J O U RN A L OF P ROT EO M IC S 7 7 ( 2 01 2 ) 5 2 1 –5 30
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
Novel nucleolar isolation method reveals rapid response of human nucleolar proteomes to serum stimulation Yi Min Liang, Xian Wang1 , Rajkumar Ramalingam, Kin Yan So2 , Yun Wah Lam⁎, Zhou Fang Li⁎⁎ Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China
AR TIC LE I N FO
ABS TR ACT
Article history:
The nucleolus is the location of ribosomal biogenesis, and plays crucial regulatory roles
Received 2 August 2012
in nuclear responses to stress. Here, we report a new and improved nucleolar isolation
Accepted 24 September 2012
method, which is simpler and more efficient than the traditional method. The purity of
Available online 31 October 2012
nucleoli obtained by using the new protocol is comparable to that by using the classical method, as judged by electron microscopy, Western blotting and SILAC-based quantitative
Keywords:
proteomics. Moreover, the improved efficiency of cell harvesting in the new method,
Nucleolar isolation
biochemical events in the nucleolus could be “frozen” and captured at precisely controlled
Quantitative proteomics
time points. Time-lapse nucleolar proteomics after serum stimulation in HeLa cell revealed
Stable isotope labelling by amino
for the first time that some nucleolar proteins respond to serum stimulation within a time
acids in cell culture
period as short as the first 5 min of serum re-stimulation. Proteins involved in ribosomal biogenesis and in DNA damage repair are among the most dynamic proteins during the first 10 min after serum replenishment. Notably, the proliferation marker Ki-67 is also found to enter the nucleolus after serum replenishment. To our knowledge, this is the first study that demonstrates such fast responses in the nucleolus, further confirming the rapid plasticity of this organelle. © 2012 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleolus is the largest and most prominent organelle in eukaryotic cells. It is the major location for the synthesis and assembly of the 60S and 40S ribosome subunits [1–3]. The importance of this organelle has been recognised in diverse fields of cell biology. For example, rapidly dividing cells have enormous demands for protein synthesis, necessitating the massive production of ribosomes [4,5]. It was estimated that in proliferating mammalian cells, up to 50% of the transcription
effort was dedicated to processes involved in ribosomal biogenesis [6], making the nucleolus one of the most metabolically active organelles in these cells. In fact, cancer cells usually exhibit larger or more nucleoli, and nucleolar size, number and morphology are often used as diagnosis tools for cancer [7,8]. In addition to the functional link of the nucleolar structure and cell proliferation, interest in the nucleolus has been kindled in the past two decades by the discovery of many non-ribosomal factors in this organelle, leading to the concept of “plurifunctional nucleolus” [9,10]. Many studies link the nucleolus to stabilization
Abbreviations: DTT, Dithiothreitol; IAA, Iodoacetamide; Min, Minutes; MS, Mass spectrometry; SILAC, Stable isotope labelling by amino acids in cell culture; TEM, Transmission electron microscopy. ⁎ Corresponding author. Tel.: + 852 3442 6347; fax: + 852 2784 4091. ⁎⁎ Correspondence to: Z.F. Li, Department of Biology, South University of Sciences and Technology of China, Shenzhen 518055, China. Tel.: +86 755 86245687, +86 18603026712. E-mail addresses:
[email protected] (Y.W. Lam),
[email protected] (Z.F. Li). 1 Present address: Sir Runrun Shaw Hospital, Zhejiang University, Hangzhou 310016, China. 2 Present address: School of Public Health, The University of Hong Kong, Hong Kong, China. 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2012.09.031
522
J O U RN A L OF P ROTE O M IC S 7 7 ( 2 01 2 ) 5 2 1 –53 0
and activation of p53 [11]. Recently, the nucleolus has also been implicated in RNA editing and mRNA maturation [12,13], telomere metabolism [14,15], viral infection response [16] and non-coding RNA functions. Because of the involvement of the nucleolus in so many crucial functions, this organelle has been the centre of biological research for decades. The nucleolus is an experimental model on which many fundamental principles in cell biology were first discovered. It is one of the first cellular structures studied by modern live cell time-lapse microscopy, which reveals the precisely coordinated dismantling and re-assembly of this organelle during cell division [17]. Quantitative fluorescent microscopy also uncovers the dynamics of protein flux through the nucleolus, leading to the paradoxical principle of selforganisation in mammalian nuclei [18]. The nucleolus is also one of the first cellular organelle to be purified. Analyses of isolated nucleoli have generated invaluable information on the functions of this nuclear structure. Recently, the combination of nucleolar isolation and mass spectrometry has created the field of nucleolar proteomics [19,20]. To date, more than 4500 proteins have been detected in the nucleoli purified from various cell lines [21]. These proteomic approaches reveal that the biochemical composition of mammalian nucleoli is unexpectedly dynamic. Distinct responses of the nucleolar proteome are detected when cells are treated with different metabolic perturbations, such as transcriptional inhibition [3], viral infection [22], senescence [23] and DNA damage [24]. To date, all nucleolar isolation methods are derived from the original protocol, first published in 1963 [25]. This protocol requires the prior isolation of cell nuclei, which are then disrupted by sonication in a low salt buffer. The purification of nucleoli, the densest structure in the nucleus, is achieved by using density cushions (Fig. 1A). However, this protocol is quite tedious, especially for adherent cells, which need to be dissociated from the culture dishes before nuclear and nucleolar isolation. This is particularly problematic for experiments that require the comparison of nucleolar compositions between two very close time points. Because of this limitation, no time course studies that use isolated nucleoli have chronicled molecular events within 20 min. This is a technical obstacle which creates an unfortunate void in our understanding of nucleolar functions, as numerous live cell microscopy experiments have suggested that nucleolar changes are extremely rapid. Although “timelapse proteomics” for the nucleolus is an appealing concept [20,26], the time resolution permitted by this technique is still orders of magnitude inferior to that of live cell microscopy. Here, we suggest a remedy to this problem, by developing a new protocol that vastly simplifies the process of nucleolar isolation from adherent cultured cells. This new protocol yields nucleoli of superior purity to the classical method, and enables the characterisation of nucleolar changes within minutes.
