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1 Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan
2 Department of Biology, Faculty of Science, Chiba University, Chiba, 263-8522, Japan
3 Department of VCAPP, Washington State University, WA-99164, USA
4 Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim 68167, Germany
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Abstract |
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 Top Abstract IntroductionResultsDiscussionConclusionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionConclusionExperimental proceduresReferences |
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10 000-fold compared to the length of naked DNA and form highly compact structures, metaphase chromosomes (Sumner 2003). Subsequently, at the M/G1 transition, the chromosomes return to their decondensed interphase structure. Despite extensive structural analyses (Finch & Klug 1976; Sedat & Manuelidis 1978; Marsden & Laemmli 1979), the mechanism of chromatin condensation and the structure of metaphase chromosomes still remain unclear (Earnshaw 1991; Belmont 2002). This can be attributed to the following two reasons. First, chromosome structures are so flexible that they show various appearances, depending on the preparation and observation methods employed. As a result, several different models of the higher order structure have been proposed (Ushiki et al. 2002; Adkins et al. 2004). Second, there is little information available regarding the components of metaphase chromosomes, except for two major non-histone chromosomal proteins (NHCPs), topoisomerase II
and condensin proteins (Gasser et al. 1986; Hirano & Mitchison 1994; Belmont 2002,). Also, several proteins were sporadically discovered to localize to chromosomes. In order to clarify the protein composition of human metaphase chromosomes, we first investigated the relationships between the isolation conditions of human metaphase chromosomes and their morphologies. As a result, it revealed that the isolation conditions are critical for the chromosome morphology and also protein composition (Sone et al. 2002). Subsequently, we reported that polyamine (PA) chromosomes (PA-Chs) are composed of approximately 300 different proteins, as revealed by two-dimensional (2-D) electrophoretic analysis combined with Coomassie Brilliant Blue (CBB) staining (Uchiyama et al. 2004). In the latter paper, we also pointed out differences in the protein compositions between chromosomes isolated from BALL-1 and HeLa cells, and suggested that these chromosomes include not only common chromosome proteins but also cell line-specific chromosome proteins. Recently, we reported a comparative proteome analysis between PA-Chs and the chromosomes purified by sucrose density gradient centrifugation (Uchiyama et al. 2005). As a result, we quantitatively identified 158 different chromosome proteins that constitute PA-Chs isolated from BALL-1 cells (BALL PA-Chs). Furthermore, a proteome analysis of chromosomes isolated by glycerol and Percoll density gradient centrifugation (PG-Chs) produced a list of 107 essential chromosome proteins. Based on these proteome analyses together with localization data for representative proteins, a four-layer model of a metaphase chromosome was proposed, in which chromosome proteins were classified into four groups: designated chromosome coating proteins (CCPs), chromosome peripheral proteins (CPPs), chromosome structural proteins (CSPs) and chromosome fibrous proteins (CFPs). However, in spite of these intense efforts to identify chromosome proteins, there is little information about the character of identified proteins and the relationship in terms of chromosome structure. Furthermore, we couldn't identify macro molecular weight (MW: > 500 kDa) chromosome proteins in our previous proteomic analysis because of the difficulties in protein preparation and separation.
In the present paper, we performed a comparative proteome analysis between PA-Chs isolated from HeLa S3 cells (HeLa PA-Chs) and BALL-1 cells (BALL PA-Chs), which have been widely used in chromosome structural studies. The newly identified 189 proteins in HeLa PA-Chs were compared with the proteome of BALL PA-Chs. Furthermore, since the giant elastic protein titin (MW: 3300–3700 kDa; also called connectin; Maruyama 1976) has been intriguing as a possible structural component of metaphase chromosomes by some researchers (Machado et al. 1998), we performed titin array analyses and confirmed no detectable expression of titin in HeLa cells. We also characterised the proteome of metaphase chromosomes in the ultra-high MW region (> 500 kDa). Based on results at both transcript and protein levels, we concluded that titin having higher MW than 1000 kDa is absent in metaphase chromosomes as well as in HeLa cells. The present data strongly suggest that most of the CFPs, which are composed of cytoskeletal proteins, are not essential structural components of metaphase chromosomes. Our current data confirm and refine the four-layer model. Finally, based on our proteome analyses together with the previous information, we provide the list of proteins that are essential for the structural organization of metaphase chromosomes.
