Line-FRAP, a versatile method based on fluorescence recovery after photobleaching to measure diffusion ratesin vitroandin vivo

Abstract

<jats:title>Abstract</jats:title><jats:p>A cell is a densely packed conglomerate of macromolecules, where diffusion is essential for their function. The crowded conditions may affect diffusion both through hard (occluded space) and soft (weak, non-specific) interactions. Multiple-methods have been developed to measure diffusion rates at physiological protein concentrations within cells, however, each of them has its limitations. Here, we introduce Line-FRAP, a method based on measuring recovery of photobleaching under a confocal microscope that allows diffusion rate measurements in versatile environments using standard equipment. Implementation of Line mode to the classical FRAP technique improves the time resolution in data acquisition. We also introduce an updated method for data analysis. Together, these contribute towards better estimation of Diffusion coefficients in a wide range of environments. We evaluated the method using different proteins either chemically labelled or by fusion to YFP. The calculated diffusion rates were comparable to literature data as measured <jats:italic>in vitro</jats:italic>, using artificial crowders, in HeLa cells and in <jats:italic>E.coli</jats:italic>. Diffusion coefficients in HeLa was ~2.5-fold slower and in <jats:italic>E.coli</jats:italic> 15-fold slower than measured in buffer. Moreover, we show that increasing the osmotic pressure on <jats:italic>E.coli</jats:italic> further decreases diffusion by 2-3 fold. The method presented here is easy to apply on a standard confocal microscope, fits a large range of molecules with different sizes and provides robust results in any conceivable environment and protein concentration.</jats:p>