Abstract
The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to γ-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.
- centriole
- centromatrix
- centrosomal protein 57 (Cep57)
- centrosome
- electron microscopy
- microtubule formation nucleation
INTRODUCTION
The centrosome is a small organelle that nucleates and regulates MTs (microtubules) of animal cells [1]. It includes a core structure consisting of a pair of centrioles with surrounding accessory proteins referred to as pericentriolar material [2,3]. In addition, Schnackenberg et al. [4] have proposed a salt- or chaotrope-insoluble internal substructure called the ‘centromatrix.’ Centrosomes are the predominant MT organization centre of animal cells and a central component is the γ-tubulin ring complex, which contains MT nucleation activity. After nucleation, the minus ends of some MTs remain anchored at the centrosome. In addition to a role as an MT organization centre, recent studies have demonstrated a significant role for centrosomes in signal transduction.
In the present study, Cep57 (centrosomal protein 57) was initially identified as one of the positive candidates through a yeast two-hybrid screen. Cep57 used to be denoted as KIAA0092, the code given by a coding-sequence-prediction project of the human genome [5], until it appeared in the list of salt-insensitive components of purified centrosomes and was denoted as Cep57 [6]. Bossard et al. [7] showed that suppression of endogenous Cep57 resulted in hindrance of translocation of the 18 kDa FGF2 (fibroblast growth factor 2) isoform from the membrane to the nucleus and called the protein Translokin. Kim et al. [8] suggested a role for Cep57 in the post-meiotic phase of sperm cell differentiation based on the observation that Cep57 mRNA is up-regulated between day 21 and 25 of post-natal testicular development.
Because of the importance of centrosomes in cell division and the incomplete understanding of their role, the distinct features of Cep57 prompted us to further perform a structure–function analysis of Cep57 as a possible key player in the centrosomes' function. Recent in silico screening suggests that two forms of Cep57 exist in Xenopus, human and mouse. We find that Cep57 has distinct functional domains to strictly target it to centrosomes, and to induce nucleation and stabilization of MTs.
MATERIALS AND METHODS
Cloning of mouse Cep57 cDNA and plasmid construction
cDNA of mouse Cep57 with flanking restriction sites was cloned from QUICK-Clone™ cDNA (mouse smooth muscle; Clontech) by PCR with a set of primers: 5′-CGCGGATCCCATGGCGGCAGCTCCGGTCTCGGCGGCTT-3′ and 5′-GCTCTAGAATTCAGTAATCCCAACACAGATTACTGCTCT-3′. The overall sequence of the PCR product was matched with the reported sequence in GenBank® (accession number AY225093) except for a few mismatches: A9T, G10C, A884G and A887C. The full-length and different segments of Cep57 cDNA were PCR-amplified from the initial PCR product and introduced into various expression vectors; e.g. pGST-Parallel1 [9], pEGFP-C and pDsRed-Express-C (Clontech) and pJRed-C (Evrogen). JRed is a monomeric red fluorescent chromoprotein from a jellyfish of the suborder Anthomedusae [10].
For low-level expression in mammalian cells, a mammalian expression vector, from which an N-terminally humanized Renilla reniformis GFP (green fluorescent protein) (hrGFP; Stratagene)-fused protein is expressed under the regulation of hUbC (human ubiquitin C) promoter, was constructed. The DNA sequence encoding hrGFP was PCR-amplified with the flanking restriction sites 5′-HindIII and 3′-EcoRI and the Kozak translation initiation sequence adjacent to the initiation codon, and recombined between HindIII and EcoRI sites of pUB/V5-His vector (Invitrogen). The DNA sequence encoding either truncated or full-length Cep57 was subsequently introduced to express N-terminally hrGFP-fused protein. An expression test of hrGFP–N-Cep57 (Cep57 N-terminus) promoted by the hUbC promoter showed 5–10-fold lower expression than that promoted by the CMV (cytomegalovirus) promoter (results not shown).
Production of GST (glutathione transferase)–C-Cep57 (Cep57 C-terminus) proteins in Escherichia coli and purification
E. coli BL21-CodonPlus (Stratagene) was transformed with pGST-Parallel1-C-Cep57, precultured to an attenuance (D600) of 2.0 at 37 °C and further cultured at 16 °C in Terrific Broth containing 100 mg/ml ampicillin and IPTG (isopropyl β-D-thiogalactoside; 1 mM) for 24 h. Bacteria were harvested by centrifugation at 6000 g for 10 min, resuspended in ice-cold PBS containing protease inhibitors (complete protease inhibitor cocktail tablets; Roche) and lysed by using a French press. Cell lysate was cleared by a two-step centrifugation, first at 20000 g for 10 min and then at 65000 g for 1 h (both at 4 °C). The resulting supernatant was mixed with Glutathione–Sepharose 4 Fast Flow beads (Amersham) and rocked overnight at 4 °C. After a thorough wash with PBS and then with 50 mM Pipes buffer (pH 7.0), the beads were packed in a column, and GST–C-Cep57 was eluted by using 10 mM GSH in 50 mM Pipes buffer (pH 7.0).
