Molecular dynamics in the nucleus
Important processes occur in the nucleus, including replication, transcription and ribosome assembly, yet the molecular dynamics within it remain poorly understood, according to a review written by researchers from the University of Dundee in the UK.
Although initially hampered by technological limitations, nuclear studies have been eased by advancements in proteomic methods and in live-cell imaging using fluorescent proteins. These techniques provide complementary information. Whereas imaging using fluorescent proteins can reveal time-dependent changes in the subcellular location of individual proteins within the cell, mass spectrometry has been limited to characterizing global populations of proteins, either in the whole cell or in a purified structure, without regard to changes over time. New “second generation” proteomics methods based on differential isotope labeling under two or more experimental conditions are providing further information, such as protein dynamics, turnover and interactions.
In the nucleus, fluorescent proteins have been used to monitor processes such as transport by cargo proteins and to characterize chromatin volume and density and other nuclear properties. Förster resonance energy transfer using pairs of fluorescent proteins has been used to elucidate protein-protein interactions, and fluorescent proteins used with lac or tet operon systems have been used to study gene expression. Photobleaching and photoactivation have been exploited to determine rates of protein movement, including promoter binding. Photobleaching over time also has been used to study DNA repair proteins. Proteomic methods have been used to identify transcription factors, DNA-associated proteins and components of the nucleolus.
As imaging and proteomics technology improve, nuclear studies will benefit. Current research aims to improve the spatial resolution of optical microscopy to a level approximating that of electron microscopy and to improve the sensitivity of proteomics down to the level of a single cell. (Science, Nov. 30, 2007, pp. 1402-1407.)
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