penicillin (100 IU/ml). The cells were typically grown to 80% confluence before experiments.
2.2.
Classical method for isolating nucleoli was conducted as published [3]. The new isolation method is described below. Prior to conducting the experiments, all of the solutions were pre-cooled at −20 °C. The high percentage of sucrose in the solutions prevents them from freezing at this temperature. Just before nucleolar isolation, the culture medium was decanted from the dishes, and a cold Solution I (0.5 M sucrose with 3 mM magnesium chloride (MgCl2), Roche's complete Protease Inhibitor Cocktail [50 ml/tablet]) was added to quench all metabolic activities. Typically, 30 ml of Solution I was used for a 10 cm dish. The solution was quickly decanted, followed by 3 rapid rinses with the same volume of Solution I. After the removal of the last wash, the cells were quickly scraped from the plate (on ice, or in a cold room) into a 1.5 ml Eppendorf tube. Solution I was added into the collected cells to make up a volume of 0.5 ml. To break down the cells and release the nucleoli, the cells were sonicated on ice at 50% amplitude, 10 s on, 10 s off, 5 times (Sonics, VCX130). The sonicated cells were checked under a microscope, in order to ensure that more than 90% of the cells were broken. Then, the cell lysate was underlaid with 0.7 ml of Solution II (1.0 M sucrose, 3 mM MgCl2). The tube was centrifuged at 1800 ×g for 5 min at 4 °C. The resulting pellet contained isolated nucleoli. The supernatant was carefully removed, so that the sucrose layers were not disturbed. The pellet was transferred to a new tube.
2.3.
Experimental procedures
2.1.
Cells
HeLa cells were obtained from ATCC and grown in a Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% foetal calf serum, streptomycin (100 mg/ml) and
Microscopy
The cells were seeded on glass coverslips one day before the experiments. After a brief rinse with a phosphate buffered saline (PBS), the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The cells were then permeabilised in PBS that contained 0.5% Triton X-100 for 10 min. Then, they were blocked by a signal enhancer (Invitrogen) for 1 h and incubated with rabbit anti-fibrillarin (Santa Cruz Biotechnology, Inc., 1:25) and mouse anti-Ki67 (Abcam, 1:100) antibodies, respectively. After washing three times with PBS that contained 0.5% Tween-20, the cells were then labelled with a secondary antibody conjugated with tetramethylrhodamine isothiocyanate (TRITC, Molecular Probes) and mounted on slides with a fluorescent mounting medium (Dako). The fluorescent labelling of transcription sites by incorporating 5-fluorouridine was carried out as described in [23]. The fluorescence imaging was performed on a laser confocal scanning microscope (Leica SPE). Transmission electron microscopy (TEM) was performed as described in [27].
2.4.
2.