Production of anti-Cep57 antibodies
The recombinant His6–C-Cep57 protein was used as an antigenic peptide for inoculation of rabbits for polyclonal antibody production (Biosource, Hoplinton, MA, U.S.A.). After the epitope region was narrowed to between amino acid residues 332 and 500 by verifying the reactivity of the crude serum to different regions of Cep57, the antibody was affinity-purified against the recombinant GST–Cep57332–500 protein. To raise monoclonal antibodies in mice, the recombinant GST–Cep57332–500 and GST–N-Cep57 proteins were used as antigenic peptides (A&G Pharmaceutical, Columbia, MD, U.S.A.).
Analysis of MT stabilization and homo-multimerization by co-localization
NIH 3T3 cells were cultured on glass coverslips in DMEM (Dulbecco's modified Eagle's medium; Invitrogen–Gibco) supplemented with 10% (v/v) FBS (fetal bovine serum; Invitrogen–Gibco) at 37 °C in 5% CO2. Plasmids for the ectopic expression of fluorescent-tagged either truncated or full-length Cep57 were transfected into cells using Lipofectamine™ 2000 (Invitrogen) following the manufacturer's standard protocol. At 24 h after transfection, cells were either fixed immediately in methanol at −20 °C or, for MT stabilization analysis, mixed with the medium containing nocodazole (5 μM) for 30 min at 37 °C and fixed. The cells were washed with PBS, blocked in 3% (w/v) BSA in PBS and stained with either a primary antibody or a conjugated fluorescent antibody in the blocking buffer. Conjugated fluorescent antibodies used for epifluorescence confocal microscopy are Cy3 (indocarbocyanine)-conjugated anti-β-tubulin antibody (Sigma) and Cy3-conjugated anti-Myc antibody (Sigma) at the dilution of 1:2000. Combinations of the primary antibody [i.e. anti-γ-tubulin antibody (Abcam; Cambridge, MA, U.S.A.) or polyclonal anti-Cep57 antibody at a dilution of 1:2000 or monoclonal anti-Cep57 antibody (hybridoma supernatant) at a dilution of 1:250] and the secondary antibody [i.e. Alexa Fluor® 488- or 594-conjugated anti-rabbit or anti-mouse IgG antibody (Invitrogen–Molecular Probes) at a dilution of 1:2000] were also used for epifluorescence confocal microscopy. The cells were washed between and after the application of the antibodies with PBS, mounted in Aqua Poly/Mount (Polysciences) and viewed under an Olympus FV300 epifluorescence confocal microscope. When cells were co-stained by the monoclonal anti-Cep57 antibody and Cy3-conjugated anti-β-tubulin antibody (mouse monoclonal), Cy3-conjugated anti-β-tubulin antibody was applied after the staining process with the monoclonal anti-Cep57 antibody had been completed.
RNAi (RNA interference)
HeLa cells were cultured in DMEM supplemented with 10% FBS (Invitrogen–Gibco) at 37 °C in 5% CO2. For transfection, HeLa cells were prepared in six-well dishes with 1.7 ml of serum- and antibiotic-free DMEM and transfected by adding OptiMEM (Invitrogen–Gibco) containing 6 μl of DharmaFECT 1 siRNA (small interfering RNA) transfection reagent (Dharmacon) and siGENOME SMARTpool reagent (Dharmacon), and a pool of four RNAi oligonucleotides targeting segments of human Cep57 sequence: 5′-GAUAAAGCAUGCCGAAAUGUU-3′, 5′-GGAAACGCAUGCAAGCUAAUU-3′, 5′-CAACAGCAGAGCCAUAUUUUU-3′ and 5′-AGUAAGAAGUUGUCAGUAAUU-3′. The final concentration of siGENOME SMARTpool reagent was 50 nM; the subconcentration of each RNA oligonucleotide was 12.5 nM. After 4 h of transfection in serum-free DMEM, 1 ml of DMEM supplemented with 30% FBS was added. After 24 h of transfection, the transfectant was trypsinized and sparsely replated in new six-well dishes with glass coverslips. The cells were fixed, immunostained and subjected to epifluorescence confocal microscopy 4 days after the transfection.
