Article |
Address correspondence to Geert Carmeliet, Legendo, Onderwijs & Navorsing, Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-345974. Fax: 32-16-345934. email: geert.carmeliet{at}med.kuleuven.ac.be
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Abstract |
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Key Words: mitotic spindle apparatus; microtubules; nucleus; mitosis; nucleolus
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Introduction |
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NuMA and TPX2 have been identified only in vertebrates and both show a cell cycledependent localization, being nuclear during interphase and spindle-associated during mitosis. Ran and the importins, the nuclear transport receptors, regulate NuMA and TPX2 activity, sequestering them from cytoplasmic tubulin until mitotic breakdown of the nuclear envelope (for review see Dasso, 2001; Kahana and Cleveland, 2001). During mitosis, Ran's function in spindle formation has been proposed to rely on elevated concentrations of its GTP-bound form (Ran-GTP) in the vicinity of chromosomes, thereby activating mediators of spindle assembly like NuMA and TPX2 through disassembly of inhibitory importin complexes. This would result in a local environment favorable for microtubule nucleation and stabilization around chromosomes (Gruss et al., 2001; Nachury et al., 2001; Wiese et al., 2001).
The proper assembly and function of the spindle is essential for genomic stability, and therefore, knowledge of key factors involved in this process and their mechanisms of action is crucial. In this paper, we have identified a novel protein, nucleolar spindleassociated protein (NuSAP), which is well conserved in vertebrates, and shows a cell cycledependent localization and microtubule-binding properties similar to NuMA and TPX2. Interestingly, NuSAP specifically associates with spindle microtubules in close contact with chromosomes in metaphase/anaphase. Depletion of NuSAP from cells by RNA interference results in evident mitotic defects that interfere with normal cell cycle progression.
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Results |
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To characterize NuSAP further, we generated pAbs against a peptide (anti-NuSAPp) and recombinant protein (anti-NuSAPr). These antibodies specifically recognized the endogenous protein in MC3T3E1 cells and other cell lines of mouse, hamster, monkey, and human origin, as well as endogenous and epitope-tagged NuSAP expressed in COS1 cells (Fig. 1, D and E).
NuSAP expression is up-regulated during the G2/M phase of the cell cycle
To confirm the initial observation that NuSAP expression is proliferation related, MC3T3E1 cells were specifically arrested in their growth by serum withdrawal and analyzed for NuSAP expression by Northern blot analysis. An 2.4-kb band was identified as the major transcript, and as expected, NuSAP RNA levels were reduced more than 10-fold. When quiescent cells were resupplemented with serum, NuSAP RNA levels increased again, displaying an expression pattern similar to the growth marker histone H4 (Stein et al., 1990b; Fig. 2 A). At the protein level, NuSAP expression was reduced more than threefold, 48 h after serum withdrawal, and increased again in cells that subsequently resumed growth (Fig. 2 B).
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Cell cycledependent localization of NuSAP to the nucleus and mitotic spindle
The subcellular distribution of NuSAP was followed throughout the cell cycle by immunostaining and in vivo imaging. Immunofluorescence microscopy of MC3T3E1 cells revealed that during interphase, endogenous NuSAP was confined to the nucleus and concentrated in nucleoli (Fig. 3 A, S/G2; Fig. 3 D). Cell fractionation experiments confirmed this nuclear localization (Fig. 3 E). At prometaphase, NuSAP redistributed from nucleoli to the vicinity of the chromosomes, forming bundles that became progressively more defined at metaphase and early anaphase (Fig. 3 A). These NuSAP bundles colocalized with microtubules of the central spindle (Fig. 3 C). Later in anaphase, NuSAP was intensely localized to the spindle in the chromosomal area. At telophase, NuSAP localized to "spikes" around the decondensing chromosomes, which disappeared toward the end of cytokinesis (Fig. 3 A). Consistent with the reduced protein levels in G1 cells seen by Western blot (Fig. 2 D), very little NuSAP could be detected in G1 cells (Fig. 3 A), indicating a rapid degradation of NuSAP after mitosis. Interestingly, the localization of NuSAP to chromosomes in mitotic cells was not sensitive to nocodazole treatment, indicating that part of its mitotic localization may be independent of microtubules (unpublished data).
