title: Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein.
abstract: All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 microm2/s up to approximately 100 microm2/s, and found significant changes in free protein movement during cell-cycle progression.
authors.affiliation: Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Kita-12 Nishi-6 Kita-ku, Sapporo, Hokkaido, 060-0812, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Matsuda
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
mesh_terms: D002453:Cell Cycle / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D011487:Protein Conformation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013347:Subcellular Fractions / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins