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1 Department of Molecular Pharmacology, and
2 Department of Gastroenterological Surgery, and
3 Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, 1-1-1 Honjo, Kumamoto 860-8556, Japan
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Abstract |
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Introduction |
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Several approaches have been developed to identify MAPs. These include biochemical purification methods (Vallee et al. 1984; Olmstead 1986; Cassimeris & Spittel 2001). One biochemical purification approach involves the polymerization of endogenous tubulin in the presence of an MT-stabilizing agent, taxol, followed by co-sedimentation of MTs and their bound proteins. This procedure is often followed by cycles of polymerization and depolymerization to enrich the MAPs. MAP2 and 
were originally identified from brain tissues by this MT co-sedimentation method (Vallee et al. 1984; Olmstead 1986; Cassimeris & Spittel 2001). The addition of exogenous MTs has been used as an alternative method to facilitate the recovery of MAPs (Richard & Kreis 1990). Most MAPs that have been identified by this MT co-sedimentation method are relatively abundant proteins that bind along the entire length of MTs.
None of the MAPs identified by this method has been found to be localized at MT-based structures, such as centrosomes and cilia. This may be due to the low abundance of components of these MT-based structures. These low abundant proteins may not easily be detected by conventional MT co-sedimentation methods. To enrich and detect low abundant MAPs, MT affinity column chromatography was developed (Kellogg et al. 1989). This chromatography method identified components of centrosomes, spindles and kinetochores, although it was not determined whether they were true MAPs.
The recent development of mass spectrometry (MS) allowed several groups to analyze components of isolated MT-based structures, such as centrosomes, cilia and mitotic spindles (Ostrowski et al. 2002; Andersen et al. 2003; Keller et al. 2005; Pazour et al. 2005; Sauer et al. 2005; Nousiainen et al. 2006; Reinders et al. 2006). In this study, we prepared MT co-sedimented proteins; exogenous MTs were added to the soluble and extract fractions from rat brain, followed by co-sedimentation of MTs and their bound proteins. MT co-sedimented proteins were then subjected to BioAssist Q anion exchange column chromatography to enrich and detect low abundant proteins, followed by tandem MS (MS/MS) analysis. We identified low abundant MAPs and novel components of centrosomes and cilia.
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Results |
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Rat brains were homogenized in the presence of Triton X-100, followed by ultracentrifugation. The supernatant and pellet fractions were employed as the soluble and insoluble fractions, respectively. From the insoluble fraction, proteins were extracted with a high concentration of NaCl, and the extract was diluted to reduce the concentration of NaCl and used as the extract fraction. When the soluble and extract fractions were incubated with MTs, followed by ultracentrifugation, bound proteins were recovered with MTs in the pellet (P1) (Fig. 1A and B). After the pellet was washed, the resultant pellet (P2) was treated with a high concentration of NaCl and ATP to release the bound proteins from MTs. The released proteins (S3) were collected as MT co-sedimented proteins and subjected to BioAssist Q column chromatography to enrich and detect low abundant proteins. Each fraction was then resolved by polyacrylamide gel electrophoresis (PAGE) (Fig. 2). After staining, each protein band was excised and then in-gel digested with trypsin. Extracted peptides were identified by MS/MS. The sequences obtained were subjected to a search for sequence similarity against the non-redundant Swiss-Prot database. As an example for the quality of MS/MS, Fig. 3 shows a fragmentation spectrum of one peptide derived from EB3, an MAP. This spectrum showed excellent signal : noise ratio and good coverage of the expected fragment mass.
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A total of 1237 protein bands (soluble fraction, 505 bands and extract fraction, 732 bands) were excised and subjected to MS/MS analysis. Database searches resulted in the identification of a total of 391 different proteins (soluble fraction, 235 proteins and extract fraction, 222 proteins) (Table 1). The proteins identified were grouped into 12 different categories on the basis of functions and/or localization: 57 MT cytoskeletal proteins, including MAPs and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins (Fig. 4). This multiplicity implies that our present method is not specific for identifying only MAPs.