Co-immunoprecipitation
HEK-293 cells (human embryonic kidney-293 cells) were cultured in minimum essential medium (Invitrogen–Gibco) supplemented with 1% non-essential amino acids, 1% sodium pyruvate and 10% (v/v) horse serum at 37 °C at 5% CO2 and transiently co-transfected with (i) FLAG–C-Cep57 with Myc–N-Cep57,(ii) FLAG–N-Cep57 with Myc–N-Cep57, (iii) FLAG–N-Cep57 with Myc–Cep5758–269 or (iv) FLAG–N-Cep57 with Myc–Cep571–239 expression vectors by a calcium phosphate method. Following incubation for 36 h, the transfectants were lysed in a buffer [1% Triton X-100 (Sigma), 150 mM NaCl, 50 mM Tris, pH 7.5, and 2% protease inhibitor cocktail (Sigma)], and the lysate was cleared by centrifugation at 18000 g for 10 min. The supernatant was diluted with an equal volume of the lysis buffer without Triton X-100 and protease inhibitor cocktail; hence, the buffer condition for immunoprecipitation was 0.5% Triton X-100, 150 mM NaCl, 50 mM Tris (pH 7.5) and 1% protease inhibitor cocktail. The diluted supernatant was mixed with 15 μl of EZview Red anti-FLAG M2 Affinity Gel (Sigma). Following overnight incubation at 4 °C, the gel was washed three times with the immunoprecipitation buffer. Proteins were solubilized in sample buffer [1% SDS, 15% (v/v) glycerol, 15 mM dithiothreitol, 62.5 mM Tris, pH 6.8, and 0.008% Bromophenol Blue] and subjected to Western-blot analysis with anti-FLAG M2 monoclonal antibody or anti-Myc antibody. The antibody dilutions were 1:20000 and 1:10000 respectively.
EM (electron microscopy)
EM analysis of cells overexpressing Cep57
NIH 3T3 cells overexpressing EGFP (enhanced GFP)–Full-Cep57 (full-length Cep57) were pelleted and trapped in a collagen matrix. Following fixation in glutaraldehyde, tannic acid and osmium tetraoxide and en-block staining with 4% uranyl acetate, the cells were embedded in Spurr's resin, and ultrathin sections at 80 nm were prepared.
EM of the samples used for in vitro tubulin-Cep57 polymerization assays
A drop of sample solution containing MTs and/or free tubulin and/or recombinant GST–C-Cep57 protein was applied on to a carbon-coated copper grid, proteins were allowed to settle and the grid was negatively stained with 4% uranyl acetate for 1 min. Excess solution was drained by lens paper. EM analyses were performed with an electron microscope (Philips CM12) at 80 keV.
In vitro tubulin polymerization assay
Tubulin polymerization was monitored at 35 °C by light absorbance at 350 nm (A350) using a spectrophotometer (Beckman DU7400). Formation of MTs was confirmed by EM. In all of the experiments, the concentration of tubulin was maintained below the critical concentration where spontaneous polymerization occurs. Purified bovine brain tubulin (obtained from Cytoskeleton Inc., Denver, CO, U.S.A.) was suspended in PME buffer (80 mM Pipes, 1 mM MgCl2 and 2 mM EGTA, pH 6.9; 60 μl) with 1 mM GTP, pre-equilibrated in a sample cuvette, and then either Taxol and/or recombinant GST–C-Cep57 protein was added. Taxol stock solution (2 mM) was prepared in DMSO, and the final concentration of DMSO in the reaction solution did not exceed 5%. Calibration plots (A350 against [tubulin]) were constructed for tubulin polymerized with 10 μM Taxol and was shown to be linear up to 12 μM tubulin. All of the experiments using GST–C-Cep57 were carried out in parallel with control experiments with GST only.
Assessment of the mitotic index and cell-cycle analysis by flow cytometry using FACS
NIH 3T3 cells transiently expressing either EGFP–N-Cep57 or Myc–N-Cep57 were fixed, immunostained for β-tubulin and/or Myc epitope tag respectively. Chromosomes were visualized by staining with ToPro3 (Invitrogen–Molecular Probes). Mitotic cells were identified by the typical mitotic appearance of MTs and chromosomes and counted. The cells expressing N-Cep57 were identified by either EGFP fluorescence or positive staining of the Myc epitope tag. Six independent preparations transfected with the expression plasmid for EGFP–N-Cep57 and three independent preparations for Myc–N-Cep57 were assessed. The P value was calculated by a two-tailed Student's t test.
NIH 3T3 cells transfected by Myc–N-Cep57 expression plasmid were fixed in cold 2% (w/v) paraformaldehyde in PBS followed by −20 °C 70% (v/v) ethanol, blocked in 3% BSA in PBS and immuno- and DNA-labelled by FITC-conjugated anti-Myc antibody (1:1000 dilution; Sigma) and propidium iodide buffer [0.1% Triton X-100 (Sigma) respectively, 100 μg of DNase-free RNase (Sigma) and 10 μg of propidium iodide (Sigma) in PBS]. The FACS analysis was performed by the BD FACSCalibur™ system (BD Biosciences). The gate for the non-transfected control cells was defined as below the ‘gap’ in signal intensity from immunolabelled Myc epitope tag. The Myc–N-Cep57-positive cells were defined as ‘all of the above’ population.