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Results |
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BALL PA-Chs and HeLa PA-Chs showed the similar authentic morphologies to each other under the optical microscope (Fig. 1A). A 92.4% of isolated PA chromosomes maintained their authentic morphology (> 1000 chromosomes were counted). Chromosome proteins extracted by the acetic acid method were judged to be completely solubilized in both the RFHR 2-DE and Laemmli sample buffers, since no precipitates were observed after centrifugation at 17 400 g. Approximately 1 mg of chromosome proteins was obtained from 6.4 x 1010 HeLa PA-Chs prepared from 8.0 x 108 HeLa S3 cells.
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As shown in Fig. 1B, 51 and 58 bands were detected from BALL PA-Chs and HeLa PA-Chs, respectively. An enlargement of the SDS-PAGE pattern of HeLa PA-Chs is shown in Fig. 2. Several of the bands (indicated by arrows in Fig. 1C) showed similar intensities to each other. The intensities of the core and linker histones of HeLa PA-Chs were similar to those of BALL PA-Chs, respectively. In general, the intensities of the non-histone proteins in HeLa PA-Chs were 20% lower than the corresponding proteins in BALL PA-Chs, when the histone H4 intensities were defined as 100%. Apparent differences were observed for bands indicated by the arrow heads in Fig. 1C. In particular, striking differences in the PA-Ch band patterns were observed in the MW range from 40 to 70 kDa. In the BALL PA-Chs, cytoskeletal proteins, such as actin, were identified in this region (Uchiyama et al. 2005). In addition, differences in the band intensities were also observed in the high MW region (> 200 kDa) and low MW region (16 kDa < MW < 25 kDa).
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PA-Chs are primarily composed of basic proteins. In our previous report, 47% of the total number and 94% of the total amount of BALL PA-Ch proteins had pI values higher than 9.0 (Uchiyama et al. 2004). Generally, the separation of basic proteins by isoelectric focusing (IEF) 2-DE is not very efficient due to the difficulty associated with pH gradient formation. On the other hand, 1-D SDS-PAGE leads to inaccurate estimation of the amount of each protein, and furthermore in the case of chromosome proteins, the intense histone bands interfere with proteins with similar MW to the histones (Fig. 1). The RFHR 2-DE method is technically superior to the conventional IEF 2-DE for separating proteins with basic pI values and/or low MW. Thus, RFHR 2-DE was employed for separating the HeLa PA-Ch proteins. Figure 3 shows the RFHR 2-DE pattern of the HeLa PA-Ch proteins. The chromosome proteins were separated into approximately 200 different spots with high resolution and high reproducibility. Consistent with the 1-D SDS-PAGE pattern, differences between the BALL PA-Ch and HeLa PA-Ch separation patterns were observed in the region from 40 to 70 kDa, and these proteins were found to be concentrated in the acidic region upon RFHR 2-DE. In contrast, the separation patterns of both cell lines in the basic region were well conserved.
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The names of the identified proteins, along with their corresponding numbers in Figs. 2 and 3 and their database accession numbers, MWs, pI values, MASCOT scores, sequence coverage and related information if available, are indicated in the Supplementary Tables. The identified proteins were classified into three categories, namely: (i) proteins identified in both BALL PA-Chs and HeLa PA-Chs (Table S1), (ii) proteins identified only in HeLa PA-Chs (Table S2) and (iii) proteins identified only in BALL PA-Chs (Table S3). Samples of the identified proteins are shown in Tables 1 and 2. A total of 240 different proteins were identified in BALL PA-Chs and HeLa PA-Chs, among which 153 were identified in BALL PA-Chs in our previous study (Uchiyama et al. 2005), and 189 were newly identified in HeLa PA-Chs in the present study. In total, 102 proteins were commonly identified in both cell lines. The highest MW proteins identified were a DNA-dependent protein kinase catalytic subunit (465 kDa) in 1-D SDS-PAGE and topoisomerase I (90 kDa) in RFHR 2-DE. Figure 4A shows the pI and MW distributions of the identified proteins. In addition to proteins identified in HeLa PA-Chs, those previously identified in BALL PA-Chs and PG-Chs are also shown. The identified proteins were distributed in the acidic (4 < pI < 6) and basic (10 < pI < 12) regions, and especially concentrated in the basic and low MW (< 30 kDa) region. In contrast, there were only a few neutral (6 < pI < 8) proteins. The distribution patterns of the identified proteins showed similar distribution patterns between the BALL PA-Chs and HeLa PA-Chs.
