Line-FRAP, a versatile method to measure diffusion rates in vitro and in vivo

• Verfasst von: Dey, Debabrata [VerfasserIn]
• Verfasst von: Marciano, Shir [VerfasserIn]
• Verfasst von: Nunes-Alves, Ariane [VerfasserIn]
• Verfasst von: Kiss, Vladimir [VerfasserIn]
• Verfasst von: Wade, Rebecca C. [VerfasserIn]
• Verfasst von: Schreiber, Gideon [VerfasserIn]
• Titel: Line-FRAP, a versatile method to measure diffusion rates in vitro and in vivo
• Verf.angabe: Debabrata Dey, Shir Marciano, Ariane Nunes-Alves, Vladimir Kiss, Rebecca C. Wade and Gideon Schreiber
• E-Jahr: 2021
• Jahr: 27 February 2021
• Umfang: 20 S.
• Fussnoten: Gesehen am 09.06.2021
• Titel Quelle: Enthalten in: Journal of molecular biology
• Ort Quelle: Amsterdam [u.a.] : Elsevier, 1959
• Jahr Quelle: 2021
• Band/Heft Quelle: 433(2021), 9, Artikel-ID 166898, Seite 1-20
• ISSN Quelle: 1089-8638
• DOI: doi:10.1016/j.jmb.2021.166898
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: eukaryotic cell
• Sach-SW: FRAP
• Sach-SW: macromolecular crowding
• Sach-SW: prokaryotic cell
• Sach-SW: protein diffusion
• K10plus-PPN: 1760107808

Abstract

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.