The Nucleolus Enter Mitosis

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Cell Science at a Glance 1335 The nucleolus enter mitosis. Owing to the difference in most convenient subcellular structures to density between the nucleolus and the purify. Thus, when mammalian nuclei Yun Wah Lam*, Laura Trinkle- surrounding nucleoplasm, it is readily are physically disrupted (e.g. by Mulcahy*,‡ and Angus I. Lamond visible in either live or fixed cells viewed sonication) in a solution of low salt Wellcome Trust Biocentre, MSI/WTB Complex, by phase contrast or differential concentration, nucleoli remain intact University of Dundee, Dundee, DD1 5EH, UK interference contrast (DIC) optics even under conditions that disintegrate *These authors contributed equally to this work ‡Author for correspondence (e-mail: (upper-left panel). Thanks to the advent most other subnuclear structures. [email protected]) of fluorescent protein (FP) technology, Nucleoli can therefore be isolated in nucleoli can also be detected by essentially pure form by centrifuging Journal of Cell Science 118, 1335-1337 fluorescence microscopy in cell lines sonicated nuclei through a density Published by The Company of Biologists 2005 doi:10.1242/jcs.01736 expressing FP-tagged nucleolar proteins. cushion (middle-left panel). The isolated An example is shown in the inset of the nucleoli are intact, similar in size and The nucleolus is the most prominent upper-left panel, in which PP1γ, a morphology to the nucleoli in live cells structure in a cell nucleus. It is the site protein phosphatase that accumulates in and even retain transcriptional activity to of ribosomal RNA (rRNA) transcription, the nucleolus, is tagged with YFP and some extent (Cheutin et al., 2002). pre-rRNA processing and ribosome stably expressed in HeLa cells (Trinkle- subunit assembly (reviewed by Olson et Mulcahy et al., 2003). Thanks to the ability to isolate large al., 2002). The nucleolus is a dynamic amounts of purified nucleoli, recent structure that assembles around the Like all other intranuclear structures, the studies have analysed the nucleolar clusters of rRNA gene repeats during nucleolus is not membrane enclosed, but protein composition in great detail using late telophase, persists throughout the combination of its unique density high-throughput mass-spectrometry interphase and then disassembles as cells and robust structure makes it one of the (MS)-based proteomic techniques

Yun Wah Lam, Laura Trinkle-Mulcahy and Angus I. Lamond

HeLa cells Nucleolar transcription 'Christmas tree' model s cleu Terminal Nu knobs Nucleolus Nucleolus rRNA Journal of Cell Science

rDNA Nucleolus rDNA transcription

Isolated nucleoli Subnucleolar compartments Granular Interpha component Dense (GC) B23 fibrillar se component rRNA processing (DFC) Fibrillarin

Ribosome assembly Fibrillar centre (FC) RPA39

DFC sis Mito Nucleus GC Mass spectrometry FC

Transmission electron microscopy

Nucleolus RPA39

Cytoplasm jcs.biologists.org Fibrillarin

B23

Small ribosomal subunit Large ribosomal subunit Nucleolar proteome: ~700 proteins www.lamondlab.com/nopdb Fluorescence microscopy

(See poster insert) 1336 Journal of Cell Science 118 (7)