Nucleolar isolation
Western blotting
The protein samples were electrophoresed (see below), and transferred to nitrocellulose membranes at 30 V for 1 h and blocked by incubation in 5% milk for another hour at room temperature. Different primary antibodies were incubated for 1 h at room temperature followed by secondary antibodies with extensive PBS washes in each step. The antibodies were
J O U RN A L OF P ROT EO M IC S 7 7 ( 2 01 2 ) 5 2 1 –5 30
523
Fig. 1 – Comparison of the two nucleolar isolation methods. (A) Schematic diagrams of the new and the existing nucleolar isolation protocols, which show the difference in steps and amount of time required. Morphologies of the scraped HeLa cells (B) and nucleoli (C) isolated by using the new protocol. Transmission electron micrographs of nucleoli isolated by using the existing (D) and new (E) protocols. (F) Coomassie Blue stained SDS-PAGE of the subcellular fractions (Wc: whole cells, Cp: cytoplasm, Nu: nuclei, Np: nucleoplasm, CN: cytoplasm and nucleoplasm and No: nucleolus). Note that each lane is loaded with proteins from fractions from the same cellular equivalents. Lower panel: Western blotting detection of fibrillarin (nucleolar marker), α-Tubulin and β-actin (cytoplasmic markers) and FUS/TLS (nucleoplasmic marker) in each fraction.
detected by using enhanced chemiluminescence (ECL plus, Amersham) horseradish peroxidase (HRP)-based assays and the signal captured by an LAS 4000 chemiluminescent imager (Fuji).
2.5.
Cell cycle analysis
The DNA content of the cells was determined by propidium iodide staining and flow cytometry. Ethanol-fixed cells were stained with 0.05 mg/ml of propidium iodide in the presence of 0.05 mg/ml ribonuclease (RNase). The fluorescence signals were measured with a flow cytometer (FACSCalibur, Becton Dickinson, CA), and the data analysed with CellQuest software (Becton Dickinson, CA).
2.6.
SILAC labelling
The cells were grown for at least five cell divisions in “light” R0K0 DMEM and “heavy” R6K4 DMEM media, respectively, as
previously described [23]. To compare the traditional and new methods, the light and heavy isotope-encoded cells were separately processed by the two methods to obtain their nucleoli (Fig. 2A). The nucleoli were lysed in 8 M urea, and the protein concentrations of the resulting lysates were measured by BCA assay (Pierce Biotechnology Ltd.). 30 μg of proteins from each method was mixed, separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by liquid chromatography/mass spectrometry (LC–MS/MS) (below). For time-lapse nucleolar proteomics, cells with different stable isotope labelling by amino acids in cell culture (SILAC) encoding (“light” R0K0; “medium” R6K4,;“heavy” R10K8, from Lamond's lab protocol) were cultured in a foetal bovine serum (FBS)-free medium for 48 h, and then restimulated by 20% FBS. The cells were harvested in Solution I at different time intervals. The same number of collected cells was mixed together and the nucleoli were then isolated from the mixed cell populations by using the new method (Fig. 3A).
524
J O U RN A L OF P ROTE O M IC S 7 7 ( 2 01 2 ) 5 2 1 –53 0
Fig. 2 – Comparison of the two nucleolar isolation methods. (A) Nucleoli of HeLa cells are purified either by the new or classical protocol. Proteins are extracted from the nucleoli with the two protocols and mass spectrometry analysis is performed. (B) The SILAC ratios of the detected proteins by nucleolus isolation through the two protocols are plotted. The enrichment of the gene oncology terms (cellular components) for proteins in various bins is analysed.
2.7.
Mass spectrometry
The protein samples were incubated in 10 mM dithiothreitol (DTT, GE Healthcare) in 8 M urea (USB) at 56 °C for 40 min, followed by incubation in 55 mM Iodoacetamide (IAA, GE Healthcare) in 8 M urea at room temperature in the dark for another 40 min. The samples were then mixed with an SDS loading buffer [0.5 M Tris Cl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 0.01% (w/v) Bromphenol blue, 5% (w/v) DTT] and incubated for 10 min at room temperature. The proteins were separated by electrophoresis on a 10% Novex precast gel (Invitrogen). The gel was then fixed, stained with colloidal Coomassie Blue (Invitrogen) according to the manufacturer's instructions. The
gel lane was excised into 8–15 slices, and each gel slice was cut into 1 mm3 pieces which were subsequently treated with a destaining solution (Sigma) according to the manufacturer's instructions. This was followed by incubation in 50 mM ammonium bicarbonate in MilliQ water that contained proteomic grade Trypsin (Sigma, 50 ng/gel slice) overnight at 37 °C. The resulting peptides were extracted from each gel slice as previously described [28], separated by high-performance liquid chromatography (HPLC, Dionex) on a commercial C18 reverse phase column (inner diameter 75 μm, 5 μm Acclaim PepMap100 medium; Dionex) over an 80 min gradient (mobile phase A: 0.1% fluoroacetic acid (FA) in 2% acetonitrile (ACN) in MilliQ water, mobile phase B: 0.1% FA in 98% ACN) and then analysed by a
J O U RN A L OF P ROT EO M IC S 7 7 ( 2 01 2 ) 5 2 1 –5 30
525
Fig. 3 – Effect of serum starvation and replenishment on HeLa cells. (A, E and I) Distribution of cell cycle stages in HeLa cells as analysed by flow cytometry. (B–D, F–H and J–L) Transcription assay by 5-fluorouridine incorporation (5-FU). Scale bar, 25 μm.
