Localization of Human Mcm10 Is Spatially and Temporally Regulated during the S Phase
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m314017200
ISSN1083-351X
AutoresMasako Izumi, Fumio Yatagai, Fumio Hanaoka,
Tópico(s)Microtubule and mitosis dynamics
ResumoMcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although 2the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30–60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30–60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex. Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although 2the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30–60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30–60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex. To maintain the integrity of the genome, eukaryotic DNA replication is tightly regulated to ensure that the initiation of replication occurs only once per cell cycle. Recent analyses using different systems show that eukaryotes share a common mechanism that coordinates the initiation of DNA replication from a large number of origins (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Google Scholar). The initiation of DNA replication consists of two steps, namely the formation of a prereplication complex (pre-RC) 1The abbreviations used are: pre-RC, prereplication complex; Orc, origin recognition complex; PCNA, proliferating cell nuclear antigen; GFP, green fluorescent protein; BrdU, bromodeoxyuridine; CSK, cytoskeleton; HA, hemagglutinin; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4′,6-diamidino-2′-phenylindole-dihydrochrolide. 1The abbreviations used are: pre-RC, prereplication complex; Orc, origin recognition complex; PCNA, proliferating cell nuclear antigen; GFP, green fluorescent protein; BrdU, bromodeoxyuridine; CSK, cytoskeleton; HA, hemagglutinin; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4′,6-diamidino-2′-phenylindole-dihydrochrolide. and the activation of pre-RC. The pre-RC is formed by the sequential assembly of the heterohexameric origin recognition complex (Orc), Cdc6, Cdt1, and the Mcm2-7 complex on chromatin in telophase (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Google Scholar, 2Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Google Scholar, 3Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Google Scholar). This process is also referred to as replication licensing. At the G1/S transition, the pre-RC is activated by S phase cyclin-dependent kinases and the Cdc7/Dbf4 kinase, which mediate the association of Cdc45 to a preformed pre-RC at each origin with programmed timing (4Aparicio O.M. Stout A.M. Bell S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9130-9135Google Scholar, 5Zou L. Stillman B. Mol. Cell. Biol. 2000; 20: 3086-3096Google Scholar, 6Walter J.C. J. Biol. Chem. 2000; 275: 39773-39778Google Scholar). Subsequent to Cdc45 loading, the DNA is unwound at the replication origin (7Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Google Scholar) and the single-stranded DNA-binding protein (RPA) and DNA polymerases are recruited (7Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Google Scholar, 8Mimura S. Takisawa H. EMBO J. 1998; 17: 5699-5707Google Scholar). Eukaryotes have several means to prevent the reinitiation from origins in the S, G2, and M phases. For example, cyclin-dependent kinase phosphorylates Cdc6 and causes either its degradation (yeast) or export from the nucleus (mammalian cells), which ensures that the pre-RC is not reassembled until the segregation of chromosomes in mitosis (9Drury L.S. Perkins G. Diffley J.F.X. EMBO J. 1997; 16: 5966-5976Google Scholar, 10Saha P. Chen J. Thome K.C. Lawlis S.J. How Z. Hendricks M. Parvin J.D. Dutta A. Mol. Cell. Biol. 1998; 18: 2758-2767Google Scholar). Moreover, Cdt1 is regulated by its expression levels and by its interaction with geminin, which guarantee that it is only functional in the G1 phase (11Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Google Scholar, 12Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Google Scholar). Mcm10 was first identified in Saccharomyces cerevisiae in screens for genes that are required for chromosomal DNA replication (13Solomon N.A. Wright M.B. Chang S. Buckley A.M. Dumas L.B. Gaber R.F. Yeast. 