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A quantitative comparative analysis was performed for the identified proteins in BALL PA-Chs and HeLa PA-Chs (for the molar ratios of the respective proteins, see Table 1 and Table S1). The most abundant proteins were the four core histones, and they represented 60% of the total chromosome proteins below 100 kDa. This composition was common for BALL PA-Chs and HeLa PA-Chs. Histone H1 commonly occupied about 10% of the chromosome proteins in both cell lines, although the ratio of histone H1 subtypes (histone H1.X/histone H1.d) was increased in HeLa PA-Chs. The quantitative analysis indicated the existence of histone proteins at 170 bp intervals in the DNA. Other than histones, the proteins present in significant amounts were HMG (high-mobility group) A, HMGN, ubiquitinated histone H2A and BAF (barrier to autointegration factor). These proteins were present at 1/5–1/20 the amounts of the core histones. As indicated in Fig. 4B and Table S1, their amounts were well conserved in both cell lines. Our quantitative and comparative analysis suggests that some identified proteins that amounts are conserved among three kinds of chromosomes, such as HMGA and boreallin (Fig. 4B), are involved in the restricted chromosomal structure (e.g., kinetochore and chromosome peripheral region).
Several proteins, such as fibrillarin, heterogeneous nuclear ribonucleoprotein G (hnRNP G) and some ribosomal proteins, were present in larger amounts in HeLa Chs. These proteins were generally abundant in both cell lines and could not be removed by sucrose density gradient centrifugation or even by Percoll density gradient centrifugation (Fig. 4B). The amounts of almost all the mitochondrial and cytoplasmic proteins differed greatly between BALL PA-Chs and HeLa PA-Chs, and moreover some of these were identified only in a certain cell line.
Titin array analyses
Since a previous study reported the existence of giant titin in mitotic chromosomes (Machado et al. 1998) and a potential chromosomal titin isoform may correspond to a specialized spliced isoform, we searched for titin transcripts using a recently described titin array that displays all 363 titin exons (Lahmers et al. 2004). When biotinylated HeLa cDNA was hybridized to the array, no significant hybridization above the background intensity was identified for any of the 363 titin exons, despite an 10-fold increase (84 µg) in the starting total HeLa RNA (upper panel in Fig. 5A). In contrast, hybridization of biotinylated skeletal muscle cDNA reverse-transcribed from 7 µg of total skeletal muscle RNA produced positive hybridization intensities (25-fold greater than those observed for HeLa cDNA) for 313 of the titin exons, consistent with the skeletal muscle titin isoform (Lahmers et al. 2004) (lower panel in Fig. 5A). We thus conclude that titin expression was not detectable at the sensitivity of our array method, and is either absent from HeLa RNA or represents a transcript of low abundance.
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In order to identify ultra-high MW proteins using mass spectrometry (MS), 2%–6% gradient SDS-PAGE was carried out. Titin T1 (MW: 3000 kDa), titin T2 (MW: 2000 kDa) and nebulin (MW: 800 kDa) from rabbit skeletal muscle were applied as positive controls for good separation and as size markers (Fig. 5B). Titin T1 is full size conventional titin which is rapidly degraded to titin T2 during sample preparation. MS/MS ion searching after in-gel digestion followed by LC-MS/MS measurements identified the protein in the highest band (titin T1 position) as the titin of rabbit skeletal muscle accompanied by a significantly high score (Mascot score of 5088 with a sequence coverage of 6%). Similarly, the MS analysis of the band at titin T2 position also provided successful identification as titin. Then, the gels at the same positions as the rabbit titin bands T1 and T2 in HeLa S3 cells (lane 3 in Fig. 5B) and HeLa PA-Ch proteins (lane 4 in Fig. 5B) were excised, followed by in-gel digestion, peptide extraction and subjection to MALDI-TOF MS or ESI LC-MS/MS analyses, although no bands were visible in these regions. We performed these analyses because protein detection by MS is generally more sensitive (< 5 ng) than that by CBB staining (> 50 ng). However, no proteins were identified in HeLa PA-Chs or HeLa S3 cell extracts through these MS analyses. The highest MW protein identified in HeLa PA-Chs was DNAPK1 (MW: 465 kDa), which was also identified in the 5%–20% gradient gel shown in Fig. 2.
