Line-FRAP, A Versatile Method to Measure Diffusion Rates In Vitro and In Vivo
| Autor: | Ariane Nunes-Alves, Gideon Schreiber, Vladimir Kiss, Rebecca C. Wade, Shir Marciano, Debabrata Dey |
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| Rok vydání: | 2021 |
| Předmět: |
Materials science
Microscope Time Factors Confocal Green Fluorescent Proteins In Vitro Techniques law.invention Diffusion 03 medical and health sciences 0302 clinical medicine Bacterial Proteins Structural Biology In vivo Confocal microscopy law Osmotic Pressure Escherichia coli Osmotic pressure Humans Diffusion (business) Molecular Biology 030304 developmental biology 0303 health sciences Molecular diffusion Proteins Solutions Luminescent Proteins Protein Transport Macromolecular crowding Biological system 030217 neurology & neurosurgery Fluorescence Recovery After Photobleaching HeLa Cells |
| Zdroj: | Journal of molecular biology. 433(9) |
| ISSN: | 1089-8638 |
| Popis: | The crowded cellular milieu affect molecular diffusion 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, combined with rigours data analysis, is able to determine diffusion coefficients in a variety of environments, from in vitro to in vivo. The use of Line mode greatly improves time resolution of FRAP data acquisition, from 20-100 Hz in the classical mode to 800 Hz in the line mode. This improves data analysis, as intensity and radius of the bleach at the first post-bleach frame is critical. We evaluated the method on different proteins labelled chemically or fused to YFP in a wide range of environments. The diffusion coefficients measured in HeLa and in E. coli were ~2.5-fold and 15-fold slower than in buffer, and were comparable to previously published data. Increasing the osmotic pressure on E. coli further decreases diffusion, to the point at which proteins virtually stop moving. The method presented here, which 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. |
| Databáze: | OpenAIRE |
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