RESULTS
To characterize mammalian Cep57, we cloned the mouse Cep57 from a smooth-muscle cDNA library. Mouse and human Cep57 are composed of 500 amino acid residues (GenBank® accession numbers Q8CEE0 and Q86XR8 respectively). The secondary structure prediction of Cep57 was performed using COILS (http://www.ch.embnet.org/software/COILS_form.html) [11] as well as the PredictProtein Server (http://cubic.bioc.columbia.edu/predictprotein/) [12]. The secondary-structure prediction indicated that Cep57 is composed of two α-helical coiled-coil segments connected by a flexible linker region and this structural information was used to determine where to truncate Cep57, and we generated a set of deletion mutants of Cep57 to study its function and define domains (Figure 1A). Unless otherwise stated, truncations were made at the double proline residues (residues 267 and 268) or their proximate residues in the middle flexible linker region. We denote the N-terminal-half truncates as N-Cep57, the C-terminal-half truncates as C-Cep57 and full-length Cep57 as Full-Cep57. Of note, the capability of producing the recombinant N-terminal-half and the C-terminal-half Cep57 proteins in E. coli separately helped to increase the production efficiency and retain the solubility of the products.
(A) A schematic diagram of the computationally predicted structure of Cep57 and its empirically defined functional domains. Mouse and human Cep57 include 500 amino acid residues and the residue numbers in this diagram are based on mouse Cep57. The N-terminal half and C-terminal half are separated at the double proline residues (residues 267 and 268) and thus each domain was independently characterized. The α-helices in the cartoon represent the consensus of different protein structure prediction algorithms. N-terminal-half: representative truncates used to define the centrosomal localization and multimerization domain. Not all truncates were used interchangeably in different experiments; therefore some items are marked as N/A where the exact truncate was not used for the specific assay. C-terminal-half: representative truncates used to define the MT localization and stabilization domain. Co-Ip w/N-Cep57, co-immunoprecipitation with N-Cep57. (B) Polyclonal antibody reacting with a protein (approx. 60 kDa) in NIH 3T3 whole cell lysate (upper panel; left-hand lane) at the same size as overexpressed FLAG–Full-Cep57 in HEK-293 cells (upper panel; middle lane). The overexpressed FLAG–Full-Cep57 is shown to react with both the polyclonal anti-Cep57 and anti-FLAG antibodies (upper and lower panels; middle lane). The polyclonal antibody does not react with HEK-293 whole cell lysate (upper panel; right-hand lane). (B′) Polyclonal antibody reacting with a protein (approx. 60 kDa) in NIH 3T3 whole cell lysate (left-hand panel) and the reaction being blocked by addition of recombinant GST–C-Cep57 protein (right-hand panel). Molecular-mass markers are indicated on the left-hand side (in kDa). (C) Endogenous Cep57 expression profile among different tissues in mouse and rat in Western-blot analysis using polyclonal anti-Cep57 antibody. Molecular-mass markers are indicated on the right-hand side (in kDa). (D) Representative confocal combined Z-stack images of mitotic HeLa cells with and without a treatment for RNAi targeting Cep57. The cells were immunostained for Cep57 and β-tubulin. The signals for Cep57 in the untreated cell (arrows) are absent in the treated cell. The pole-to-pole distance of mitotic spindles (lines) is indicated.
Rabbit polyclonal and mouse monoclonal anti-Cep57 antibodies were produced using truncated Cep57 recombinant protein as an antigen. Following affinity purification against recombinant C-Cep57, the specificity of the polyclonal antibody was verified by (i) specific reactivity to C-Cep57 recombinant protein and overexpressed C-Cep57 in mammalian cells (results not shown), (ii) size agreement between endogenous Cep57 and overexpressed Full-Cep57 (Figure 1B) and (iii) blockage of reactivity to endogenous Cep57 in the presence of recombinant C-Cep57 (Figure 1B′). Specificity of the monoclonal antibody was verified in a similar manner as for the polyclonal antibody. A tissue screen using the polyclonal antibody shows ubiquitous expression of Cep57 in the tissues tested (Figure 1C). NIH 3T3 cells were immunostained with the polyclonal or monoclonal anti-Cep57 antibody and showed one or two spots adjacent to the nucleus in each cell (Figure 2A). These spots were also stained by anti-γ-tubulin antibody, a marker for the centrosomes. The immunostain by monoclonal antibody in HeLa cells disappeared when the cells were transfected with RNA oligonucleotides for RNAi targeting Cep57 mRNA (Figure 1D). Taken together, we concluded that both the polyclonal and monoclonal antibodies specifically recognize Cep57. Although kinetochore staining is also observed on purified chromosomes from Xenopus cells, we did not see it in either Xenopus, human or mouse cells [13]. These results suggest that Cep57 is primarily localized to the centrosome in somatic cells.