(lower-left panel). For example, initial of cellular activities linked to the The nucleolus is a structure in which the proteomic studies of HeLa nucleoli nucleolus include RNA editing (Sansam interactions and translocation of a large reported the identification of over 400 et al., 2003), DNA damage repair (van number of proteins and RNAs are proteins (Andersen et al., 2002; Scherl et den Boom et al., 2004), telomere conducted and coordinated (reviewed by al., 2002). Ongoing improvements in metabolism (Kieffer-Kwon et al., 2004; Fromont-Racine et al., 2003). During protein separation methods prior to MS, Zhang et al., 2004), tRNA processing ribosome subunit biogenesis, pre-rRNA and in the sensitivity of MS, continue to (Paushkin et al., 2004) and regulation of transcripts are transcribed by RNA expand the number of nucleolar proteins protein stability (Mekhail et al., 2004; polymerase I from the repeated clusters identified. An online database describing Rodway et al., 2004). of rDNA genes. This process is vividly close to 700 human proteins detected in demonstrated when eukaryotic cells are purified nucleoli is now available (www. The internal structure of the nucleolus swollen and spread in an alkaline, lamondlab.com/Nopdb) (Andersen et al., has been studied in detail by both hypotonic solution in the presence of 2005). A proteome of the Arabidopsis transmission and scanning electron detergent (e.g. Chooi and Leiby, 1981; thaliana nucleolus has also been microscopy. For example, field-emission Mougey et al., 1993; Osheim et al., identified recently (http://bioinf.scri.sari. scanning electron microscopy (FESEM) 2004). Under these conditions (Miller ac.uk/cgi-bin/atnopdb/home) (Pendle et analysis of isolated HeLa nucleoli spreads), the rDNA repeats are fully al., 2005). One striking discovery from provides a high-resolution (~1 nm) view extended and, along this central DNA these proteomic studies is that up to 30% of the 3D contour of the nucleolar axis, nascent growing rRNA transcripts of the nucleolar proteins are encoded surface and the interface between the can be seen emerging from each rDNA by previously uncharacterised genes nucleolus and the nucleoplasm (main unit. As transcription proceeds from the (Andersen et al., 2002; Andersen et al., central image). The internal structure of initiation point, the rRNA transcripts 2005). This suggests that despite the the nucleolus is revealed by transmission become increasingly longer, resulting in extensive previous research on the electron microscopy (TEM) of thin a ‘Christmas tree’ model (reviewed by nucleolus extending over almost two sections cut through nucleoli. Based on Trendelenburg et al., 1996). It is not centuries, there is still much to be the morphology revealed by such TEM clear how this complex structure is learned about its structure and function. images, three subcompartments have assembled in the nucleolus. The Moreover, with the new availability of been identified within the interior of the initiation of transcription probably human and plant nucleolar proteomes, nucleolus. These include fibrillar centres occurs either within the FCs or at the FC- bioinformatic studies have already (FCs), which are surrounded by dense DFC boundary. The resulting pre-rRNA started to reveal new insights concerning fibrillar components (DFCs), and the transcripts then emerge into the DFC common motifs found in nucleolar FC-DFC complexes are embedded in the region, where they are cleaved and proteins (Leung et al., 2003) and the granular component (GC). Immuno-EM modified by the small nucleolar RNPs evolution of this nuclear organelle experiments show that many nucleolar (snoRNPs) and other processing (Staub et al., 2004). proteins accumulate in one or two of enzymes. The rRNAs also begin the Journal of Cell Science these subcompartments, suggesting they pathway of assembly with ribosomal Examination of the proteome provides a each have distinct protein compositions proteins in the DFC and continue this as glimpse into the functional complexity and functions (Schwarzacher and they pass through the GC and are of the nucleolus. Its central role in Mosgoeller, 2000). For example, the exported to the cytoplasm. The ribosome subunit biogenesis is RNA polymerase I subunit RPA39 is sequential movement of rRNA through confirmed by the presence of many predominantly localised in the FC, the FC, DFC and GC subcompartments proteins (more than one third of the whereas fibrillarin, a protein that is can be demonstrated when cells are nucleolar proteome) involved in involved in ribose 2′-O-methylation of labelled with a short pulse of different steps in rRNA transcription, rRNA, accumulates in the DFC, and halogenated nucleotide, which reveals a rRNA processing and modification, as nucleolar phosphoprotein B23 is found wave of nascent rRNA spreading from well as the large and small ribosome in the GC. Recently, cell lines have been the FC-DFC complexes to the GC subunit proteins themselves (Andersen established that simultaneously express regions (this is shown in three stages et al., 2005). However, there are also these three proteins tagged with different in the panel labelled ‘Nucleolar many proteins that have no obvious fluorescent proteins. This allows the transcription’). relationship with these ‘classical’ assembly and disassembly of each nucleolar processes. For example, many subnucleolar compartment to be Despite major advances in recent years, proteins related to cell cycle regulation analysed in live cells during mitosis by many important questions remain to be (about 3.5% of the identified proteome), high-resolution light microscopy (Leung answered about the nucleolus. For DNA damage repair (about 1%) and pre- et al., 2004). Recent data have also example, it is still unclear what mRNA processing (about 5%) are revealed that some factors in nucleoli are components provide the structural detected in isolated nucleoli. This is localised in subnucleolar regions that do integrity of the nucleolus. It is also not consistent with the idea that the not precisely correlate with any of the known how nucleolar assembly and nucleolus performs additional roles three well-known subcompartments disassembly is regulated during mitosis, beyond generating ribosomal subunits (Politz et al., 2002), which suggests that and much remains to be learned (reviewed by Olson et al., 2002; the nucleolus is more complex than a concerning the full range of biological Pederson, 1998). More recent examples sum of FCs, DFCs and the GC. processes that either occur within, or else Cell Science at a Glance 1337

