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Living cells are 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 coefficients at physiological protein concentrations within cells, each with its limitations. Here, we show that Line-FRAP, a method based on measuring recovery of photobleaching under a confocal microscope that allows diffusion coefficient measurements in a variety of environments, from in vitro to in vivo. The use of Line mode greatly improves the time resolution in of FRAP data acquisition, from 20-50 Hz in the classical mode to 800 Hz in the line mode. We also introduce an updated method for data analysis to obtain diffusion coefficients in various environments, with the number of pixels bleached at the first frame after bleaching being a critical parameter. 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 in vitro, in HeLa cells and in E.coli. Diffusion coefficients in HeLa was ~2.5-fold slower and in E.coli 15-fold slower than measured in buffer and were comparable to previously published data. Moreover, we show that increasing the osmotic pressure on E.coli further decreases diffusion, to the point at which proteins virtually stop moving. Next, we investigated the diffusion behavior of small organic molecule drugs. The diffusion rates of these molecules differed greatly in crowding conditions and living cells from the expected, pointing towards interactions of the small molecules with the surrounding. Micrographs have shown many of these molecules to accumulate in the lysosomes of cells, explaining their extremely slow diffusion. These findings are relevant for drug design, as the observed phase separation would make the small molecules not accessible to their targets. The method presented here requires a confocal microscope equipped with dual scanners, can be applied to study a large range of molecules with different sizes, and provides robust results in a wide range of environments and protein concentrations for fast diffusing molecules.
Reference: [1] D. Dey, S. Marciano, A. Nunes-Alves, V. Kiss, R. C. Wade and G. Schreiber, Line-FRAP, a versatile method to measure diffusion rates in vitro and in vivo, Journal of Molecular Biology, 433, 9, 2021, 166898.