micrOTOF-Q2 ESI-Qq-TOF mass spectrometer (Bruker Daltonics). The resulting peak lists were generated by using Data Analysis, Version 4.0 (Bruker Daltonics). The MS data were searched against the Uniprot database for Homo Sapiens (12-2011 release, 20249 sequences searched), by using the MASCOT search engine, version 2.2 (Matrix Science). Fixed modification was carbamidomethyl (C) and variable modifications used were oxidation (M) as well as the appropriate SILAC modifications. Trypsin specificity was used, allowing for two missed cleavages, and a mass tolerance of 0.1 Da was used for MS precursors and 0.2 Da for fragment ions. Peptide charges of + 2 and + 3 were selected. Individual ions with mascot scores higher than 20 were used. Twenty is a threshold commonly used for confident protein identification from tandem MS data [22]. Only rank 1 peptides were considered, thus removing duplicate homologous proteins from the results. Under these conditions, the estimated false positive rate was less than 5%, according to a previous analysis (26). SILAC quantitation was done by WarpLC software (Bruker Daltonics), which measures the averaged MS peak heights of isotopic pairs. Proteins with heavy/light isotopic ratios lower than 0.01 were discarded as they mostly represented environmental contaminants. Only proteins with standard deviations (SDs, the variations among the measured peptide ratios for the same protein) less than 70% of the averaged ratios were accepted for further analysis.
2.8.
Data analysis
The functional annotation of the identified proteins was performed by using the web-based DAVID bioinformatic
tools (http://david.abcc.ncifcrf.gov/). To compare the proteomes of nucleoli isolated by two different protocols, we divided the detected proteins into bins according to their SILAC ratios. The enrichment of Gene Ontology (GO) Slim (cellular component) of the proteins in each bin compared to general human proteomes was calculated. The significance of the enrichment data was presented as log2 (1/p-value). In some experiments, the proteins were organised according to their GO Slim metabolic pathway.
3.
Results and discussion
3.1.
Limitation of current nucleolar isolation protocols
The nucleolus is one of the most well studied organelles, largely because this organelle can be conveniently isolated from cultured cells. Virtually all of the existing nucleolar isolation methods are modifications of the original protocol, developed almost 50 years ago [25]. This protocol is established on the principle that cell nuclei can be efficiently shattered by sonication in the presence of a low concentration of magnesium ions, but the nucleolar structure remains intact [29]. The released nucleoli can be readily pelleted through a sucrose cushion (Fig. 1A). Although this protocol and its variations remain highly popular, we envisaged several limitations. First, this protocol requires the prior purification of cell nuclei. The need for nuclear purification inevitably affects the yield of nucleolar isolation. Typically, relatively large amount of cells, up to 2 × 107, is required to harvest enough nucleoli for a proteomic experiment [3,20]. This makes nucleolar isolation impossible for cell types that cannot be cultured at a large scale. Secondly, nuclear purification is commonly achieved by
526
J O U RN A L OF P ROTE O M IC S 7 7 ( 2 01 2 ) 5 2 1 –53 0
Dounce homogenisation on hypotonically swollen cells, and unsuitable for cell types with very strong cytoskeletons. Thirdly, hypotonic swelling only works for cells with intact plasma membranes. Therefore, if an experiment requires the use of adherent cell cultures, the cells must be dissociated from the flasks by using gentle methods, such as trypsinisation, a process that inevitably causes artifactual effects on cell metabolism. Furthermore, trypsinisation is an enzymatic step that necessitates a warm working temperature. It usually takes 5–10 min for all the cells to detach from the substratum, before the harvested cells can be transferred to a cold buffer where metabolism is stopped. This delay tends to introduce inaccuracies in time course experiments on the effect of a treatment over a defined period of time, especially when the desired treatment time is not significantly longer than 5–10 min. Hence, it remains virtually impossible to characterise biochemical events in a nucleolus within less than 20 min after a particular treatment.
3.2.