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We identified many proteins that had not been previously characterized. These proteins are intriguing candidates for being involved in MT-based functions and structures. Homology analysis indicated that these uncharacterized proteins, except MAP7 domain-containing proteins 1 and 2, did not show obvious homology to known proteins and did not contain functional motifs or domains. MAP7 domain-containing proteins 1 and 2 possessed a domain with a sequence similar to the MT-binding site of MAP7 (data not shown). It is plausible that these proteins function as MAPs. Apart from these MAP candidates, six novel gene products were chosen for further characterization because their cDNAs were available (Table 2). These gene products were expressed as N-terminal enhanced green fluorescent protein (EGFP)-fused proteins in the retina pigment epithelium RPE-1 cells. The localization of expressed proteins was examined by indirect immunofluorescence microscopy. Of the six proteins analyzed, FLJ21438 and the hypothetical protein (Q96MC5) showed a vesicular staining pattern whereas LRRC40 showed a cytoplasmic staining pattern (data not shown). On the other hand, KIAA1505 and LRRC45, both of which contained coiled-coil domains, were co-localized with the centrosomal protein 
-tubulin (Figs 5 and 6). These results indicate that these two proteins are novel components of centrosomes.
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Discussion |
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MT cytoskeletal proteins
Our analysis detected many MAPs previously shown to be biochemically associated with MTs. These included not only side-binding proteins (e.g. MAP1A/B, MAP2 and ) but also plus-end-binding proteins (e.g. CLIP-115, CLIP-170 and EB1). However, we failed to identify several MAPs, such as MAP4, APC, NuMA and CENP-E. These MAPs may have been missed by MS because of an extremely low abundance in the brain or weak interaction with MTs (removed by washing). Furthermore, a minus-end-binding protein, -tubulin, was not identified. -Tubulin not only associates with centrosomes but also exists as a cytosolic form (Moudjou et al. 1996). The cytosolic form is shown to be capable of binding to taxol-stabilizing MTs. This binding of -tubulin to MTs is resistant to salt, ATP and GTP treatment. Thus, a possible explanation for the absence of -tubulin in our identified proteins is that -tubulin was co-sedimented with MTs but was not released from MTs with NaCl and ATP.
Other cytoskeletal proteins
We detected actin, intermediate filaments and septin cytoskeletal proteins. MTs, actin microfilaments and intermediate filaments are the three main cytoskeletal systems (Rodriguez et al. 2003; Chang & Goldman 2004). Although these systems are composed of distinctly different proteins, they are assumed to interact with each other. Septins are also regarded as cytoskeletal components that associate with the actin and MT cytoskeleton (Spiliotis & Nelson 2006). We identified several molecular linkers for mediating structural interactions of these cytoskeletal systems. MAP2 possesses both actin- and MT-binding sites and is regarded as a linker protein between actin and MTs (Rodriguez et al. 2003). Intermediate filaments are assumed to be critical in mediating structural interactions between MTs and actin, functioning through plakins, a family of cytoskeletal cross-linkers. Plakins are large multidomain proteins that bind intermediate filaments and are essential for maintaining tissue integrity (Chang & Goldman 2004). Several plakins, including plectin, bullous pemphigoid antigen 1 and MT-actin cross-linking factor, also contain actin- and MT-binding sites. Identification of MAP2 and plakins, together with the actin and intermediate filament proteins, suggests that the three cytoskeletal systems are interlinked by these linker proteins.
Centrosomal proteins
chTOG, which we identified, is an MAP predominantly localized at centrosomes (Kinoshita et al. 2002), although this protein was classified here into MT cytoskeletal proteins. Identification of this MAP indicates that our method is useful for detecting low abundant MAPs localized at MT-based structures. Several other components of centrosomes were also identified. It is not clear how they were co-sedimented with MTs, but chTOG is known to interact with centrosomal proteins (Ohkura et al. 2001). chTOG may mediate interaction of these centrosomal proteins with MTs.
Chaperones
We identified several chaperones. HSP70s were originally characterized as MAPs before they were identified as molecular chaperons (Liang & MacRae 1997). HSP90 was also shown to associate with MTs in cultured cells. Furthermore, it was shown that the chaperons directly associate with , an MAP, and increase the association of with MTs (Dou et al. 2003). On the basis of these observations, it is proposed that molecular chaperones have roles in MT organization (Liang & MacRae 1997). Our results are consistent with this proposal.