involve, the nucleolus. Similarly, a TBP-related factor 2. Mol. Biol. Cell 15, 4356- forms of human p14(ARF). Oncogene 23, 6186- clearer picture is needed at the molecular 4368. 6192. Leung, A. K., Andersen, J. S., Mann, M. and Sansam, C. L., Wells, K. S. and Emeson, R. B. level between subnucleolar structure and Lamond, A. I. (2003). Bioinformatic analysis of (2003). Modulation of RNA editing by functional speciﬁc functions. Although a detailed the nucleolus. Biochem. J. 376, 553-569. nucleolar sequestration of ADAR2. Proc. Natl. description of the protein content of the Leung, A. K., Gerlich, D., Miller, G., Lyon, C., Acad. Sci. USA 100, 14018-14023. nucleolus is now emerging, we still do Lam, Y. W., Lleres, D., Daigle, N., Zomerdijk, Scherl, A., Coute, Y., Deon, C., Calle, A., J., Ellenberg, J. and Lamond, A. I. (2004). Kindbeiter, K., Sanchez, J. C., Greco, A., not know what functions many of these Quantitative kinetic analysis of nucleolar Hochstrasser, D. and Diaz, J. J. (2002). proteins perform, and less is known breakdown and reassembly during mitosis in live Functional proteomic analysis of human nucleolus. about the full spectrum of RNAs and human cells. J. Cell Biol. 166, 787-800. Mol. Biol. Cell 13, 4100-4109. DNA sequences that associate with Mekhail, K., Gunaratnam, L., Bonicalzi, M. E. Schwarzacher, H. G. and Mosgoeller, W. (2000). Ribosome biogenesis in man: current nucleoli. The nucleolus is therefore and Lee, S. (2004). HIF activation by pH- dependent nucleolar sequestration of VHL. Nat. views on nucleolar structures and function. likely to remain a source of interesting Cell Biol. 6, 642-647. Cytogenet. Cell Genet. 91, 243-252. new discoveries and undoubtedly some Mougey, E. B., O’Reilly, M., Osheim, Y., Miller, Staub, E., Fiziev, P., Rosenthal, A. and more surprises for the foreseeable future. O. L., Jr, Beyer, A. and Sollner-Webb, B. Hinzmann, B. (2004). Insights into the evolution (1993). The terminal balls characteristic of of the nucleolus by an analysis of its protein domain repertoire. Bioessays 26, 567-581. eukaryotic rRNA transcription units in chromatin We thank Anthony K. L. Leung and Carol E. Lyon Trendelenburg, M. F., Zatsepina, O. V., spreads are rRNA processing complexes. Genes for providing some of the images, and other Waschek, T., Schlegel, W., Troster, H., Dev. 7, 1609-1619. members of the Lamond lab for help and Rudolph, D., Schmahl, G. and Spring, H. (1996). Olson, M. O., Hingorani, K. and Szebeni, A. discussions. Multiparameter microscopic analysis of nucleolar (2002). Conventional and nonconventional roles of structure and ribosomal gene transcription. the nucleolus. Int. Rev. Cytol. 