(A) Confocal images of NIH 3T3 cells immunostained by the polyclonal and monoclonal anti-Cep57 antibodies (Ab; top and bottom rows respectively). The centrosomes are identified by γ-tubulin stain except for the low-magnification image of the cells immunostained with the monoclonal anti-Cep57 antibody, which is co-stained with anti-β-tubulin antibody and thus the MT network is shown. Insets show the mitotic cells corresponding to each treatment. The centrosomes at high magnification shown in the bottom row are in different cells from those shown at low magnification. (B) Confocal images showing centrosome localization of hrGFP–Full-Cep57, hrGFP–N-Cep57 and hrGFP–Cep5758–239 expressed at low levels in NIH 3T3 cells (top, middle and bottom rows respectively). The centrosomes are identified by γ-tubulin stain (middle column). Insets show the mitotic cells corresponding to each sample except the one for hrGFP–Cep5758–239 showing the centrosomes at high magnification; the centrosomes shown in the insets are in different cells from the ones shown at low magnification.
The cells showing no signal from Cep57 following the transfection for RNAi had reduced pole-to-pole distance of the mitotic spindles. The average pole-to-pole distance of the treated cells (n=47) was 8.70 μm as compared with 9.87 μm in the untreated cells (n=47; P≪0.05). This suggests that Cep57 plays a role in mitotic spindle formation in mammalian cells.
The first α-helical coiled-coil segment of Cep57 contains a centrosome-targeting domain
Cells that express low levels of ectopic expression of hrGFP–Full-Cep57 in NIH 3T3 cells showed localization at the centrosomes (Figure 2B). Both ectopically expressed Cep57 and endogenous Cep57 appeared to be at a cylindrical internal structure in the centrosomes indicated by immunostaining with an anti-γ-tubulin antibody (Figure 2). This suggests that Cep57 is indeed localized at the centromatrix and/or centrioles and is consistent with its biochemical identification in proteomic analysis of a salt-resistant centrosomal fraction [6].
The centrosome-targeting domain is localized in the N-terminus of Cep57. hrGFP–N-Cep57 was expressed in cells localized to the centrosome (Figure 2B). When highly overexpressed, this protein could also be found in the cytoplasm (Figures 3B, panel a, and 5A, panel m). This centrosomal localization was recapitulated by hrGFP–Cep5758–239, whereas hrGFP–C-Cep57 did not show apparent localization to the centrosomes. This strongly suggests that the amino acid residues between 58 and 239 are responsible for centrosome localization of Cep57 (Figures 1 and 2B). Thus we define this amino acid stretch as the CLD (centrosome localization domain) of Cep57.
(A) FLAG–C-Cep57 and Myc–N-Cep57 (lane 1), and FLAG–N-Cep57 and Myc–N-Cep57 (lane 2) or Myc–Cep5758–269 (lane 3) or Myc–Cep571–239 (lane 4) co-expressed in HEK-293 cells, immunoprecipitated (IP) by anti-FLAG antibody and blotted (IB) by anti-FLAG and anti-Myc antibodies, showing co-immunoprecipitation of Myc–N-Cep57 (lane 2), Myc–Cep5758–269 (lane 3) and Myc–Cep571–239 (lane 4) with FLAG–N-Cep57, but not with FLAG–C-Cep57 (lane 1). (B) EGFP–N-Cep57 is co-expressed either with DsRed–C-Cep57 (a and b) or DsRed–Full-Cep57 (c and d) in NIH 3T3 cells, showing co-localization of EGFP–N-Cep57 with DsRed–Full-Cep57, but not with DsRed–C-Cep57.
Ectopic expression of the CLD resulted in reduction in the mitotic index and G1 arrest. For the cells showing ectopic expression of EGFP–N-Cep57, the mitotic index was 0.51% (n=2744), in contrast with that for the control cells (3.82%; n=12971, P≪0.05). This result is consistent where Myc–N-Cep57 was ectopically expressed, resulting in a mitotic index of 0.91% (n=1694) as compared with 4.21% (n=6240) in control cells (P<0.004). To determine how this cell-cycle hindrance occurs, we further analysed changes in cell cycle by flow cytometry using FACS. The control cells showed a typical distribution among different phases in the cell cycle, whereas the cells ectopically expressing Myc–N-Cep57 were almost exclusively in G1 phase (results not shown) (G1 arrest).
The N-terminus of Cep57 contains a multimerization domain
Dimerization of Cep57 has been suggested by Bossard et al. [7]. A portion of the N-terminal half of Cep57 is homologous with a central region of EGF (epidermal growth factor) receptor substrate 15 protein and EGF substrate 15-related protein. The central region of these proteins contains a coiled coil that is involved in homo- or hetero-dimerization [14,15]. To test the hypothesis that the N-terminal half is a dimerization domain in Cep57, co-immunoprecipitation and co-localization assays were performed. Myc–N-Cep57 was co-expressed in HEK-293 cells either with FLAG–N-Cep57 or with FLAG–C-Cep57 and the whole cell lysate of each treatment was subjected to anti-FLAG antibody-conjugated agarose. As a result, Myc–N-Cep57 was co-immunoprecipitated with FLAG–N-Cep57, but not with FLAG–C-Cep57 (Figure 3A, lanes 1 and 2). Co-localization of ectopically co-expressed various Cep57 truncations also suggested homo-multimerization of Cep57 via its N-terminal half. As discussed below, high-level expression of Full-Cep57 induces a massive fibrous ‘basket-like’ structure around the nucleus, and this feature is retained in C-Cep57, which is shown using a EGFP-fused protein (Figure 5A). As expected, DsRed (Discosoma corallimorpharian red fluorescent protein)–C-Cep57 also induced the basket-like structure (Figure 3B). EGFP–N-Cep57 co-expressed with DsRed–Full-Cep57 co-localized to the basket-like structure, but the one co-expressed with DsRed–C-Cep57 did not. The results of immunoprecipitation and indirect fluorescence microscopy together suggest homo-multimerization of Cep57 through its N-terminal half. The crucial amino acid residues involved in multimerization were narrowed by co-immunoprecipitation of further truncated N-terminal-segments. When co-expressed with FLAG–N-Cep57, both Myc–Cep5758–259 and Myc–Cep571–239 were co-immunoprecipitated by anti-FLAG antibody-conjugated agarose (Figure 3A, lanes 3 and 4). Therefore the N-terminal amino acid residues between 58 and 239, the coiled-coil and α-helical segment, namely CLD of Cep57, are also crucial in multimerization of Cep57.