Other proteins
MTs not only play a role in sister chromatid segregation, but also serve as major tracks for the intracellular transport of ribosomes, mitochondria, membrane vesicles and RNA granules. RNA granules transport a subset of mRNAs to axons or dendrites through RNA-binding proteins, referred to as heterogeneous nuclear ribonucleoproteins (hnRNPs), along MTs (Carson & Barbarese 2005; Anderson & Kedersha 2006). MTs are furthermore involved in the migration and positioning of the nucleus and Golgi, although it is poorly understood how the nucleus and Golgi interact with MTs (Barr & Egerer 2005; Starr 2007). Several proteins localized at these organelles were consistently identified, suggesting that these proteins may directly or indirectly interact with MTs. In addition, we identified many proteins with diverse cellular functions and/or localization. Similar multiplicity has also been reported in MS screening of MT-based structures, spindles and cilia (Ostrowski et al. 2002; Sauer et al. 2005). Consistent with these previous reports, our results suggest that many more proteins are involved in MT-based functions and structures than previously assumed.
A major concern is nonspecific contamination by proteins unrelated to MTs. Such contamination would contribute artifactually to the observed multiplicity of MT co-sedimented proteins. However, we identified hnRNP A2, a component of RNA granules, which is shown to bind to chTOG and proposed to mediate the association of hnRNP A2-positive granules with MTs (Kosturko et al. 2005). Furthermore, KARP-1-binding protein 2/KAB1/Cep170 was originally identified as a binding partner of KARP-1, a DNA-binding protein (Do et al. 2003), but it has been found to associate with the mature mother centrioles and spindle MTs during mitosis (Guarguaglini et al. 2005). It is, therefore, plausible that at least some of presumptive impurities actually reflect a specific association with MTs or a role as MAPs.
Of 42 uncharacterized proteins, three proteins with coiled-coil domains (KIAA1505, LRRC45 and CCDC40) were novel components of centrosomes and cilia. In addition, we have found another novel centrosomal protein (Q9NXG0) and named it centlein (Makino et al. 2008). This protein also contains coiled-coil domains. Centrosomes are known to be made up of numerous proteins with coiled-coil domains (Doxsey et al. 2005). Increasing evidence indicates that this molecular structure may be well designed for the organization of protein complexes, such as MT-based structures. Of the uncharacterized proteins, those with coiled-coil domains are highly expected to be components of MT-based structures.
Compared to previous MS analyses of isolated MT-based structures, such as centrosomes, cilia and mitotic spindles (Ostrowski et al. 2002; Andersen et al. 2003; Keller et al. 2005; Pazour et al. 2005; Sauer et al. 2005; Nousiainen et al. 2006; Reinders et al. 2006), our method is characterized by anion exchange column chromatography used to enrich low abundant proteins. It is likely that this chromatography contributes to the identification of novel components of centrosomes and cilia, which have not been previously identified. In conclusion, our present method is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future analysis. It is important to follow up the proteomics approach with more focused experiments to examine whether these candidate proteins show a specific association with MTs and/or MT-based structures and, if so, what their physiological functions are?
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Experimental procedures |
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Tubulin was purified from fresh porcine brains by three cycles of polymerization and depolymerizaiton (Shelanski et al. 1973) followed by DEAE-Sephadex column chromatography (Williams & Lee 1982). Purified tubulin was stored at –80 °C until use. MTs were prepared by incubation of purified tubulin (4 mg/mL) for 20 min at 37 °C in polymerization buffer (80 mM PIPES–NaOH, pH 6.8, 1 mM MgCl2, 1 mM EGTA, 1 mM GTP and 10% glycerol) (Yamamoto et al. 2002; Uezu et al. 2007). After the incubation, taxol was added to give a final concentration of 15 µM.