We further investigated chromosomal titin in our samples by immunoblotting and immunostaining. Three anti-titin antibodies directed against three distinct epitopes within titin were used: 9D10 andibody recognizes conserved PEVK repeats that make up most of the elastic PEVK regions of titin (Trombitas et al. 1997). MIR antibody was used because this epitope is highly conserved, and immunoblotting using MIR antibody provides high sensitivity that allows us to detect 1 ng of conventional titin (Fig. S4). Moreover, the MIR antibody recognizes the chromosomal D-titin (Machado et al. 1998). The 5460 antiserum was raised against recombinant M-is6 fragment corresponding to the C-terminal region of human titin. This serum was reported to recognize recombinant M-is6 fragment as well as giant titin in HeLa cells (Zastrow et al. 2006). The nuclear localization of titin has been also reported using this serum (Zastrow et al. 2006). In addition, the -KZ antiserum raised against expressed D-titin protein was used (Machado et al. 1998). This serum was previously employed for the detection of the chromosomal titin in human cell (Machado et al. 1998).
None of the four antibodies detected any conventional titin with its MW of 3000 kDa in either whole HeLa cell extracts, HeLa S3 extracts or HeLa PA-Chs by immunoblotting (Fig. 5C, Fig. S3A and data not shown for -KZ). Furthermore, immunostaining analyses of fixed HeLa cells and spread chromosome specimens revealed that titin was not localized to mitotic chromosomes, even when the antibodies specifically recognized titin (Fig. 5D and Fig. S3B). Although no signal corresponding to titin was detected in HeLa cell extracts and HeLa PA-Chs by immunoblotting, the MIR antibody produced weak signals on the surface of isolated chromosomes and strong signals in the cytoplasm of fixed cells (Fig. 5D). However, these signals presumably originate from cross-reactivity of the anti-titin antibody. Cross-reactivity of this anti-titin antibody with desmoyokin has also been reported (Wernyj et al. 2001) and actually this antibody recognizes desmoyokin (AHNAK, data not shown in our immunoblotting experiments). Regarding -KZ, no signals were detected in any of the present experiments. As for immunostaining analyses using 5460 antiserum of HeLa cells fixed with 4% paraformaldehyde or 3.7% formaldehyde, the localization patterns similar to that observed in the previous study were obtained (Fig. S3B), indicating that titin was not localized to mitotic chromosomes even if this antiserum reacts with titin molecules.
Classification of the identified proteins
The identified HeLa PA-Ch proteins were first classified into the six groups by their known subcellular localizations at interphase. The population of each group was calculated based on the number of identified proteins (Fig. S1). Among the six groups, the largest contained the nuclear proteins (45.0%, 85/189). HeLa PA-Chs also contained large numbers of mitochondrial (16.9%, 32/189) and ribosomal (15.3%, 29/189) proteins. Cytoplasmic and cytoskeletal proteins represented 11.6% and 7.9%, respectively. The populations based on the molar amounts of the identified proteins in HeLa PA-Chs produced a different view for the ratios of the six groups, as predicted in our previous study. The ratios of the six groups of chromosome proteins in HeLa PA-Chs were similar to those in BALL PA-Chs (Uchiyama et al. 2005). The nuclear proteins occupied a large proportion (79.3%) of the amount of chromosome proteins (Fig. S1). The identified proteins were then re-classified into four groups, CCPs, CPPs, CSPs and CFPs, as described in our previous study (Uchiyama et al. 2005). The percentages of the classified proteins in terms of their numbers and molar amounts are shown in Fig. 6A. In this figure, CSPs constitute the largest population, followed by CPPs that are bound to chromosomes periphery. Although the number of CSPs was rather small, they represented the largest amount of chromosome proteins. Although some containment proteins presently identified might be involved in chromosome structure, these results suggest that the chromosome structure is maintained by small numbers of CSPs. Additionally, some of identified proteins (mainly CPPs) are transferred on the chromosomes as reported previously (Haraguchi et al. 2000; Van Hooser et al. 2005).
