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Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells

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Item Type:Article
Title:Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells
Creators Name:Gruenwald, D. and Spottke, B. and Buschmann, V. and Kubitscheck, U.
Abstract:Uridine-rich snRNPs are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single molecule sensitivity. The single particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5-8 microm(2)/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, respectively U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.
Keywords:Single particle tracking, Single molecule tracking, Single molecule detection, Nuclear architecture, Intracelluar trafficking, Pre-mRNA splicing, U snRNPs
Source:Molecular Biology of the Cell
Publisher:American Society for Cell Biology
Page Range:5017-5027
Date:1 December 2006
Additional Information:Copyright (c) 2006 by The American Society for Cell Biology
Official Publication:https://doi.org/10.1091/mbc.E06-06-0559
PubMed:View item in PubMed

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