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NuSAP is a microtubule-associated protein (MAP)
NuSAP's specific localization to central spindle microtubules in mitotic cells led us to study whether it interacts with pure prepolymerized microtubules in a sedimentation assay. First, we used in vitro translated NuSAP in the assay, and as shown in Fig. 4 A, NuSAP was recovered in the microtubule pellet when microtubules were present, but remained soluble in the absence of microtubules (Fig. 4 A). This interaction was specific, as a well-characterized MAP (MAP2) also bound to microtubules, whereas BSA did not (unpublished data). To estimate the affinity of NuSAP for microtubules, NuSAP was mixed with different concentrations of microtubules in the same assay. Plotting of bound NuSAP versus the microtubule concentration and direct hyperbolic curve-fitting yielded an equilibrium dissociation constant of 1 µM (the tubulin concentration required to bind 50% of NuSAP; Fig. 4 B), indicating microtubule binding with moderately high affinity. To determine whether NuSAP interacts directly with microtubules, the sedimentation assay was repeated in a more defined system using purified recombinant NuSAP protein and pure prepolymerized microtubules. Recombinant NuSAP co-pelleted to large parts with microtubules, and only little remained in the microtubule-unbound supernatant fraction (Fig. 4 C). These results indicate that NuSAP not only colocalizes, but also directly binds to microtubules, fulfilling the criteria of a bona fide MAP.
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The region within NuSAP that mediates binding to microtubules was determined using the permeabilization/fixation assay described above. A series of GFP-tagged NuSAP fragments were expressed in COS1 cells (Fig. 4, E and F) and assayed for binding to cytoplasmic microtubules after permeabilization. First, however, the intracellular distribution of the various NuSAP fragments was analyzed in nonpermeabilizing conditions. As shown in Fig. 4 F, all NuSAP fragments localized to the nucleus in interphase, and some showed a prominent nucleolar staining as observed for full-length NuSAP (for a detailed summary, see Table I). In permeabilized cells, only NuSAP fragments 243427 and 129367 showed localization to cytoplasmic microtubules similar to full length NuSAP. Thus, the minimal microtubule-binding domain is contained toward the COOH terminus of NuSAP, between residues 243367 (Fig. 4 E).
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Suppression of NuSAP by small interfering RNA (siRNA) causes delayed entry into mitosis
To study the effect of loss of NuSAP function, NuSAP protein levels were suppressed in HeLa cells using siRNA duplexes (Elbashir et al., 2001). Immunoblot and immunofluorescence analysis showed that at 24 h after transfection, the level of endogenous NuSAP could be reduced by more than ninefold in cells transfected with duplexes directed to NuSAP compared with control cells, which were transfected with the nonspecific duplex (transfection efficiencies were typically 80%; Fig. 6 A; unpublished data).
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Interestingly, suppression of NuSAP also altered nuclear morphology of nondividing HeLa cells (Fig. 6 C) already after 24 h transfection. Staining for nucleic acids (Fig. 6 C) and B-type lamins (unpublished data) revealed that nuclei of depleted cells were frequently folded or invaginated (28% compared with 9% in controls; n = 200, three independent experiments; Fig. 6 E).
Suppression of NuSAP results in defective anaphase and cytokinesis
Some of the NuSAP-depleted mitotic cells with highly disorganized spindles appeared to be able to proceed to chromosome segregation. A more than threefold increase in abnormal anaphase cells was observed (n = 100, three independent experiments; Fig. 7, A and B). Again, these cells showed a less dense and disorganized microtubule array, often with aberrant segregation of less condensed chromosomes. At later stages, cells often displayed defective cytokinesis with both chromosome sets in one daughter cell (Fig. 7 A). Consistent with this, we observed a threefold higher number of binucleated cells 40 h after transfection relative to control cells (n = 300, three independent experiments; Fig. 7 C). These cells frequently contained four centrosomes, each containing a pair of centrioles as determined by centrin staining (Fig. 7 D). 50 h after transfection, a time sufficient for cells to complete two cell cycles with reduced levels of NuSAP, we observed a more than threefold increase in the number of multipolar spindles compared with control cells (n = 100, three independent experiments; Fig. 7 E). This agrees well with the increase in binucleated cells at 40 h, and was confirmed by DNA content analysis, which showed a more than threefold increase in cells with a larger than tetraploid DNA content (particularly 6N and 8N DNA content) at both 48 and 72 h after transfection (Fig. 6 B; unpublished data). 72 h after transfection, we could also detect an increase of hypodiploid cells, indicating that NuSAP-depleted cells eventually became apoptotic (unpublished data).