The C-terminus of Cep57 binds, nucleates and bundles MTs
As mentioned above, high-level expression of EGFP-fused Cep57 in mammalian cultured cell lines, such as NIH 3T3 cells, generated massive fibrous basket-like structure around the nucleus. Immunostaining for MTs revealed that the basket-like structure was composed not only of EGFP-fused Cep57, but also of MTs, and EM revealed convoluted thick-cable structures with deposition of dense material on the periphery of MTs (Figure 5B). Formation of the basket-like structure is also in agreement with a similar observation reported by [7]. This feature, the formation of the basket-like structure, was retained in the C-terminal half of Cep57. Therefore, to study how C-Cep57 can induce the massive MT network, in vitro tubulin polymerization assays were carried out in the presence and absence of the recombinant protein.
A typical time course of tubulin (0.5 mg/ml) polymerization induced by 10 μM Taxol, monitored by light absorption at 350 nm, is shown in Figure 4(B). The critical concentration (minimum [tubulin] capable of initiating tubulin polymerization) was estimated to be <1 μM, and MT formation was confirmed by EM (Figure 4A). Two distinct phases were observed during Cep57-induced MT formation (Figure 4B). Addition of GST–C-Cep57 to tubulin initiated a prompt increase in A350, implying nucleation of MT formation although it takes approx. six times longer to reach the theoretical MT-origin maximum A350 (the first phase) as compared with Taxol-induced polymerization. The theoretical MT-origin maximum A350 is the absorbance achieved when all tubulin molecules in the sample participate in the MT structure. Surprisingly, a second phase occurred with a much higher rate surpassing the MT-origin maximum A350. The increase eventually levelled off at substantially higher A350 than the MT-origin maximum A350, i.e. 1.25 compared with 0.4 at 0.25 mg/ml tubulin.
(A) Electron micrographs of each sample at maximal A350: (a) Taxol-induced MTs; (b) MTs with recombinant C-Cep57 protein (MT bundles are indicated by arrows); and (c) the mixture of tubulin and GST treated in the equivalent condition as the sample with recombinant C-Cep57 protein. (B) Transition of A350 (OD350), reflecting the quantity of MTs present in each sample: Taxol-induced MTs, Cep57-induced MTs and tubulin+10% glycerol.
At high [tubulin]/[Cep57], only the first phase was observed (results not shown). Subsequent addition of GST–C-Cep57 triggered the second phase, which far exceeded the MT-origin maximum A350. This is also observed when GST–C-Cep57 was added to MTs pre-assembled with Taxol. The source of light absorbance in the second phase was explored by EM. In the sample with GST–C-Cep57, we observed side-to-side aggregation of MTs that was not observed among MTs promoted by Taxol (Figure 4A). Among 50 GST–C-Cep57-induced MTs, 48 MTs appeared to participate in side-to-side aggregation, whereas among 50 Taxol-induced MTs, no formation of side-to-side aggregation was observed. Taken together, we concluded that GST–C-Cep57 weakly nucleates MT formation and also introduces strong side-to-side aggregation of MTs. These findings were consistent with the side-to-side aggregation of MTs observed by EM in NIH 3T3 cells where Cep57 was overexpressed (Figure 5B). The MTs formed in the presence of GST–C-Cep57 were stable, tolerating prolonged storage at room temperature (22 °C). Although formation of the basket-like structure is physiologically irrelevant, localization to and stabilization of MTs by overexpression of Cep57 may reflect an important functional feature of Cep57.