The new nucleolar isolation protocol
In this study, we have sought to provide a technical solution that can close this research gap. In this new protocol (Fig. 1A), the need for nuclear purification is completely bypassed. Adherent HeLa cells were directly washed in a sonication buffer and then harvested by scraping. The low working temperature and high concentration of sucrose appeared to help preserve the integrity and morphology of the scraped cells (Fig. 1B). The concentrations of sucrose and magnesium ions in the buffer were carefully adjusted so that whole cells were disrupted by sonication while the nucleoli remained intact. The released nucleoli were then pelleted through a density cushion (Fig. 1C). TEM indicates that the nucleoli isolated by the new protocol (Fig. 1E) exhibit a similar ultrastructure to the ones isolated by the classical protocol (Fig. 1D). We analysed the subcellular fractions collected from both the classical and new protocols by Western blotting analysis (Fig. 1F). The new protocol produced isolated nucleoli that were virtually devoid of cytoplasmic and nucleoplasmic markers, while retaining most of the cellular content of fibrillarin. This indicates that the nucleoli obtained by this new method are of similar purity to those produced by the classical protocol. The new procedure is robust and reproducible, possibly as a result of its simplicity. The yield and purity of nucleolar isolation are highly consistent across experiments, and are relatively insensitive to changes in the starting cell number. We reckon that this new protocol offers three major advantages over the classical version. (1) It is a much simpler procedure, allowing the isolation of nucleoli in a period of time as short as 20 min. (2) We found that this protocol can produce a higher yield of isolated nucleoli. The increase in yield is reflected in the Coomassie Blue stained gel showing the proteins collected from each fraction (Fig. 1F). To obtain a similar amount of nucleolar proteins, significantly more starting materials (lane whole cells (WC)) are required for the existing protocol. By using the new method, we can routinely isolate nucleoli from HeLa cells cultured on a single 10 cm dish (about 106 cells). (3) Because of the high percentage of sucrose, solutions used in this protocol can be pre-cooled to well below 0 °C. Cell metabolism can now be stopped
instantaneously on the addition of a sonication buffer, hence very precisely controlling the timing of cell harvesting.
3.3. Comparison of nucleolar proteomes isolated by two procedures To further establish the quality of the nucleoli isolated by the new procedure, we systematically compared the proteomes of nucleoli purified by this protocol and those by the classical method. HeLa cells were metabolically encoded with different SILAC labels, and independently subjected to two subcellular fractionation procedures. The resulting nucleoli were mixed and analysed by LC–MS/MS (Fig. 2A). In total, 422 proteins were identified and quantified. As shown in Fig. 2B and Supplementary Table 1, the majority of the identified proteins have SILAC ratios between −0.4 and −1.2, which suggest that most of the proteins are commonly detected in nucleoli isolated by both methods. The slight deviation from the ideal ratio of 0 may be due to errors in equalising protein amounts before mixing. As expected, functional classifications show that this common group is enriched with proteins that function in ribosomal biogenesis and other nucleolar functions. Therefore, these nucleolar factors appear to be isolated by both protocols with similar efficiency. Interestingly, many proteins that are over-represented in nucleoli isolated by the classical method (low SILAC ratios) are related to the mitochondria. Mitochondrial proteins are common contaminants in traditional nucleolar isolation procedures, and the new protocol appears to overcome the problem of mitochondrial contamination. It is possible that increases of sucrose concentration in the density cushion may help separate mitochondria from the nucleoli. There seem to be a few proteins that are preferentially purified by the new method (high SILAC ratios). Taken together, we conclude that the new method is not only faster and better in yield compared to the classical protocol, but the resulting nucleoli are also of more superior purity.
3.4. Rapid changes in nucleolar proteomes in response to serum stimulation We envisaged that the improved effectiveness of cell harvesting afforded by the new nucleolar isolation protocol will allow for more efficient sample collection, and therefore better time resolution, in the biochemical analyses of nucleolar dynamics. As proof of the concept, we applied this protocol on a study of nucleolar proteome responses immediately after the restimulation of serum-deprived HeLa cells. During serum withdrawal, cell growth and proliferation are suppressed. Serum starvation is known to induce changes in nucleolar morphology [30–32]. The cellular responses to serum stimulation are very rapid, with the initial wave of signalling events occurring within the first 30 min [33]. Since the nucleolus is the key site of ribosomal biogenesis and nuclear stress responses, it is interesting to investigate how the nucleolar structure and composition change within this time window. Consistent with the published results [34], serum-starvation and replenishment do not induce significant differences in the distribution of cell cycle stages (Fig. 3A, E, I), but dramatic changes in RNA synthesis. An in vivo transcription assay, based on the metabolic incorporation of 5-fluorouridine [35], showed
J O U RN A L OF P ROT EO M IC S 7 7 ( 2 01 2 ) 5 2 1 –5 30