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Discussion |
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Sequestration of NuSAP to the nucleus during interphase prevents any interactions with microtubules until after breakdown of the nuclear membrane, at the beginning of mitosis. The localization and activity of NuSAP could be regulated in a similar manner as NuMA and TPX2, where nuclear localization is governed by asymmetric distribution of the GTPase Ran, and binding to the nuclear transport receptors, importin- and -ß (for review see Dasso, 2001; Kahana and Cleveland, 2001). NuSAP could interact with nuclear transport receptors via the two different domains that were sufficient for nuclear targeting. The first is the potential bipartite NLS, which could interact with importin-
and -ß. Interestingly, the novel ChHD also conferred nuclear localization. It has recently been suggested that importins might act as chaperones for exposed basic domains (Jakel et al., 2002), and ChHD is extremely basic with an isoelectric point >10. Thus, importins might bind to this region, stabilizing an inactive state by shielding of basic patches, and simultaneously might import NuSAP into the nucleus.
NuSAP is a cycling protein essential for cell cycle progression
Protein levels of NuSAP were cell cycle regulated and restricted to late S/G2 and M phases with undetectable expression in G1, similar to the expression pattern of human TPX2 (Heidebrecht et al., 1997; Gruss et al., 2002). Although the increased expression in late S phase correlated well with transcription levels, the rapid disappearance of the protein in G1 suggests rapid degradation after cytokinesis. This is consistent with the potential KEN boxes and PEST sequence in NuSAP, which could target NuSAP to the ubiquitin/proteasome pathway (Rechsteiner and Rogers, 1996; Pfleger and Kirschner, 2000). This destruction may be important for proper exit from mitosis, to prevent any excess NuSAP from associating and bundling cytoplasmic microtubules in interphase. Consistent with this, increased levels of NuSAP inhibited cell proliferation apparently by irreversibly bundling interphase microtubules (unpublished data), indicating that endogenous NuSAP levels have to be tightly controlled.
It is interesting to note that proteins like NuSAP or TPX2 (Heidebrecht et al., 1997) that show little or no expression in G1 and G0 may be reliable histochemical markers for proliferation and could therefore be useful for cancer prognosis. Consistent with this, we identified NuSAP based on its abundance in proliferating cells, and a significant number of human NuSAP ESTs are derived from cancer tissues. Furthermore, the defects in spindle organization seen in NuSAP-depleted cells could contribute to genomic instability, which is also observed in aggressive tumor cells (Brinkley, 2001).
A role for NuSAP in spindle formation
We used the method of RNA interference knockdown to study the function of NuSAP in cells. NuSAP levels could be efficiently depleted within 24 h, suggesting that cells only needed to exit one mitosis after transfection of the siRNA to severely affect protein levels. The predominant early consequence of NuSAP depletion was a 50% increase of cells arrested in G2-M (Fig. 6). Several aspects of mitotic spindle formation and function were impaired in NuSAP-depleted cells. For example, in >50% of prometaphase cells, chromosome condensation and congression appeared to be delayed or incomplete. Furthermore, the metaphase arrangement of chromosomes often appeared less compact than in normal cells, displaying unaligned chromosomes that indicated a deficiency in chromosome congression or maintenance of a stable metaphase configuration. The bipolar spindles produced in NuSAP-depleted cells showed a reduction in the content of microtubules in the spindle midzone, suggesting that NuSAP might be involved in the stabilization or production of spindle microtubules associated with chromosomes at the kinetochore and/or at chromosome arms. This is entirely consistent with the localization of NuSAP to chromatin and the spindle midzone.