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Chromosome proteins commonly identified in both HeLa PA-Chs and BALL PA-Chs
Several proteins present in large amounts were identified in both PA-Chs (Table 1). Most of these proteins were classified as CSPs. In addition, proteins commonly present at similar amounts and supposed to contribute to chromosome structure were also classified as CSPs. Histone variants and post-translationally modified histones were included in CSPs among the identified histones. HMGN and HMGA were identified as the second and third most abundant proteins, respectively, after the histones. The amount of HMGB was much lower. These results are consistent with our previous results for BALL PA-Chs (Uchiyama et al. 2005). Among the CFPs, which are mainly composed of cytoskeletal proteins, only ß-actin (MW: 40 kDa) and vimentin (MW: 50 kDa) were identified in PA-Chs from both cell lines. ß-actin was previously reported to have a nuclear localization (Capco et al. 1982), and an actin network is required for chromosome congression in animal oocytes (Lenart et al. 2005). In mitotic HeLa cells, vimentin was associated closely with the mitotic spindle (Zieve et al. 1980). In addition, vimentin and tubulin were identified in Chinese hamster metaphase chromosomes isolated under physiological conditions (Peters et al. 1982). There is a possibility that these CFPs are related to chromosome structure, but we suggest the idea that CFPs are not essential for chromosome structure, because chromosome purification by Percoll density gradient centrifugation reduced the amounts of CFPs. These CFPs may tightly bind to metaphase chromosomes presumably in order to sustain the chromosomes at definite positions during mitosis. Thus, it is quite difficult to completely remove CFPs from isolated chromosomes due to a technical limitation.
Proteins commonly identified but present at different amounts in BALL PA-Chs and HeLa PA-Chs
The proteins with different amounts in HeLa PA-Chs and BALL PA-Chs were mainly CPPs. Generally, CPPs are more weakly bound to chromosomes than CSPs, and most CPPs are nuclear envelope and nucleolar proteins, such as fibrillarin, hnRNPs, BAF and lamin A/C. After the disappearance of the nuclear membrane and nucleoli, their components move to the chromosome periphery, a layer of material covering the chromosome arms during mitosis, and some of these nucleolar components, such as B23, nucleolin and fibrillarin, form nucleolus organizing regions (NORs). CPPs would be essential for reorganization of the nuclear envelope and nucleolus for the onset of the cell cycle after cytokinesis. Several ribosomal proteins are possibly distributed at the chromosome periphery, because they are present in significant amounts in the metaphase chromosomes. Actually, chromosome peripheral localization of ribosomal protein S1 was reported (Hugle et al. 1985).
Proteins identified in either HeLa PA-Chs or BALL PA-Chs
As shown in Fig. 1, the band pattern of BALL PA-Chs in the MW region from 40 to 70 kDa showed marked differences from that of HeLa PA-Chs. Considering that the chromosome morphologies of PA-Chs between epithelial-like cells and lymphoma-type cells were indistinguishable, even when observed under the electron microscope (Fig. S2), these results indicate that PA-Chs include proteins that are not essential for maintaining the chromosome morphology. In this region, several CFPs, such as keratins and septins, were identified only in HeLa PA-Chs (Table 2). These results are consistent with a previous report in which their expressions were limited to specific tissues (Coulombe & Omary 2002).
Regarding CCPs, some of the mitochondrial and cytoplasmic proteins were identified in PA-Chs from only one of the two cell lines (Tables S2 and S3). Many basic proteins (8 < pI < 10) were also removed from PG-Chs (Fig. 4A). Consistent with our previous study (Uchiyama et al. 2004), most of the spots that disappeared in BALL PA-Chs were mitochondrial proteins. Thus, these proteins are most likely to be contaminants originating from the cytoplasm during chromosome isolation.
Proteins identified in the >100 kDa region
The proteins identified in the >100 kDa region included CFPs (myosin, myoferlin and plectin), chromatin remodeling proteins (SPT16 and SNF2 h) and well-known chromosome proteins (topoisomerase II and SMC proteins) (Yokomori 2003).
A previous study indicated that titin was localized both in the nucleus and condensed chromatids in human and Drosophila cells (Machado et al. 1998). It was also reported that titin has functions in the nucleus (Zastrow et al. 2006) and is involved in chromosome condensation (Machado & Andrew 2000). In addition, an analysis of the molecular forces in condensed chromatin suggested the existence of titin-like elastic molecules in the chromosomes (Houchmandzadeh & Dimitrov 1999). It should be, however, pointed out that none of these studies has positive control using conventional titin in the biochemical analyses.
