Pictures in cell biology

pictures in cell biology Pictures in cell biology Moving right along in the nucleus One of the long-standing open questions regarding the mammalian cell nucleus has been: how do proteins move within the nucleus? While it is intuitive to think that proteins in the cytoplasm ‘float’ through space, it has been difficult to imagine how proteins make their way through a nucleus that is densely packed with DNA, RNA and proteins. Determining the speed and the mechanisms of movement in the nucleus would go a long way towards understanding how nuclear processes are organized and coordinated – but would also be key to understanding how nuclear compartments, such as the nucleolus, form. Significant steps towards uncovering how proteins move in the nucleus have been made recently1,2. The key advance is the ability to make movement of nuclear proteins visible. This can be achieved by a photobleaching technique termed fluorescence recovery after photobleaching (FRAP). In cells expressing fusion proteins between the green fluorescent protein (GFP) and a nuclear protein of interest, an area containing fluorescent protein is rapidly bleached and the kinetics of fluorescence signal recovery recorded by repeated imaging of the bleached area. If a protein moves rapidly, the bleached area will rapidly fill up with mobile molecules that are moving in from the vicinity of the bleached spot. Therefore, the recovery of fluorescence intensity will be rapid. On the other hand, recovery of fluorescence signal is slow for slowly moving proteins. FRAP analysis of proteins that are involved in entirely different nuclear functions (chromatin remodelling, pre-mRNA splicing, rRNA processing) reveals that they all move rapidly within the nucleoplasm. Importantly, movement is independent of energy, suggesting that nuclear proteins move by a passive, diffusion-based mechanism. However, the proteins move more slowly than would be expected based on their molecular mass. This cannot be due to ‘bumping’ of molecules into any intranuclear structures such as components of a static nuclear matrix or chromosomes because a GFP molecule alone, which is very unlikely to interact specifically with any nuclear component, moves as expected for its molecular mass. This observation demonstrates powerfully that free diffusion within the nucleus is possible despite the assumed high packing density. Similarly high mobility has been reported previously for microinjected dextrans3. Free movement within the nucleus is corroborated by the observation that large overexpression-induced protein aggregates can move rapidly2 and that RNP particles move freely within the nucleus4. It seems likely therefore that nuclear proteins continuously roam the nucleus in search of appropriate binding partners, and the slowing down is due to the continuous interaction of proteins with binding partners such as other proteins, RNA or DNA. Similar photobleaching techniques have also been used to investigate the morphogenesis of subnuclear compartments. Time-lapse microscopy has previously established that the compartments do not move around very much. It has now been demonstrated that proteins continuously and rapidly associate and dissociate with nuclear compartments and that the morphology of the compartments is dictated by the function of the proteins1,2. In other words, although the nuclear compartments appear stable, there is a continuous flux of proteins through each compartment (Fig. 1). Photobleaching experiments are a novel, powerful tool in cellbiological studies of nuclear proteins. Their relative simplicity makes it likely that they will become widely used and will contribute greatly to answering many important questions in the field of nuclear architecture and nuclear function. References 1 Phair, R.D. and Misteli, T. (2000) High mobility of proteins in the mammalian cell

nucleus. Nature 404, 604–609 2 Kruhlak, M.J. et al. (2000) Mobility of the ASF splicing factor in live cells. J. Cell Biol. 150, 41–51 3 Seksek, O. et al. (1997) Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138, 131–142 4 Politz, J. et al. (1999) Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells. Curr. Biol. 9, 285–291

FIGURE 1 Pseudocolour representation of fluorescence intensity during a fluorescence recovery after photobleaching (FRAP) experiment. A small area (red circle) is bleached with a high-power laser pulse. Recovery is monitored by time-lapse imaging. The fluorescence signal from a fusion protein between the green fluorescent protein (GFP) and the chromatin-binding protein HMG-14 or GFP and the nucleolar protein B23 recovers rapidly after a short bleach pulse in a small nuclear area (red circle). Areas outside of the bleached spot become dimmer over time owing to movement of fluorescent protein into the bleached area. The rapid reappearance of GFP–B23 fluorescence in the nucleolus directly demonstrates that this apparently stable nuclear subcompartment is in continuous flux.

Contributed by Tom Misteli, National Cancer Institute, NIH, 41 Library Drive, Bldg 41, Bethesda, MD 20892, USA. E-mail: [email protected]

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trends in CELL BIOLOGY (Vol. 10) September 2000