Despite malformed spindles and misaligned chromosomes, a significant fraction of NuSAP-depleted cells eventually progressed to anaphase and cytokinesis. At these later mitotic stages, the cells also showed striking defects. The central spindles between the separating chromosomes in NuSAP-depleted cells often contained significantly fewer microtubules than in normal anaphase cells. Possibly as a consequence of this weak central spindle, the chromosomes were typically segregated only incompletely and by a much shorter distance than in control cells. Also at cytokinesis, NuSAP-depleted cells frequently failed to cleave between the partially segregated chromosomes, resulting in binucleated cells that also contained additional centrosomes. These binucleate cells were apparently able to enter at least one subsequent cell cycle, as even long after NuSAP suppression, >30% of the cells exhibited multipolar spindles. These spindle poles contained essential structural components like -tubulin and NuMA, as well as the centriolar marker centrin. Interestingly, a similar multipolar spindle phenotype has recently been described after suppression of TPX2 (Garrett et al., 2002). However, unlike for TPX2, where multiple poles were suggested to arise from fragmentation of centrosomal material, it is likely that the appearance of multipolar spindles is an indirect consequence of NuSAP suppression. After depleting NuSAP for one cell cycle, only aberrant spindle organization (but no increase in multipolar spindles) was observed. Multipolar spindles arose only much later, and are therefore most likely caused by the observed defects in the preceding round of chromosome segregation and cytokinesis.
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Materials and methods |
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Cloning of mouse NuSAP cDNA
Total RNA extracted from MC3T3E1 cultures at the stage of proliferation or differentiation (mineralizing) was used in a differential display analysis, which was performed essentially as described previously (Liang et al., 1993). To obtain the full-length cDNA, a 5'-RACE kit (Invitrogen) was used. All PCR products were ligated into the pGEMTEasy cloning vector (Promega) and subsequently sequenced.
Northern blot analysis
Total RNA extracted from cell cultures using TRIzol® LS (Invitrogen) was separated by electrophoresis, transferred to nylon membranes, and hybridized with cDNA probes as follows: pFO002 to detect histone H4 (Pauli et al., 1989), which was provided by Gary Stein (University of Massachusetts Medical School, Worcester, MA), and a 0.8-kb NuSAP BglII/ AspI restriction fragment. To assess equal loading, blots were rehybridized with a radiolabeled 18 S ribosomal probe.
Construction of NuSAP vectors
The NuSAP-GFP vectors were designed to contain the coding sequences of NuSAP fused upstream to the GFP sequence of the pEGFP-N1 plasmid (CLONTECH Laboratories, Inc.). Specific primers incorporating a 5' SacI and a 3' BamHI site were used to obtain the insert. Vectors containing YFP- and CFP-tagged NuSAP were constructed by subcloning the full-length NuSAP cDNA from pNuSAP-EGFP into pEYFP-N1 and pECFP-N1 (CLONTECH Laboratories, Inc.). For construction of the Myc-tagged NuSAP vector, the GFP tag was removed by digesting with BamHI and NotI, and by inserting a Myc adaptor duplex containing these restriction sites. The vector used in the transcription and translation reaction was constructed using XhoI and BamHI restriction fragments derived from the pNuSAP-EGFP vector, which was inserted into the complementary sites of the pcDNA3.1MycHisB plasmid (Invitrogen). The GST-NuSAP-C fusion vector used for protein expression in Escherichia coli for antibody generation was designed to contain the COOH-terminal coding sequence (530 bases) of mouse NuSAP fused downstream to the GST sequence of the pGEX5X1 plasmid (Amersham Biosciences). Specific primers incorporating a 5' BamHI and a 3' XhoI site were used. The zzNuSAP vector used for recombinant protein expression in the microtubule-binding assay was constructed by amplifying the coding region of NuSAP from pNuSAP-EGFP and cloning into the SphI-BamHI sites of zzpQE80, a derivative of pQE (QIAGEN) with two consecutive z-tags (IgG-binding domain from protein A) at the NH2 terminus and a COOH-terminal His tag. All constructs were sequenced to verify junctions and to ensure the proper NuSAP sequence.
Recombinant protein expression and purification
zzNuSAP was expressed from zzpQE80 in E. coli BL21 Rosetta (Novagen) and purified on Nickel-NTA agarose (QIAGEN). GST and GST-NuSAP-C were expressed from pGEX5X vectors in E. coli BL21-Codon Plus (DE3)-RIL (Stratagene).
In vitro transcription and translation
For in vitro NuSAP protein production, plasmid DNA was transcribed and translated using the TNT T7 reticulocyte lysate system (Promega). In the radioactive assays translation grade L-[35S]cysteine (1,000 Ci/ mmol; Amersham Biosciences) was used, and translation products were separated by SDS-PAGE and further processed for fluorography. Luciferase DNA was used as a positive control. Treatment with calf intestine alkaline phosphatase was for 30 min at 37°C. In the nonradioactive assays, protein samples were further processed for Western blot analysis.