an obvious decrease in transcription foci in the serum-starved cells (Fig. 3F–H). Interestingly, the level of 5-fluorouridine incorporation, especially in the nucleolus, dramatically increases as soon as 5 min had passed upon the re-addition of serum (Fig. 3J–L). This indicates that the early cellular response to the re-stimulation of serum-deprived cells occurs in a time frame even shorter than previously envisaged. Here, by combining our new nucleolar isolation protocol with SILAC proteomics, we monitored the proteomic changes in the nucleolus during the first 10 min of serum stimulation after a 48-hour serum withdrawal (Fig. 4A). Since the new protocol enables a precise fixation of cell metabolism prior to nucleolar isolation, we were able to harvest nucleoli after a time period as short as 5 min after serum replenishment. The relative abundance of proteins co-purified in nucleoli isolated from HeLa cells 5 and 10 min after serum replenishment, as compared to that in unstimulated serum-starved cells, was monitored (Fig. 4B, and Supplementary Table 2). We interpreted that the proteome dynamics detected in this time window is the consequence of protein homeostasis or intracellular redistribution of proteins, as the effect of gene expression changes would take a longer time to manifest. Like other nucleolar proteome dynamic studies [3], the SILAC-encoded cells were mixed immediately after cell harvesting, and nucleoli were isolated
527
from the mixed populations. Any variations associated with nucleolar isolation, protein extraction and peptide preparation were internally controlled. Our data reveal the rapid response of nucleolar proteomes to serum re-stimulation. We clustered the detected nucleolar proteins in terms of their dynamics (Fig. 4B). About half of the detected proteins remained unchanged during the first 10 min of serum replenishment (Cluster 1). The nucleolar abundance of some of the proteins increased as early as 5 min but returned to the pre-stimulation level quickly afterwards (Cluster 2). Another wave of proteins accumulated in the nucleolus at 10 min (Cluster 4). There are also proteins that exited the nucleolus 5 min (Cluster 3) or 10 min (Cluster 5) after serum stimulation. We examined the dynamics of several groups of nucleolar proteins (Fig. 4C). Some of the proteins involved in ribosomal biogenesis and snoRNA metabolism, such as IF6, NIP7, IMP4, NHP2 and PWP2, are among the proteins that were depleted from the nucleolus as early as 5 min after serum addition. Unlike other hnRNPs, hnRNP-R rapidly accumulated in the nucleolus after serum replenishment. This hnRNP has been identified as an interaction partner of the survival motor neuron protein [36], a protein essential for the assembly of snRNP. Interestingly, arginine methylation of hnRNP-R is highly
Fig. 4 – Rapid changes in the nucleolar proteome in response to serum stimulation. (A) HeLa cells are cultured in a serum-free medium that contains different isotopes, named “light”, “medium” and “heavy” for 48 h and replenished with serum for either 0, 5 or 10 min. The cells are collected and mixed together. Nucleoli are isolated from the mixed cells and subjected to mass spectrometry analysis. (B) Proteome changes within 5 min and 10 min of serum replenishment are clustered in terms of their dynamics. (C) Groups that belong to specific cellular components are used as examples to show the dynamic changes of each protein group. (D) Changes in nucleolar proteins after 5 min of serum replenishment. The box highlights the 5 proteins with the highest accumulation.
528
J O U RN A L OF P ROTE O M IC S 7 7 ( 2 01 2 ) 5 2 1 –53 0
correlated with cell proliferation [37]. The rapid accumulation of this protein after serum stimulation may reflect its previously unknown role in nucleolar functions. NOG2, a GTP-binding protein involved in 60S ribosomal subunit assembly, also accumulates in the nucleolus on serum stimulation. This protein is known to regulate cell cycle control and timing during embryogenesis [38]. Some ribosomal proteins, especially the components of the large subunit (e.g., RPL31), show early nucleolar accumulation. Another group of proteins that exhibit significant changes is the chromatin proteins, such as many histone proteins. Notably, two components of the FACT (FAcilitates Chromatin Transcription) complex, SSRP1 and SP16H, exhibited a strong nucleolar accumulation after serum addition. Interleukin enhancer-binding factor 2 (ILF2), DNAdependent protein kinase (PRKDC) and XRCC6, members of the Ku-dependent DNA damage repair pathway, also showed nucleolar accumulation. SSRP1, PRKDC and components of the Ku pathway have been shown to distribute between the nucleolus and the nucleoplasm in a DNA damage-dependent manner [39]. This observation further suggests the link of chromatin modification and DNA repair in the nucleolus and serum stimulation. Our data offer a previously unavailable glimpse on some of the earliest responses of the nucleolar proteome to serum stimulation. Since this is the first report of this kind, the functional significances of these changes are unknown at this stage, and warrant future investigations.
3.5.
Ki-67 protein rapidly responds to serum stimulation
One of the proteins that displayed the most dramatic increase during the first 5 min of serum stimulation is Ki-67 (Fig. 4D). The Western blotting analysis confirmed that an abundance of this protein is quickly upregulated in both whole cells and isolated nucleoli after serum replenishment (Fig. 5A–B). Immunofluorescence also showed that the upregulated Ki-67 mostly accumulates in the nucleolus. Interestingly, in serum-starved cells, Ki-67 was detected in the peripheries of nucleoli (Fig. 5C).