(A) High-level expression of EGFP–Full-Cep57, EGFP–C-Cep57 and EGFP–N-Cep57 and appearance of MTs in NIH 3T3 cells. Confocal images of EGFP–Full-Cep57 (a and d), EGFP–C-Cep57 (g and j) and EGFP–N-Cep57 (m and p) vigorously expressed in NIH 3T3 cells with corresponding β-tubulin immunostains (b, e, h, k, n and q) and merged images (c, f, i, l, o and r). Cells treated with nocodazole (d–f, j–l and p–r). The cells expressing either EGFP–Full-Cep57 or EGFP–C-Cep57 showing the basket-like phenotype (a, d, g and j) and its co-localization with MTs (c, f, i and l), which is absent in cells expressing EGFP–N-Cep57 (m, o, p and r). Stabilized nocodazole-resistant MTs (e and k) as a result of overexpression of either EGFP–Full-Cep57 or EGFP–C-Cep57. Increased signal of EGFP is observed at the centrosomes along with cytosolic diffusion when EGFP–N-Cep57 was overexpressed at high levels (m and o, arrows). (B) Electron micrographs from ultra-thin sections (80 nm) of NIH 3T3 cells overexpressing EGFP–Full-Cep57, showing the convoluted cable-like MT structure: (a) low-magnification image; (b) high-magnification image of the boxed area in (a). (C) A confocal image of the basket-like structure in a cultured cell overexpressing EGFP–Full-Cep57 (a) and a nocodazole-pretreated cell overexpressing EGFP–Full-Cep57 (b).
Overexpression of the C-terminus of Cep57 reorganizes MTs into nocodazole-resistant basket-like MT structures
To further explore the function of C-Cep57 in vivo, EGFP–C-Cep57 was expressed ectopically in NIH 3T3 cells. As a result, the same phenotypic basket-like structure as in the cells overexpressing EGFP–Full-Cep57 at high levels was observed (Figure 5A, panel g). Therefore we concluded that the C-terminal half is involved in association with MTs in vivo. The MT-association domain of Cep57 was further narrowed by fusing different tags; that is, Cep57278–491 localized to MTs when it was fused to JRed (Figure 1).
We found that, after the NIH 3T3 cells overexpressed EGFP–Full-Cep57 for an extended period of time, the cells eventually died, but the basket-like structures remained intact. Furthermore, the basket-like structure consisting of EGFP–Full-Cep57 or EGFP–C-Cep57 and MTs remained intact after the exposure to 5 μM nocodazole for 30 min, whereas the MTs not co-localized with EGFP–Full-Cep57 or EGFP–C-Cep57 were disrupted by this MT-depolymerizing drug (Figure 5A). EGFP alone does not lead to MT stabilization because MTs were completely disrupted when cells overexpressing EGFP alone were subjected to nocodazole (results not shown). Therefore we concluded that exogenous Cep57 overexpressed in NIH 3T3 cells associates with MTs through its C-terminal half and promotes a nocodazole-resistant MT network. Inhibition of MT formation in culture medium supplemented with nocodazole prior to plasmid transfection for ectopic expression of EGFP–Full-Cep57 resulted in no basket-like structures, but random aggregation in the cytoplasm (Figure 5C). The different outcomes with pre- and post-treatment of nocodazole in the presence of the basket-like structure imply that overexpression of Cep57 hinders MT dynamics through inhibition of depolymerization.
DISCUSSION
The present study focuses on the domains of a recently described centrosomal protein, Cep57, and provides insights into their distinct roles and functions. The N-terminal coiled-coil domain localizes to the centrosome, internal to γ-tubulin, demonstrating that it is associated with both the centrioles or the centromatrix component. Cep57 also directly interacts with MTs through its C-terminal half, which, based on the lack of similarity to known MT-binding domains, represents a novel MT-binding domain. The ability of this domain to both nucleate and bundle MTs in vitro and in vivo suggests a role for it in organizing the centrosomal MTs. Thus the two domains of Cep57 have different functional roles, one domain to target the protein to the centrosome and the other domain perhaps for anchoring and organizing MTs at the centrosomes of mammalian cells.
Through the generation of highly specific antibodies to Cep57 as well as expression of hrGFP–Full-Cep57, we have confirmed that Cep57 is indeed a centrosomal protein. Its localization to the core of the centrosome and its ability to induce side-to-side aggregation of MTs suggest its importance in the structural organization of centrosomal MTs. We did not see kinetochore staining of Cep57 in either Xenopus, human or mouse cells, although it is clearly visible on purified chromosomes from Xenopus cells [13]. New sequence data have been deposited in the Xenopus databases that identified a protein more closely related to Cep57. We propose to call the protein characterized in Xenopus the Cep57R (Cep57-related protein). A Cep57R family member was also found in mouse and human cells. This recent finding suggests a more complex picture of Cep57 or the Cep57 family members and probably accounts for the differences in localization and phenotypes observed in mammalian and Xenopus cells. For example, we are currently testing whether Xenopus Cep57R reported by Emanuele and Stukenberg [13] is another member of the Cep57 protein family that functions at both the kinetochore and the centrosome.
