"All routes into the cell have characteristic pH profiles," Bruchez said. "Our pH-biosensor allows us to determine whether the tagged protein in this case a surrogate antigen is moving through neutral compartments into the cell, or through acidic compartments into the cell. Those sorts of things determine whether the antigen enters the cell through an active endocytic process, a phagocytic process, or a caveolar uptake process."
In the current study, researchers tagged a surrogate antigen on the surface of a dendritic cell with the FAP. They added the pH sensitive dye, causing the FAP antigen to glow green, an indication of a neutral pH. As the antigen and its bound dye passed to a separate dendritic cell, the antigen/FAP complex glowed red, indicating it used an acidic pathway to enter the new cell. This change in pH from neutral to acidic reveals that antigens are passed between cells through an active endocytic process.
"Once it's nibbled by the acceptor cell, the antigen goes through this endocytic pathway where it can potentially then be reprocessed and re-displayed on the surface of the receptor cell," Bruchez said.
The new biosensor's activity is novel, Bruchez said, because it binds to its target with nanomolar affinity, becomes fluorescently activated, and then is carried into the cell under endocytic conditions, reporting on the pH as it goes. The researchers are hopeful that this technology is the first in a platform of targetable environmental sensors. The current biosensor can read out pH, but this approach could be extended to measure calcium or other ion fluctuations in living cells. According to Bruchez, there are many ways that this basic chemical concept can be extended.
|Contact: Jocelyn Duffy|
Carnegie Mellon University