Diffusion and Exchange of Non-Integral Membrane Associated Fluorophores During Fluorescence Recovery After Photobleaching with the Confocal Laser Scanning Microscope: ROI Size Analysis of EGFP:Ras2 Plasma Membrane Diffusion in Saccharomyces cerevisiae

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein localization in cellular membranes. In this study, we developed numerical simulations of lateral diffusion of a fluorophore in a membrane with arbitrary bleach geometry and exchange of the fluorophore with cytosol during Fluorescence Recovery after Photobleaching (FRAP) experiments. Based on our model simulations, we designed and performed FRAP experiments with varying bleach region sizes on plasma-membrane localized EGFP:Ras2 in live yeast cells to investigate the mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated from a 1 micron x 1 micron bleach region-of-interest (ROI) size successfully predicted the 0.5 micron x 0.5 micron bleach ROI experiment without additional fitting. Successful prediction of the second experiment without data fitting shows the agreement of the experiment with the theory and excluded alternative models including both diffusion and binding, which were also tested against the experimental data. We also performed Fluorescence Correlation Spectroscopy (FCS) experiments as an independent method to measure the mobility of EGFP:Ras2. We show that simulation of FRAP experiments based on the mobilities and fluorophore fractions derived from FCS model fits enables the validation of the FCS model. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane associated fluorophores using FRAP on widely available commercial confocal laser scanning microscopes.