Homology searches were unable to detect similarity to known MT-binding domains; thus the C-terminal half of Cep57 represents a novel MT-binding domain. The CLD of Cep57 may also contain a novel centrosomal-targeting motif. The CLD has no recognizable similarity to previously known centrosomal targeting signals, i.e. the PACT [pericentrin/AKAP450 (A-kinase-anchoring protein of 450 kDa) centrosomal targeting] domain and cyclin E centrosomal localization signal motif [16,17]. On the other hand, a homology search using CLD implied its homology with a segment of either CNN (centrosomin; Drosophila) or SPD-5 (spindle-defective protein-5; Caenorhabditis elegans): both centrosomal proteins. Conserved amino acid sequences among Cep57, CNN and SPD-5 are segmental and, therefore, it is unlikely that these proteins are homologues as a whole protein. Nonetheless, the fact that the CLD amino acid sequence is conserved among different species implies a common centrosome localization motif and, more importantly, functional importance. CNN and SPD-5 both are proposed to recruit γ-tubulin to the centrosomes and to an alternative ‘centrosome-like’ structure and thus participate in nucleation of tubulin polymerization [18,19].
Ectopic expression of the CLD resulted in reduction in the mitotic index and G1 arrest. In addition, we found that the CLD of Cep57 also plays a role in multimerization of Cep57 and, therefore, the reduction in the mitotic index and the G1 arrest could reflect disrupted multimerization of endogenous Cep57 and perturbed proper structural organization of the centrosomes. Reduction in the mitotic index and G1 arrest induced by ectopic expression of the CLD of Cep57 is reminiscent of the cell-cycle hindrance commonly observed with a molecular disturbance of other centrosomal proteins, such as centriolin and AKAP450 [20,21], and in physical ablation of the centrosomes [22,23]. Therefore reduction in the mitotic index and G1 arrest due to molecular disturbance of Cep57 strongly implicates participation of Cep57 in the complex of centrosomal proteins needed for the normal function of the centrosomes in the cell cycle.
Transfection of RNAi to reduce Cep57 resulted in reduced pole-to-pole distance of the mitotic spindles in cells in which no Cep57 fluorescence was detectable by immunolabelling. This suggests that Cep57 plays a role in mitotic spindle formation in tissue culture cells as has been implicated in Xenopus early embryonic cycles [13]. These phenotypes are less dramatic phenotypes than seen after depletion or addition of antibodies in Xenopus extracts. This may be a result of poor knockdown or the presence of redundant activities in somatic cells that are not present in embryos.
Little is known about how spindle control size is determined, so it is interesting that spindles assembled in the absence of Cep57 are smaller. Perhaps centrosomes depolymerize MTs at a higher rate in the absence of Cep57. Alternatively, there may be misregulation of astral MTs that anchor the centrosomes and MTOC (MT-organizing centre) to the distal ends of the cells and thereby longitudinally ‘stretch’ the mitotic spindles in mitosis. Our study clearly shows stringent centrosomal localization of Cep57 and its ability to bind and stabilize MTs. We also obtained data suggesting the involvement of Cep57 in anchoring to the centrosomes [13]. Taken together, we prefer a model that the decrease in length of mitotic spindles in Cep57-knockdown cells may reflect astral MTs having a reduced anchoring ability resulting in loss of ‘stretch’ in the mitotic spindles.
Thus our analyses have uncovered a number of important activities in Cep57. For example, our observation provides insights into the mechanism of its possible function as an MT anchor at the centrosome. This role as an MT anchor also agrees with the observation in Xenopus egg extracts where MTs dislodged from the centrosome after depletion or addition of loss-of-function antibodies [13]. Cep57 directly interacts with MTs. The ability to induce side-to-side aggregation of MTs and the localization of Cep57 to the core of the centrosome imply a role in organizing centrosomal MTs. Finally, structurally Cep57 has separate domains to target to centrosomes and bundle MTs, suggesting a mechanism for anchoring MTs. While the N-terminal domain resides in the core of the centrosomes on the centrioles or centriomatrix, the C-terminal domain directly anchors MTs to the centrosomes. Both domains on Cep57 contain numerous Aurora kinase consensus sites so it will be interesting to determine how these activities are regulated. In view of the importance of Taxol in the treatment of cancer, an understanding at the structural level of Cep57 and its targets could provide insights for future drug design.
Abbreviations: AKAP450, A-kinase-anchoring protein of 450 kDa; Cep57, centrosomal protein 57; Cep57R, Cep57-related protein; CLD, centrosome localization domain; CNN, centrosomin; Cy3, indocarbocyanine; DMEM, Dulbecco's modified Eagle's medium; DsRed, Discosoma corallimorpharian red fluorescent protein; EGF, epidermal growth factor; EM, electron microscopy; FBS, fetal bovine serum; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione transferase; HEK-293 cells, human embryonic kidney-293 cells; hUbC, human ubiquitin C; JRed, a red fluorescent protein from a jellyfish of the suborder Anthomedusae; MT, microtubule; RNAi, RNA interference; SPD-5, spindle-defective protein-5
- © The Authors Journal compilation © 2008 Biochemical Society
References
June 2008
Volume: 412 Issue: 2
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