Preparation and resolution of MT co-sedimented proteins
All the purification procedures were carried out at 0–4 °C. Twenty rat brains were homogenized in 100 mL of buffer A (50 mM HEPES–NaOH, pH 7.5, 5 mM EGTA, 2 mM EDTA, 1 mM DTT, 50 mM NaF, 1 mM Na3VO4, 0.1% Triton X-100, 1 mM PMSF, 10 µg/mL of leupeptin and 1 µg/mL of pepstatin A). The homogenate was subjected to ultracentrifugation at 100 000 g for 1 h. The supernatant and pellet were employed as the soluble and insoluble fractions, respectively. MT co-sedimented proteins from the soluble fraction were prepared as follows: MgCl2, GTP and taxol were added to the soluble fraction to give final concentrations of 4 mM, 1 mM and 20 µM, respectively. The sample was then incubated with 1 mg/mL of MTs for 3 h. The mixture was placed over a 20-mL cushion of 10% sucrose in HEM buffer (50 mM HEPES–NaOH, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 50 mM NaF, and 1 mM Na3VO4, 1 mM GTP and 20 µM taxol), followed by ultracentrifugation at 100 000 g for 30 min. The pellet (P1) was rinsed with HEM buffer and then homogenized in 20 mL of HEM buffer containing 50 mM NaCl, followed by ultracentrifugation at 100 000 g for 30 min. The pellet (P2) was rinsed with HEM buffer and then homogenized in 20 mL of HEM buffer containing 0.5 M NaCl and 0.1 mM ATP to dissociate co-sedimented proteins from MTs, followed by ultracentrifugation at 100 000 g for 30 min. The supernatant (S3, 24 mg of protein) was collected and used as MT co-sedimented proteins from the soluble fraction.
The extract fraction was prepared as follows: the insoluble fraction was re-homogenized in 20 mL of buffer A containing 1.0 M NaCl. The homogenate was mildly stirred for 30 min and centrifuged at 100 000 g for 1 h. The supernatant was diluted with 120 mL of buffer A, followed by ultracentrifugation at 100 000 g for 1 h. The supernatant was collected and the concentration of NaCl was examined by measuring the conductivity. The sample was further diluted with buffer A and brought to a final concentration of 100 mM NaCl. This sample was employed as the extract fraction. The extract fraction was then subjected to MT co-sedimentation in the same manner as described above.
MT co-sedimented proteins from the soluble and extract fractions (10 mg of protein each) were separately subjected to precipitation by a chloroform–methanol–water system (Pohl 1990) and dissolved in 5 mL of buffer B (40 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 0.6% CHAPS and 4 M urea). The sample was applied to a BioAssist Q column (4.6 x 50 mm; Tosoh, Tokyo, Japan) equilibrated with buffer B. The column was washed with 20 mL of buffer B, and elution was performed with a 12.5-mL linear gradient (0–0.25 M) of NaCl in buffer B and a subsequent 6.25-mL linear gradient (0.25–0.5 M) of NaCl in buffer B, followed by 6.25 mL of 1 M NaCl in buffer B. Fractions of 0.5 mL each were collected.
Identification by MS/MS analysis
Each fraction of BioAssist Q column chromatography was subjected to SDS-PAGE (10% polyacrylamide gel), followed by protein staining with silver (Shevchenko et al. 1996). Each protein band was excised and digested with trypsin (Promega, Madison, WI). After destaining with 100 µL of 10 mM potassium ferricyanide and 50 mM sodium thiosulfate (Gharahdaghi et al. 1999), the gel pieces were dried and swollen in digestion buffer [50 mM ammonium bicarbonate, 10% acetonitrile (ACN) and 10 ng/µL trypsin], and incubated at 37 °C for 16 h. Peptides were extracted by 30% and 80% ACN then concentrated in a vacuum centrifuge. Dried samples were dissolved in 15 µL of 0.1% trifluoroacetic acid (TFA) then desalted by Zip tips C18 (Millipore, Bedford, MA) and peptides were mixed with matrix solution (10 mg/mL a-cyano-4-hydroxycinnamic acid in 50% ACN and 0.1% TFA) and crystallized onto the 576-well target plate. Matrix-laser desorption/ionization-time of flight (TOF) and tandem TOF data were acquired in batch mode using an ABI4700 Proteomics Analyzer (Applied Biosystems. Foster City, CA). MS reflector positive ion mode with automated acquisition of 800–4000 m/z range was used with 1000 shots per spectrum. A maximum of 10 peaks were selected per spot, with a minimum signal : noise ratio of 10 and a cluster area of 200. Precursor ions were submitted for MS/MS, where a positive ion mode with a collision induced-dissociation cell and 1kV collision energy were used, and 5000 shots were accumulated per spectrum. Database searching was performed with the GPS EXPLORER SOFTWARE (Applied Biosystems) utilizing Mascot (v1.9) at the search engine (Matrix Science Ltd., London, UK) allowing ±0.3 Da as the parent tolerance and ±0.1 Da as the fragment ion tolerance. Combined peak lists were searched against the non-redundant Swiss-Prot database, and the oxidation of methionine and one possible missed cleave site was selected as a variable modification. Peptides were considered identified if their Mascot score was at or over the 95% confidence limit; meaning that the fragmentation data is of sufficient quality as to have a > 95% probability of being assigned to the proper peptide sequence. Proteins were considered identified if at least one peptide matched to it with a significant Mascot score.
