Vesicle formation processes on the Golgi significantly altered by overexpression - Duel FRAP investigation*

Overexpression of a target protein is commonly used in fluorescence-based experiments, such as fluorescence-recovery-after-photobleaching (FRAP), to boost the fluorescence intensity. Since the order of magnitude of protein concentration is artificially altered, the biochemical processes under investigation might be altered as well. We have studied how overexpression could affect the way we understand vesicle formation using FRAP experiments and kinetic Monte Carlo simulation based on continuous-time random walk. Specifically, we are investigating two proteins that are vital during initial vesicle formation on the Golgi (GBF1 and Arf1). Traditionally, either GBF1 or Arf1 would be overexpressed during FRAP, but we also performed dual FRAP where both proteins are overexpressed and measured simultaneously. Comparing the single versus dual FRAP measurements with the computer simulation and a simple mathematical model, we show a significant difference between the two cases. More importantly, the difference would affect the way vesicle formation is understood in cell biology. This shows overexpression can affect biochemical dynamics enough to alter our conclusion(s) from the data.