The cell-based assay allows for the observation and quantification of trans adhesion interactions without the need for lengthy protein purifications or specialized equipment. Although the protein interactions tested here are relevant to neurobiology, this method may be applied to any area of research where protein adhesion is occurring intercellularly. 48 hours after transfecting the HEK-293T cells, harvest the cells from the six-well plate for aggregation.
First, wash each well twice with PBS. Next, to gently disassociate the cells, add one milliliter of 10 millimolar EDTA to each well, then incubate the plate at 37 degrees Celsius. After five minutes of incubation, gently tap the plate to detach the cells and harvest each well into a separate 15 milliliter conical tube.
Centrifuge the tubes at 500 x g at room temperature for five minutes. While the cells are pelleting, prepare six incubation tubes by labeling the tops of microcentrifuge tubes with the experimental conditions. Every permutation of GFP and mCherry should be used.
Remove the supernatant from the 15 milliliter conical tubes and resuspend the cells in 500 microliters of medium. Using a hemocytometer, count the cells in each conical tube. Then aliquot 200, 000 cells from each condition into the appropriate incubation tube for a one-to-one mix in a total volume of 500 microliters.
After assessing baseline aggregation described in the next section, place the incubation tubes in a slow tube rotator at room temperature. To assess aggregation, at baseline and again after 60 minutes, pipette 40 microliters from the incubation tube onto a charged microscope slide. Baseline acquisition should be done as quickly as possible after the addition of cells to the microcentrifuge tube.
Image the slide under fluorescence in both the green and red channels. For each slide, capture images of three different fields of view in one focal plane. HEK-293T cells were transfected with a protein of interest, a mutated protein of interest, or a ligand of interest and co-transfected with a fluorescent protein.
Cell populations expressing the protein of interest were mixed with cell populations expressing the ligand of interest and assessed for aggregation after 60 minutes. In overlays of images captured in the green and red channels, aggregation appears as yellow puncta. Conditions in which cells were not expressing any synaptic ligands showed minimal aggregation.
Also, minimal aggregation was exhibited when only one of the two populations were expressing synaptic ligands. No aggregation was exhibited when the two populations of cells expressed incompatible adhesion molecules. Conditions with compatible adhesion molecules exhibited significant aggregation after a 60 minute incubation.
Surprisingly, the mutated protein of interest exhibited significantly more aggregation than its wild-type counterpart, suggesting that the point mutation enhances the proteins binding capabilities. Mutations in many adhesion molecules are commonly linked to neurodevelopmental, neuropsychiatric, and addiction disorders. With this technique, one can screen the effects of disease relevant point mutations on trans interactions.