In order to confirm the existence of the ultra-high MW proteins in chromosomes, we tried to detect giant proteins using 2%–6% SDS-PAGE. Contrary to our previous discussion, both the nucleotide homologous to titin and the expression of titin were not detected in HeLa cells (Fig. 5A). The failure of HeLa cDNA to hybridize to a titin gene covering array also denies the possibility that chromosomal titin is a specialized spliced isoform. Consistent with our titin array results, titin was not detected in either whole cell extracts or chromosome proteins by immunoblotting and MS after enzyme digestion, although we applied sufficient amounts of the chromosome proteins for detection in each experiment. In detail, we used at least 1 x 1010 chromosomes for one lane in the SDS-PAGE experiment, which would contain 50 ng of titin assuming that each chromatid contains one titin molecule. This amount of titin should be detected even with CBB staining (Fig. S4). Furthermore, it is the sufficient amount of titin to be detected by immunoblotting or identified by MS, because we could detect 1 ng of titin by immunoblotting identify or 5 ng of titin by MS (Fig. S4).
When we used 5460 antiserum for immunoblotting, intense but smeared bands were detected at the higher molecular weight position than full size conventional titin in HeLa cell extracts, PA-Chs and rabbit skeletal muscle proteins (Fig. S3A). Since these bands were not detected with the titin specific antibodies, 9D10 and MIR, and any expression of the titin isoform was not expected by the result of titin array experiment, we consider them as the aggregated proteins or non-full size conventional titins recognized by the 5460 antiserum. Although proteins having titin-like domains that are recognized by serum against titin might exist in HeLa cells, these results indicate that conventional titin does not exist in the chromosomes.
Nuclear signals of the protein having epitope recognized by 5460 antiserum were detected in interphase cells as reported previously (Fig. S3B; Zastrow et al. 2006). However, the existence of titin in chromosomes was denied by the current immunostaining using 9D10, MIR and 5460 antiserum of fixed HeLa cells, spread chromosome specimens and isolated chromosomes. These three different specimens are suitable for the localization analysis of chromosome proteins (Uchiyama et al. 2005). By using 9D10 and MIR, some signals were detected in the cytoplasm and isolated chromosomes; these would be cross-reactions between the anti-titin antibody and proteins other than titin because several bands were detected in immunoblotting using these antibodies at the position different from the conventional titin. In conclusion, our results are consistent with a model in which the elastic nature of chromosomes is not generated from a scaffold composed of full size conventional titin, but rather from other factors, such as a cross-linking network of chromatin (Poirier & Marko 2002a,b; Gassmann et al. 2005).
Novel chromosome proteins
The present study has identified several candidates for novel non-histone proteins that may contribute to chromosome structure. For example, significant amounts of hnRNP G and HSP70 were detected in PA-Chs from both cell lines. B-cell receptor-associated protein (BAP37) was present at almost equal amounts to peripheral proteins in both HeLa PA-Chs and BALL PA-Chs. BAP37 was also identified in a recent proteomic analysis of the chromosome scaffold (Gassmann et al. 2005). In addition, proteins whose localization and function have not yet been revealed were identified (Tables S1 and S2). Further studies of these proteins, such as investigations of their interactions and dynamics in vivo, will provide essential information to clarify their functions in terms of chromosome structure.
