, facilitating cell detachment. Mix the two cell populations and incubate, allowing the neighboring cells' receptors to form homotypic trans-interactions.
Place the cells under a confocal microscope. Illuminate the sample using laser sources of appropriate wavelengths, causing emission from both fluorescent labels. Locate two neighboring cells with different-colored fluorescence signals in contact.
Acquire a line scan — perpendicular to the region of cell membranes in contact — using the spectral channel of both fluorescent labels. The laser focuses on a small volume of the sample — the detection volume. As the fluorescent-labeled receptors diffuse in and out of the detection volume, the fluorescence intensity fluctuates.
If the receptors of the neighboring cells do not interact, they move individually, leading to independent fluctuations in their fluorescence intensity with a low cross-correlation.
If the neighboring cells in contact interact via their receptors, the interacting proteins diffuse together, causing a correlated fluctuation in their fluorescence intensity.