219, 199-266. Histochem. Cell Biol. 106, 167-192. Osheim, Y., French, S. L., Keck, K. M., References Trinkle-Mulcahy, L., Andrews, P. D., Champion, E. A., Spasov, K., Dragon, F., Wickramasinghe, S., Sleeman, J., Prescott, A., Andersen, J. S., Lyon, C. E., Fox, A. H., Leung, Baserga, S. J. and Beyer, A. L. (2004). Pre-18S Lam, Y. W., Lyon, C., Swedlow, J. R. and A. K., Lam, Y. W., Steen, H., Mann, M. and ribosomal rna is structurally compacted into the Lamond, A. I. (2003). Time-lapse imaging reveals Lamond, A. I. (2002). Directed proteomic SSU processome prior to being cleaved from dynamic relocalization of PP1gamma throughout analysis of the human nucleolus. Curr. Biol. 12, 1- nascent transcripts in Saccharomyces cerevisiae. the mammalian cell cycle. Mol. Biol. Cell 14, 107- 11. Mol. Cell 16, 943-954. 117. Andersen, J. S., Lam, Y. W., Leung, A. K. L., Paushkin, S. V., Patel, M., Furia, B. S., Peltz, S. van den Boom, V., Citterio, E., Hoogstraten, D., Ong, S. E., Lyon, C. E., Lamond, A. I. and W. and Trotta, C. R. (2004). Identiﬁcation of a Zotter, A., Egly, J. M., van Cappellen, W. A., Mann, M. (2005). Nucleolar proteome dynamics. human endonuclease complex reveals a link Hoeijmakers, J. H., Houtsmuller, A. B. and Nature 433, 77-78. between tRNA splicing and pre-mRNA 3′ end Vermeulen, W. (2004). DNA damage stabilizes Cheutin, T., O’Donohue, M. F., Beorchia, A., formation. Cell 117, 311-321. interaction of CSB with the transcription Vandelaer, M., Kaplan, H., Defever, B., Ploton, Pederson, T. (1998). The plurifunctional elongation machinery. J. Cell Biol. 166, 27-36. D. and Thiry, M. (2002). Three-dimensional nucleolus. Nucleic Acids Res. 26, 3871-3876. Zhang, S., Hemmerich, P. and Grosse, F. (2004). organization of active rRNA genes within the Pendle, A. F., Clark, G. P., Boon, R., Nucleolar localization of the human telomeric nucleolus. J. Cell Sci. 115, 3297-3307. Lewandowska, D., Lam, Y. W., Andersen, J., repeat binding factor 2 (TRF2). J. Cell Sci. 117, Chooi, W. Y. and Leiby, K. R. (1981). An Mann, M., Lamond, A. I., Brown, J. W. and 3935-3945.

Journal of Cell Science electron microscopic method for localization of Shaw, P. J. (2005). Proteomic analysis of the ribosomal proteins during transcription of Arabidopsis nucleolus suggests novel nucleolar ribosomal DNA: a method for studying protein functions. Mol. Biol. Cell 16, 260-269. assembly. Proc. Natl. Acad. Sci. USA 78, 4823- Politz, J. C., Lewandowski, L. B. and Pederson, Cell Science at a Glance on the Web 4827. T. (2002). Signal recognition particle RNA Electronic copies of the poster insert are Fromont-Racine, M., Senger, B., Saveanu, C. localization within the nucleolus differs from the available in the online version of this article and Fasiolo, F. (2003). Ribosome assembly in classical sites of ribosome synthesis. J. Cell Biol. at jcs.biologists.org. The JPEG images can eukaryotes. Gene 313, 17-42. 159, 411-418. be downloaded for printing or used as Kieffer-Kwon, P., Martianov, I. and Davidson, Rodway, H., Llanos, S., Rowe, J. and Peters, G. slides. I. (2004). Cell-speciﬁc nucleolar localization of (2004). Stability of nucleolar versus non-nucleolar

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