Upon the addition of serum, Ki-67 became detectable inside the nucleolus within 5 min. Ki-67 has been recognised as a proliferation marker in mammalian cells [40–42], and is associated with the rRNA transcription machinery [40]. Here, we showed that the expression level and nucleolar localisation of this protein are remarkably dynamic. In this study, we have demonstrated the isolation of nucleoli from HeLa cells by using a simplified protocol. The isolated nucleoli are comparable to those isolated by the classical method in terms of morphology and protein composition. The improvement of the yield enabled the isolation of nucleoli from a smaller number of cells. Moreover, this new protocol allows more precise control in cell harvesting, thus enabling the comparison of proteomes on nucleoli isolated at time intervals of 5 min. This time resolution is now comparable to that of typical time lapse imaging experiments. In such a time frame, we are able to reveal previously unknown dynamics of nucleolar proteins in response to growth stimulation. With the advent of fluorescent protein tagging and live cell microscopy in the late 20th century, time has emerged as a re-discovered dimension in cell biology. Cell imaging techniques allow for the quantitative analysis of the spatial and temporal distributions of biomolecules in living cells, but only a relatively small number of proteins can be studied in one experiment. In contrast, biochemical analyses, especially proteomics, allow the large-scale identification of proteins in complex biological systems, but often only produce static snapshots of organellar compositions that fail to describe and explain the dynamic nature of intracellular dynamics. Hence, microscopy and proteomics occupy two distant corners in cell biology research, and are used to tackle questions that involve different scales and time resolutions. We believe that our new protocol demonstrates a step toward the bridging of these two powerful techniques, and will be useful for the proteomic study of dynamic events in mammalian cells. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2012.09.031.
Fig. 5 – Rapid response of Ki-67 to serum stimulation. Western blotting analysis of Ki-67 in HeLa cells (A) and nucleoli (B) collected at different time points after serum replenishment. β-Actin and B23 are used as loading references for whole cells and isolated nucleoli, respectively. “S”: serum starved, “N”: FBS supplemented control. (C) Immunofluorescence of Ki-67 in serum starved (0 min) and replenished (5 min) HeLa cells. Fibrillarin is used as the nucleolar control. Scale bar, 10 μm.
J O U RN A L OF P ROT EO M IC S 7 7 ( 2 01 2 ) 5 2 1 –5 30
Acknowledgement This project was funded by a General Research Fund (project number 9041520) provided by the Research Grant Council, Hong Kong. The proteomic equipment used in this study was funded by a UGC special equipment grant (project number 874004). We thank the members of the Lam lab for their fruitful discussion and Dr. Rajkumar Ramalingam for additional support in mass spectrometry.
REFERENCES [1] Nissan TA, Bassler J, Petfalski E, Tollervey D, Hurt E. 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO J 2002;21:5539–47. [2] Ferreira-Cerca S, Poll G, Kuhn H, Neueder A, Jakob S, Tschochner H, et al. Analysis of the in vivo assembly pathway of eukaryotic 40S ribosomal proteins. Mol Cell 2007;28:446–57. [3] Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, et al. Nucleolar proteome dynamics. Nature 2005;433:77–83. [4] Korostelev A, Trakhanov S, Laurberg M, Noller HF. Crystal structure of a 70S ribosome–tRNA complex reveals functional interactions and rearrangements. Cell 2006;126:1065–77. [5] Selmer M, Dunham CM, FVt Murphy, Weixlbaumer A, Petry S, Kelley AC, et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 2006;313:1935–42. [6] Moss T, Stefanovsky VY. At the center of eukaryotic life. Cell 2002;109:545–8. [7] Derenzini M, Trere D, Pession A, Govoni M, Sirri V, Chieco P. Nucleolar size indicates the rapidity of cell proliferation in cancer tissues. J Pathol 2000;191:181–6. [8] Lorenzato M, Abboud P, Lechki C, Browarnyj F, O'Donohue MF, Ploton D, et al. Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry. Micron 2000;31:151–9. [9] Olson MO, Hingorani K, Szebeni A. Conventional and nonconventional roles of the nucleolus. Int Rev Cytol 2002;219:199–266. [10] Pederson T. The plurifunctional nucleolus. Nucleic Acids Res 1998;26:3871–6. [11] Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI. The nucleolus under stress. Mol Cell 2010;40:216–27. [12] Jellbauer S, Jansen RP. A putative function of the nucleolus in the assembly or maturation of specialized messenger ribonucleoprotein complexes. RNA Biol 2008;5:225–9. [13] Sansam CL, Wells KS, Emeson RB. Modulation of RNA editing by functional nucleolar sequestration of ADAR2. Proc Natl Acad Sci U S A 2003;100:14018–23. [14] Kieffer-Kwon P, Martianov I, Davidson I. Cell-specific nucleolar localization of TBP-related factor 2. Mol Biol Cell 2004;15: 4356–68. [15] Zhang S, Hemmerich P, Grosse F. Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2). J Cell Sci 2004;117:3935–45. [16] Wang L, Ren XM, Xing JJ, Zheng AC. The nucleolus and viral infection. Virol Sin 2010;25:151–7. [17] Leung AK, Gerlich D, Miller G, Lyon C, Lam YW, Lleres D, et al. Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells. J Cell Biol 2004;166:787–800. [18] Dundr M, Hebert MD, Karpova TS, Stanek D, Xu H, Shpargel KB, et al. In vivo kinetics of Cajal body components. J Cell Biol 2004;164:831–42.
