ReviewProtein dynamics in the nuclear compartment
Introduction
Until recently, protein movement within the eucaryotic nucleus has been viewed in terms of factor shuttling between static locations. After synthesis in the cytoplasm and transport to the nucleus, many current models envisage transcription factors and associated proteins in stable complexes that are bound to chromatin for relatively long periods of time. This view of the nucleus as a highly organized environment with many chromosome locations occupied by static complexes has been eclipsed as a result of recent findings that monitor the actual movement of proteins in real time. With the advent of fluorescent protein tags that permit the observation of proteins in living cells, it has become apparent that many, if not most, nuclear proteins are moving quickly, and exchanging rapidly with a variety of nuclear targets.
A large number of proteins have now been studied with this real-time, live-cell, approach. We discuss examples that show that the vast majority of nuclear proteins are highly dynamic, and exhibit both rapid movement in the nucleoplasmic space and fast exchange with a variety of targets. For the group of nuclear proteins involved in gene targeting and transcriptional regulation, interactions with the genome also appear to be dynamic. These views are in conflict with some aspects of current models of transcriptional regulation. Specifically, the presence of stable multi-protein complexes with long residence times appears inconsistent with the data available from living-cell experiments. We discuss this dichotomy, and suggest potential mechanisms consistent with the data.
Section snippets
Tracking nuclear proteins in real time
By tagging proteins with the green fluorescent protein (GFP), it is possible to follow the intracellular movement of these molecules in living cells. Assuming that attachment of the GFP moiety to a factor does not significantly alter the properties of the protein, this becomes an attractive method to observe protein movement and factor interactions in real time. Surprisingly, many GFP-tagged nuclear proteins and transcription factors seem to function with little impairment. For example, all of
Chromatin components
Chromosomes and the basic building blocks of the chromatin fiber also demonstrate an unexpectedly dynamic behavior. In contrast to the impression that chromosomes are immobile and fixed in certain positions, these structures undergo significant diffusive motion in the nucleus [18•]. This motion is constrained to a limited subregion of the nucleus and is resistant to metabolic inhibitors such as sodium azide, suggesting a classical Brownian motion as the basis for the movement.
The behavior of
Protein mobility on specific chromosome targets
Photobleaching studies carried out on general compartments (the cytoplasm and the nucleus) afford insight into the mobility of factors within a region of the compartment, but specific targets of interaction must be inferred. Direct targeting of a fluorescent molecule to a single-copy gene with a background of thousands of nuclear molecules is beyond the techniques available. Two systems have appeared, however, that allow visualization of binding events on repeated elements. Belmont and
Long-term residency versus rapid exchange
The results obtained with photobleaching experiments in living cells suggest that the association of some transcription factors with their binding sites is highly transient, with occupancy times of a few seconds. This view contrasts significantly with the more traditional view of promoter-bound complexes as highly stable multi-protein aggregates with long half-lives. Indeed, the enhanceasome model [28] proposes specifically that members of the small HMG-IY family associate simultaneously with
Underlying mechanisms of factor movement
Rapid exchange has now been observed for several components of the local chromosome domain, including general chromatin components 19., 21., and sequence-specific DNA-binding proteins 24., 26••.. What mechanisms are involved in the surprisingly dynamic movements of these proteins? Two models have recently emerged that offer promising leads into the mechanism of rapid exchange. Fletcher et al. [33••] report that the GR can be displaced from chromatin assembled in vitro in an ATP-dependent
Conclusions
The classic view of the nucleus as a ‘quiet’ environment, with many proteins located in static structures, is clearly in need of revision. It is now apparent that the nucleus is a maelstrom of rapidly moving proteins, most of which likely exist in metastable states with many partners, and are exchanging rapidly between many multi-protein complexes. In particular, it appears that many of the factors involved in regulating gene activity, as well as more general structural components of the
References and recommended reading
Papers of particular interest, published within the annual period of review,have been highlighted as:
• of special interest
•• of outstanding interest
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