Molecular cloning
Q8N2T9 was obtained from the Kazusa DNA Research Institute. Q8IYE0 was purchased from Invitrogen (Carlsbad, CA) (MGC clone, 5273288). Q8BI79 (RikenB930008102) was purchased from DNAFORM (Yokohama, Japan). The following cDNAs were obtained by PCR : Q9CRC8, (primers) 5'-CTG CAG CTT CTG GAC CTA GGA CTT TG-3' and 5'-ATG ACA AGG GTT ATA AAG CAA CTC CAT G-3', (template) HeLa cell cDNA; Q96MC5, (primers) 5'-GAT CAG CGA TGG AAT TAA AGC AAT C-3' and 5'-GAC TCC CTA CTG CTA CAC TCT GTA CAG-3', (template) HeLa cell cDNA; and Q8CIM1, (primers) 5'-GAA TTC ATG GAG GAG TTC CGG CGC TCC TAC-3' and 5'-GTC GAC TCA TTT AGG GGG ATC CAA GGC TCT C-3', (template) mouse brain cDNA. These cDNAs were cloned into pEGFP-C1 (Clontech. Mountain View, CA) and pMXII-Myc (Ono et al. 2000).
Cell culture, transfection and immunofluorescence microscopy
RPE-1 and MDCK cells were maintained at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection was performed using FuGene6 (Roche Diagnostics, Basel, Switzerland) transfection reagent according to the manufacturer's protocol. For cilia immunostaining in RPE-1 cells, cells were cultured in medium with 0.25% serum for 48 h (Gromley et al. 2003). For cilia immunostaining in MDCK cells, a recombinant retrovirus carrying Myc-CDCC40 cDNA was prepared with pMXII-Myc (Nishimura et al. 2002). MDCK cells were infected with the recombinant retrovirus and cultured for 1 day. After the medium was changed, the cells were grown for 7 days post-confluence on 10 mm Transwell filters as described (Fan et al. 2004).
The following antibodies (Abs) were purchased from commercial sources: mouse anti-Myc monoclonal Ab (9E10) (American Type Culture Collection, Manassas, VA); mouse anti-acetylated tubulin (clone 6-11B-1) and mouse anti--tubulin monoclonal Abs (Sigma-Aldrich, Seelze, Germany); rabbit anti-EGFP polyclonal Ab (MBL Co., Nagoya, Japan); and secondary Abs conjugated with Alexa Fluor 488 and 594 (Invitrogen). For immunofluorescence microscopy, cells were fixed in 100% methanol at –20 °C for 5 min. After blocking with 1% bovine serum albumin for 1 h at room temperature, the samples were incubated with primary Abs for 1 h, followed by incubation with second Abs for 30 min. The samples were viewed with a florescence microscope (Olympus, BX51).
Other procedures
Protein concentrations were determined with bovine serum albumin as a reference protein (Bradford 1976). SDS-PAGE was done as described (Laemmli 1970).
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: hnakanis{at}gpo.kumamoto-u.ac.jp
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References |
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Received: 20 November 2007
Accepted: 19 December 2007
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