Comprehensive list of chromosome proteins
Finally, in Fig. 6B, we proposed the model of metaphase chromosome structure with a comprehensive list of chromosome proteins based on our study and previous reports. Histone proteins including several histone variants are distributed over all of chromosomes and they form fundamental structure of chromosomes by packaging DNA into nucleosomes. There are no proteins that have the comparable amounts with histone proteins, but some proteins such as HMG proteins (Saitoh & Laemmli 1994; Pallier et al. 2003), SNF2H (Uchiyama et al. 2005), KIF4 (Mazumdar et al. 2004) and DEK (Kappes et al. 2001) are also distributed to the chromosome arm. However, their biological functions in mitotic chromosomes have not been understood well. The chromosome scaffold is mainly composed of topoisomerase II and two types of SMC and non-SMC protein complex, condensin I and condensin II (Gasser et al. 1986; Gassmann et al. 2005). The localizations of scaffold proteins are visualized in the chromosome arm by immunostaining and their coiled axial distributions are observed within the chromosome arm except for the centromeric region. The chromosome scaffold is required for maintaining condensation of chromatin during mitosis and chromosome segregation (Hirano 2006). Chromatid cohesion is preserved at the centromeric heterochromatin, and kinetochore structure is constructed on the outside of the centromere which provides the scaffold of microtubule attachment with the chromosome. Various proteins, such as CENP proteins and chromosome passenger proteins, are involved in centromere and kinetochore formation. Recently intriguing proteome and interaction analyses of the centromere (Obuse et al. 2004; Foltz et al. 2006; Okada et al. 2006) were performed and the mechanism of centromere and kinetochore formation is being clearer. The telomere is also a functional component of chromosomes that protects the chromosome end. TRF proteins, Ku proteins, several of hnRNP proteins and ASF are examples of telomere proteins (Kanoh & Ishikawa 2003; Sumner 2003). In our proteome analysis, many chromosome periphery proteins have been identified. As discussed above, chromosome periphery is mainly constructed by nucleolus components. However, despite large amounts and variety of peripheral proteins, their functions in mitosis are now controversial. The chromosome fiber is not required for chromosome structure, but it binds with chromosomes strongly in mitosis because stable interaction between chromosomes and chromosome fiber might be required for chromosome movement, such as chromosome alignment at metaphase and chromosome segregation at anaphase. Tubulin, actin and their associated proteins form this structure.
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The BALL-1 and HeLa S3 cell lines were cultured as previously described (Sone et al. 2002; Uchiyama et al. 2004). At a cell concentration of 5 x 105 cells/mL, colcemid was added at a final concentration of 0.02 µg/mL to BALL-1 or 0.1 µg/mL to HeLa S3 cells for synchronization of the cell cycle, followed by further incubation for 12 h. Approximately 60% of the mitotic index was achieved by this procedure.
Isolation of human metaphase chromosomes
Metaphase chromosomes were isolated in PA buffer (15 mM Tris–HCl pH 7.2, 2 mM EDTA, 80 mM KCl, 20 mM NaCl, 0.5 mM EGTA, 0.2 mM spermine and 0.5 mM spermidine) containing 1 mg/mL digitonin (Sigma, St. Louis, MO), 14 mM 2-mercaptoethanol and 0.1 mM PMSF according to a previously described method (Uchiyama et al. 2005).
Extraction of chromosome proteins and their separation by electrophoresis
Proteins were extracted from the isolated PA-Chs using the acetic acid method (Hardy et al. 1969). After lyophilization, the chromosome proteins were solubilized in 8 M urea and the protein concentration was determined using the Advanced Protein Assay Reagent (Cytoskeleton, Denver, CO). Next, the protein solution was mixed with an equal volume of 2x Laemmli sample buffer (125 mM Tris pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue and 2% 2-mercaptoethanol) and the mixture was boiled at 100 °C for 5 min. The samples were then electrophoresed through 5%–20% gradient SDS-PAGE gels. For radical-free and highly reducing two-dimensional electrophoresis (RFHR 2-DE; Wada 1986), the lyophilized chromosome proteins were solubilized in RFHR 2-DE sample buffer (8 M urea and 1.5% 2-mercaptoethanol) and the solution was incubated at 40 °C for 30 min. RFHR 2-DE was performed as previously described (Uchiyama et al. 2005). After electrophoresis, all gels were stained with CBB.
Titin array analysis
For titin transcription profiling, total HeLa RNA was purchased from Stratagene (La Jolla, CA), randomly labeled with MLV-RT by reverse transcription and hybridized to a titin-specific array as previously described (Lahmers et al. 2004). Skeletal muscle RNA (Stratagene) was included as a positive control.