Microtubule-binding assay
A nonradioactive crude transcription and translation reaction product was used for the sedimentation assay in the presence and absence of pure, prepolymerized microtubules. The assay was performed using the Microtubule-Associated Protein Spin-Down Assay Biochem kit (Cytoskeleton, Inc.) according to the manufacturer's instructions. Controls that were run included detecting BSA (negative control) and MAP-2 (positive control).
For the direct microtubule-binding assay, 10 µM porcine tubulin, purified according to Mitchison and Kirschner (1984), was incubated with 1 µM purified zzNuSAP in BRB-80 buffer (80 mM Pipes, 1 mM K-EGTA, and 1 mM MgCl2, pH 6.8) containing 2 mM GTP and 20 µM taxol for 20 min at 30°C. The reaction was then centrifuged through a sucrose-BRB-80 cushion at 100,000 g for 10 min. Pellet and supernatant were applied to SDS-PAGE, tubulin was detected by Coomassie blue staining, and zzNuSAP was detected by immunoblotting.
Primary antibodies
Rabbit polyclonal anti-NuSAPr antibodies were generated using purified, bacterially expressed GST fusion (GST-NuSAP-C) as antigen. The antibodies were affinity purified by sequential passage through GST and GST-NuSAP-C fusion protein columns, according to the manufacturer's instructions (GST orientation kit; Pierce Chemical Co.). The polyclonal anti-peptide rabbit antibody (anti-NuSAPp) was generated using a peptide (QENQENQDPRDTAEV) coupled to KLH as antigen in an immunization program performed at Eurogentec. The anti-NuSAPp serum was affinity purified using antigen peptide coupled to CNBr-activated Sepharose 4B (Amersham Biosciences).
Antibodies were used to detect epitope-tagged NuSAP as follows: anti-c-Myc rabbit antibody (A-14; Santa Cruz Biotechnology, Inc.) and anti-c-Myc mouse mAb (clone 9E10; Sigma-Aldrich). Mouse mAbs were used as follows: anti--tubulin (clone DM1A; Sigma-Aldrich), anti-
-tubulin (clone GTU-88; Sigma-Aldrich), anti-ß-actin (clone AC-15; Sigma-Aldrich), anti-BrdU (clone B44; Becton Dickinson), and anti-BSA (clone BSA-33; Sigma-Aldrich). In addition, goat pAbs were used as follows: anti-lamin B (M-20; Santa Cruz Biotechnology, Inc.) and anti-MAP-2 (D-19; Santa Cruz Biotechnology, Inc.). Anti-
-tubulin rabbit (AK-15; Sigma-Aldrich) antibody was also used. The mouse mAb to nucleolin was provided by Benigno Valdez (Baylor College of Medicine, Houston, TX). The anti-NuMA rabbit antibody was provided by Duane Compton (Dartmouth Medical School, Hanover, NH). The anti-centrin mAb (20H5) was provided by Jeffrey Salisbury (Mayo Clinic, Rochester, MN).
Confocal microscopy, immunofluorescence, and image analysis
For immunostaining of endogenous NuSAP and nucleolin, MC3T3E1 cells were fixed in 1% PFA for 10 min. Images of endogenous NuSAP during mitosis were primarily made using anti-NuSAPp antibody in cells that were fixed in 0.1% glutaraldehyde (containing 0.5% Triton X-100) for 15 min, followed by a 10-min incubation in 0.5 mg/ml NaBH4 to reduce free aldehyde groups. Anti-NuSAPr antibody stains weaker in this fixative condition. This fixative was also used to stain Myc-tagged NuSAP in transfected (FuGENETM; Roche) COS1 cells, using anti-c-Myc mouse antibodies (clone 9E10), and to detect incorporated BrdU, which also required the subsequent incubation of cells in 0.07 N NaOH for 2.5 min to denature genomic DNA. Methanol fixation was also used to detect lamin B, centrin, - and
-tubulin, NuSAP, and NuMA in MC3T3E1 and HeLa cells.
