Dissecting the Nucleolus

The Faculty of 1000 is a Web-based literature awareness tool published by BioMed Central. It provides a continuously updated insider's guide to the most important peer-reviewed papers within a range of research fields, based on the recommendations of a faculty of more than 1,400 leading researchers. Each issue, The Scientist publishes a review, like the one above, that examines related papers in a single field. We also publish a selection of comments on interesting recent papers from the Faculty of 1000's output. For more information visit www.facultyof1000.com. Using cutting-edge technology and creative adaptations of older techniques, two groups recently uncovered new dynamics and complexity in the nucleolus—a nuclear organelle that organizes around the genes that encode ribosomal RNA during each cell cycle. Their work provides a compelling framework for future research on the function of nucleolar proteins, as well as large-scale proteomics in general. The nucleolus has long been known to play a role in the transcription and processing of large ribosomal RNAs and ribosome assembly. However, according to Lucio Comai, assistant professor of molecular microbiology and immunology at the University of Southern California, other nucleolar functions are being revealed, including regulatory sequestering of cell cycle proteins. To shed light on these other possible nucleolar functions, researchers in the labs of Angus Lamond, professor at the University of Dundee and Matthias Mann, professor at the University of Southern Denmark, biochemically isolated nucleolar proteins, and then used a combination of mass spectrometry and sequence database searches to identify 271 nucleolar proteins.1 "So far, this approach had been applied to the identification of [proteins within] multiprotein complexes," says Comai. "In this case, the approach has been applied to identify proteins within an organelle. It is several orders of magnitude over what has been done so far." Although this catalog of proteins could keep researchers busy for years, the authors note that it is unlikely to be the complete nucleolar proteome. Comai, a Faculty of 1000 member, reviewed the Lamond et al. paper for F1000. With the proteome in hand, researchers then compared it to that of cells treated with Actinomycin D, which inhibits transcription, and found a number of differences. "The differences show that things are changing over time in the nucleolus. It's a very dynamic structure," says Marilyn Parsons, associate director of the Seattle Biomedical Research Institute. In a separate paper, which Parsons reviewed for F1000, Danyang Chen and Sui Huang at Northwestern University Medical School also demonstrated the dynamic nature of nucleolar proteins using photobleaching techniques that showed proteins shuttling in and out of the nucleolus at different rates.2 "They brought together a really interesting technique that has been rejuvenated over the past few years because of the availability of GFP [green fluorescent protein], using the fluorescent photobleaching technique coupled with GFP in live cells," says Parsons. The complementary techniques, called fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) revealed the movement of seven known nucleolar proteins that the researchers labeled with GFP. Using FRAP, researchers found that labeled proteins rapidly moved into the nucleolar region that had been photobleached, and using FLIP they determined that the nucleolar proteins were also actively moving in the nucleoplasm. "The tendency has been to think of the nucleolus as a static structure," says Parsons. "But what this paper shows is that many or most of the proteins involved in ribosome biogenesis are actually associating and disassociating from the nucleolus very rapidly. It shows how dynamic the nucleolus is, and in a way that was a big surprise." The nucleolus is different than many other organelles because it does not have a membrane, Parsons explains. "We think of the nucleolus as an organelle, but it's not membrane-bound. If you look in the literature, people have tended to look for protein localization signals similar to those for proteins that pass through into membrane-bound organelles. However, the picture that has emerged over the past several years is that the nucleolus is formed through protein-protein and protein-RNA interactions. There appears to be no consensus like a mitochondrial or peroxisomal localization sequence, therefore each nucleolar protein might have a different interaction that causes it to be localized to the nucleolus." If considered this way, she says, "you can see how proteins could associate and disassociate quite rapidly." And indeed, although researchers in the Lamond and Mann labs looked for simple nucleolar targeting motifs, they found none and speculate that localization of proteins to the nucleolus may occur through a variety of mechanisms. Of the 271 proteins identified in the proteomic effort, more than 30% were encoded by previously unknown or uncharacterized genes. Parsons says she thinks the FRAP/FLIP technique could be used to investigate the behavior of these genes. "We don't know how stably these proteins are associated with the nucleolus or if they show transient effects. We don't know if there are different classes of nucleolar proteins. [The Chen and Huang paper] show that the ribosomal subunit proteins were slower than some of the other proteins so there could be different classes of proteins, some that cycle rapidly and some that cycle slowly," she says. Demonstrating that this technique works in a system as large as an organelle opens up the possibility of using it to an even wider degree, Comai says. He sees promise in the comparative proteomic approach. "This approach can be used to go to a larger scale; you can do comparative analysis of protein expression between normal cells and cancer cells as an example." Mignon Fogarty (mignon@welltopia.com)is a freelance writer in Santa Cruz, Calif. References 1. J.S. Andersen et al., "Directed proteomic analysis of the human nucleolus," Current Biology, 12:1-11, Jan. 8, 2002. 2. D. Chen and S. Huang, "Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells," The Journal of Cell Biology, 153:169-76, April 2, 2001.

Dissecting the Nucleolus

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