Separation and identification of ultra-high MW proteins and immunostaining of titin
Ultra-high MW proteins were extracted and separated using methods developed for the largest protein, titin (Kimura et al. 1992). Cellular and chromosome proteins were extracted from HeLa S3 cells or chromosomes using SDS-PAGE sample buffer (50 mM Tris pH 8.0, 5% SDS, 2.5 mM EDTA and 25 mM DTT) and heated at 60 °C for 3 min. Rabbit skeletal muscle proteins were used as marker proteins. After centrifugation, the samples were separated in 2%–6% gradient SDS-PAGE gels. The separated proteins were detected by CBB staining and then analyzed by enzyme digestion followed by mass spectrometry (MS) as described below. The detection of titin by immunoblotting was carried out using the MIR antibody (Centner et al. 2000) at 1:50 and the 9D10 antibody (Trombitas et al. 1997) at 1:20 the -KZ antiserum at 1:200, and the 5460 antiserum at 1:2000. The -KZ and 5460 antiserum were kindly provided by Prof. D. J. Andrew and by Prof. K. L. Wilson (The Johns Hopkins University School of Medicine), respectively. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham). For the immunostaining, HeLa cells were grown on cover slips coated with polylysine, fixed with 4% paraformaldehyde and stained as previously described (Machado et al. 1998). In addition, immunostaining procedures using the same antibodies were also carried out on spread chromosome specimens and isolated chromosomes according to a previously described method for analyzing the localizations of chromosome proteins (Uchiyama et al. 2005). The primary antibodies were diluted to 1:50 for MIR antibody and 1:1000 for 5460 antiserum. All fluorescently labeled secondary antibodies were used at a dilution of 1:200.
Image analysis
The CBB stained gel images were scanned with IMAGESCAN (Amersham Biosciences, Chalfont St Giles, UK). The separated proteins after one-dimensional (1-D) SDS-PAGE were detected and quantified using IMAGEQUANT (Amersham Biosciences). The RFHR 2-DE pattern was analyzed using IMAGEMASTER 2-D ELITE (Amersham Biosciences).
Protein identification
Detected bands or spots for HeLa PA-Ch proteins were newly processed for identification, even if they were detected at the same positions in the electrophoretic patterns as the previously identified proteins in the proteome analysis of BALL PA-Chs (Uchiyama et al. 2004). Protein identification was performed according to our previous report (Uchiyama et al. 2005). Briefly, identification was primarily carried out using a peptide mass fingerprinting (PMF) method and an Autoflex or Ultraflex matrix-assisted laser desorption ionization time of flight mass spectrometer (Bruker Daltonics, Bremen, Germany). In addition, an Esquire electrospray ionization ion trap mass spectrometer (Bruker Daltonics) or micrOTOF-Q mass spectrometer (Bruker Daltonics) both equipped with Ultimate liquid chromatography (Dionex, Sunnyvale, CA) was used for protein identification by MS/MS ion searching. PMFs and MS/MS spectra were analyzed using the MASCOT software (Matrix Science, Wyndham Place, UK) employing the NCBInr database. Masses were compared with the human protein database at +/–100–200 p.p.m. for PMF analysis and 0.4 Da for both MS and MS/MS ion search mass tolerances. Identification was considered positive when high scores were obtained in more than three independent experiments, when high scores were obtained from both trypsin and Lys-C or Glu-C digestion or when more than three peptide sequences were obtained by MS/MS analysis.
Quantitative analysis of identified proteins
To obtain molar amounts of each protein, the volumes of each band in 1-D SDS-PAGE and spot in RFHR 2-DE gels were divided by their calculated MW. The volumes were normalized by histone H4 volume; thus, the molar amount of each protein was estimated as the ratio to 100 molecules of histone H4, which was well separated from the other core histones. To avoid artifactual underestimation resulting from stain saturation of the band or spots, the amounts of each protein were estimated by averaging more than six gels results obtained under the same conditions that the amount of H4 was not saturated. If several proteins were identified from a single band, the band intensity was divided by the averaged molecular masses of the identified proteins, which provided the summation of the relative molar ratio of the identified proteins.
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Acknowledgements |
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-KZ and 5460 antiserum. We also thank Takashi Nirasawa and Daisuke Higo (Bruker Daltonics) for the protein identification. This study was supported in part by a Special Coordination Fund of the Ministry of Education, Culture, Sports, Science and Technology, Japan. |
Footnotes |
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aPresent address: Institute for Microbiological Diseases, Osaka University, Suita 565-0871, Japan.
*Correspondence: E-mail: kfukui{at}bio.eng.osaka-u.ac.jp |
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Received: 18 July 2006
Accepted: 28 November 2006
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