NuSAP, NuMA, and -tubulin were also detected in permeabilized MC3T3E1 cells. In the permeabilization assay, cells were first briefly (35 s) incubated in warm (37°C) microtubule-stabilizing PEM buffer (100 mM Pipes, pH 6.9, 5 mM EGTA, and 1 mM MgCl2), then extracted for 30 s in detergent (0.1% Triton X-100)-containing PEM buffer followed by fixation in 1% PFA for 15 min. For extensive permeabilization of transfected COS1 cells, extraction was prolonged to 3 min, and cells were fixed in 0.5% glutaraldehyde.
Further processing included incubating cells in 5% BSA for 10 min before incubations with primary and secondary antibodies for 1 h at RT (diluted in 0.5% Tween 20). Secondary antibodies were conjugated to Alexa®-488 or -546 dye (Molecular Probes, Inc.). Cells were mounted in DakoCytomation mounting medium, containing TO-PRO-3 (Molecular Probes, Inc.) when nucleic acids were stained. Images of fixed cells were acquired on an inverted microscope (Diaphot 300; Nikon) (Plan Apo 60x/ 1.40 oil) connected to a confocal microscope (model MRC1024; Bio-Rad Laboratories) using LaserSharp software (version 3.2).
The four-dimensional confocal imaging system used has been described elsewhere (Gerlich et al., 2001; Beaudouin et al., 2002). In brief, imaging was performed on a customized microscope (model LSM510; Carl Zeiss MicroImaging, Inc.) equipped with a z-scanning stage, selected PMTs, a 413 nm Kr and 488 nm Ar laser, custom dichroics, and emission filters. Images were acquired using a 63x PlanApochromat NA 1.4 DIC oil immersion objective. Only images from cells that completed mitosis normally were used for subsequent analysis. Images were processed in Adobe Photoshop® version 6.0.1 (Adobe Systems).
RNA interference
For siRNA, the following target sequences in the NuSAP cDNA were used: 5'-AACTGAGATACACGTTAGCAG-3' and 5'-AAGATCTCTATGCACGGATGA-3' in the mouse, and 5'-AAGCACCAAGAAGCTGAGAAT-3' in humans. As a nonspecific control, the GL2 luciferase siRNA duplex was used (Elbashir et al., 2001). Oligonucleotides (Dharmacon Research, Inc.) were annealed and transfected using OligofectamineTM (Invitrogen) as described previously (Elbashir et al., 2001). Thymidine-synchronized HeLa cells were transfected with the siRNA duplexes 2 h after release. These synchronized cells were analyzed at different time points after transfection (10, 30, and 50 h) corresponding with successive M phases, but complete suppression of NuSAP was only manifest from the second mitosis (30 h after transfection).
Extract preparation and Western blot analysis
Nuclear extracts of MC3T3E1 cells were prepared essentially as described by Feng et al. (2000). Whole-cell extracts were prepared from the different cell lines, by washing and scraping cells in PBS containing 5 mM EDTA and 5 mM EGTA, and subsequently lysing cell pellets in ice-cold TCL buffer (50 mM TrisCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate) containing protease inhibitors. Lysed cells were subsequently sonicated for 5 s and centrifuged at 14,000 g (20 min at 4°C). Proteins were boiled in SDS sample buffer, separated by SDS-PAGE using precast 412% Bis-Tris gels (Invitrogen), and transferred to nitrocellulose membranes (Amersham Biosciences). Membranes were probed and subsequently developed by ECL (Western Lightning; Perkin Elmer).
FACS® analysis
Subconfluent MC3T3E1 and HeLa cells were stained with propidium iodide and subsequently analyzed for DNA content on a FACScanTM (Becton Dickinson) flow cytometer using CellQuestTM software (BD Biosciences).
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Acknowledgments |
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This work was supported by the Fonds voor Wetenschappelijk Onderzoek (FWO; G.0233.97), and European Commission Transnational Access to Research Infrastructures Programme (European Advanced Light Microscopy Facility at the EMBL in Heidelberg) grants, and a long-term EMBO fellowship (to K. Ribbeck). J. Beaudouin was supported by a fellowship through EMBL's International Ph.D. Program; J. Ellenberg acknowledges funding from the Human Frontiers Science Program (RGP0031/2001-M).
Submitted: 21 February 2003
Accepted: 29 July 2003
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