529
[19] Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, et al. Directed proteomic analysis of the human nucleolus. Curr Biol 2002;12:1–11. [20] Lam YW, Lamond AI, Mann M, Andersen JS. Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins. Curr Biol 2007;17:749–60. [21] Ahmad Y, Boisvert FM, Gregor P, Cobley A, Lamond AI. NOPdb: nucleolar proteome database—2008 update. Nucleic Acids Res 2009;37:D181–4. [22] Lam YW, Evans VC, Heesom KJ, Lamond AI, Matthews DA. Proteomics analysis of the nucleolus in adenovirus-infected cells. Mol Cell Proteomics 2010;9:117–30. [23] Kar B, Liu B, Zhou Z, Lam YW. Quantitative nucleolar proteomics reveals nuclear re-organization during stress-induced senescence in mouse fibroblast. BMC Cell Biol 2011;12:33. [24] Boisvert FM, Lam YW, Lamont D, Lamond AI. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage. Mol Cell Proteomics 2010;9: 457–70. [25] Busch H, Muramatsu M, Adams H, Steele WJ, Liau MC, Smetana K. Isolation of nucleoli. Exp Cell Res 1963;24(Suppl. 9): 150–63. [26] Trinkle-Mulcahy L, Lamond AI. Toward a high-resolution view of nuclear dynamics. Science 2007;318:1402–7. [27] Cheung ID, Bagnat M, Ma TP, Datta A, Evason K, Moore JC, et al. Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b. Dev Biol 2012;361:68–78. [28] Tong WY, Liang YM, Tam V, Yip HK, Kao YT, Cheung KM, et al. Biochemical characterization of the cell-biomaterial interface by quantitative proteomics. Mol Cell Proteomics 2010;9:2089–98. [29] Lam MH, Thomas RJ, Martin TJ, Gillespie MT, Jans DA. Nuclear and nucleolar localization of parathyroid hormone-related protein. Immunol Cell Biol 2000;78:395–402. [30] Bhat KP, Itahana K, Jin A, Zhang Y. Essential role of ribosomal protein L11 in mediating growth inhibition-induced p53 activation. EMBO J 2004;23:2402–12. [31] Chan PK, Aldrich M, Busch H. Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation. Exp Cell Res 1985;161: 101–10. [32] Matsumura K, Sasaki K, Tsuji T, Murakami T, Takahashi M, Shinozaki F. Changes of AgNORs in HeLa cells during serum starvation. Br J Cancer 1990;62:385–7. [33] Jones SM, Kazlauskas A. Growth factor-dependent signaling and cell cycle progression. FEBS Lett 2001;490:110–6. [34] Khammanit R, Chantakru S, Kitiyanant Y, Saikhun J. Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts. Theriogenology 2008;70:27–34. [35] So LK, Cheung SK, Ma HL, Chen XP, Cheng SH, Lam YW. In situ labeling of transcription sites in marine medaka. J Histochem Cytochem 2010;58:173–81. [36] Rossoll W, Kroning AK, Ohndorf UM, Steegborn C, Jablonka S, Sendtner M. Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum Mol Genet 2002;11:93–105. [37] Kim C, Lim Y, Yoo BC, Won NH, Kim S, Kim G. Regulation of post-translational protein arginine methylation during HeLa cell cycle. Biochim Biophys Acta 2010;1800:977–85. [38] Paridaen JT, Janson E, Utami KH, Pereboom TC, Essers PB, van Rooijen C, et al. The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish. Dev Biol 2011;355:286–301. [39] Dejmek J, Iglehart JD, Lazaro JB. DNA-dependent protein kinase (DNA-PK)-dependent cisplatin-induced loss of nucleolar facilitator of chromatin transcription (FACT) and
530
J O U RN A L OF P ROTE O M IC S 7 7 ( 2 01 2 ) 5 2 1 –53 0
regulation of cisplatin sensitivity by DNA-PK and FACT. Mol Cancer Res 2009;7:581–91. [40] Bullwinkel J, Baron-Luhr B, Ludemann A, Wohlenberg C, Gerdes J, Scholzen T. Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells. J Cell Physiol 2006;206:624–35.
[41] Kee N, Sivalingam S, Boonstra R, Wojtowicz JM. The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J Neurosci Methods 2002;115:97–105. [42] Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol 2005;23:7212–20.