1992; 8: 273-289Google Scholar, 14Merchant A.M. Kawasaki Y. Chen Y. Lei M. Tye B.K. Mol. Cell. Biol. 1997; 17: 3261-3271Google Scholar). Studies in S. cerevisiae suggest that Mcm10 plays multiple roles in DNA replication. mcm10 mutants suffer a defect in the initiation of DNA replication (14Merchant A.M. Kawasaki Y. Chen Y. Lei M. Tye B.K. Mol. Cell. Biol. 1997; 17: 3261-3271Google Scholar). In the mcm10-1 mutant, the replication forks stall when the replication machinery passes through origins that did not fire (14Merchant A.M. Kawasaki Y. Chen Y. Lei M. Tye B.K. Mol. Cell. Biol. 1997; 17: 3261-3271Google Scholar). This suggests that Mcm10 plays a unique role in DNA replication. Budding yeast Mcm10 has also been found to physically interact with the components of Orc and Mcm2-7 complexes (14Merchant A.M. Kawasaki Y. Chen Y. Lei M. Tye B.K. Mol. Cell. Biol. 1997; 17: 3261-3271Google Scholar). Moreover, it genetically interacts with Cdc45 and DNA polymerases δ and ϵ, which are necessary for the elongation steps of DNA replication (15Homesley L. Lei M. Kawasaki Y. Sawyer S. Christensen T. Tye B.K. Genes Dev. 2000; 14: 913-926Google Scholar, 16Kawasaki Y. Hiraga S. Sugino A. Genes Cells. 2000; 5: 975-989Google Scholar). These results suggest that budding yeast Mcm10 is involved in both the activation of the pre-RC and the elongation steps of DNA replication. Mcm10 homologs have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila, and Xenopus (17Aves S.J. Tongue N. Foster A.J. Hart E.A. Curr. Genet. 1998; 34: 164-171Google Scholar, 18Wohlschlegel J.A. Dhar S.K. Prokhorova T.A. Dutta A. Walter J.C. Mol. Cell. 2002; 9: 233-240Google Scholar, 19Christensen T.W. Tye B.K. Mol. Biol. Cell. 2003; 14: 2206-2215Google Scholar). Recently we identified human Mcm10 and found that human Mcm10 interacts with the mammalian Orc2 protein and the Mcm2-7 complex (20Izumi M. Yanagi K. Mizuno T. Yokoi M. Kawasaki Y. Moon K.-Y. Hurwitz J. Yatagai F. Hanaoka F. Nucleic Acids Res. 2000; 28: 4769-4777Google Scholar), similar to the budding yeast Mcm10. However, the budding yeast and vertebrate Mcm10 proteins seem to have different functions. In S. cerevisiae, removing Mcm10 from the chromatin in G1 phase releases Mcm2 from chromatin, which suggests that Mcm10 is required for pre-RC formation (15Homesley L. Lei M. Kawasaki Y. Sawyer S. Christensen T. Tye B.K. Genes Dev. 2000; 14: 913-926Google Scholar). In contrast, the human and Xenopus Mcm10 proteins bind chromatin after pre-RC formation (18Wohlschlegel J.A. Dhar S.K. Prokhorova T.A. Dutta A. Walter J.C. Mol. Cell. 2002; 9: 233-240Google Scholar, 21Izumi M. Yatagai F. Hanaoka F. J. Biol. Chem. 2001; 276: 48526-48531Google Scholar). In addition, the functions of Mcm10 seem to be controlled by different mechanisms between species. Human Mcm10 protein levels fluctuate during the cell cycle and decrease from anaphase to G1 phase (21Izumi M. Yatagai F. Hanaoka F. J. Biol. Chem. 2001; 276: 48526-48531Google Scholar). Human Mcm10 also specifically binds chromatin in S phase and dissociates from chromatin in G2/M phase, which is accompanied by protein phosphorylation (21Izumi M. Yatagai F. Hanaoka F. J. Biol. Chem. 2001; 276: 48526-48531Google Scholar). In contrast, in S. cerevisiae and S. pombe, Mcm10 remains bound to chromatin throughout the entire cell cycle and displays constant expression levels (15Homesley L. Lei M. Kawasaki Y. Sawyer S. Christensen T. Tye B.K. Genes Dev. 2000; 14: 913-926Google Scholar, 16Kawasaki Y. Hiraga S. Sugino A. Genes Cells. 2000; 5: 975-989Google Scholar, 22Gregan J. Lindner K. Brimage L. Franklin R. Namdar M. Hart E.A. Aves S.J. Kearsey S.E. Mol. Biol. Cell. 2003; 14: 3876-3887Google Scholar). Our previous studies suggest that human Mcm10 is involved in S phase progression, although the precise role played by human Mcm10 still remains unknown. To clarify the function of Mcm10 in mammalian cells, we established HeLa cell lines expressing green fluorescent protein (GFP)- and hemagglutinin (HA)-tagged Mcm10 and studied the dynamics of Mcm10 localization during the cell cycle. The subcellular localization of human Mcm10 changed during S phase progression, and it appeared to be recruited to the replication sites prior to DNA synthesis. These results indicate that human Mcm10 may participate in the activation of pre-RC. The implication of these results and the role Mcm10 may play in DNA replication are discussed. Plasmid Construction—The cDNA for human Mcm10 was first subcloned into ptetGFP (23Izumi M. Gilbert D.M. J. Cell. Biochem. 1999; 76: 280-289Google Scholar) or ptetHA11, which contains the GFP tag or HA epitope tag upstream of the multicloning sites, respectively. To construct ptetHA11, two oligonucleotides with the sequences 5′-CCGGT CGCCA CCATG GCTAG CTACC CATAC GACGT CCCAG ACTAC GCTAG CTTGA-3′ and 5′-GATCT CAAGC TAGCG TAGTC TGGGA CGTCG TATGG GTAGC TAGCC ATGGT GGCGA-3′ were annealed and cloned into the AgeI-BglII sites of ptetGFP. Human Mcm10 cDNA was amplified by PCR using the primers 5′-ATTAG TCGAC GATGA GGAGG AAGAC AATCT GTC-3′ and 5′-ATTAC CGCGG TTTTA AGGCT GTTCA GAAAT TTAGC ATG-3′, digested with SacII and SalI, and subcloned into the compatible sites of ptetGFP or ptetHA11 to construct ptetGFP-Mcm10 or ptetHA11-Mcm10, respectively. Sequence analysis confirmed that no mutations had been introduced into the amplified sequence during PCR. To construct pIREShyg/GFP-Mcm10 or pIREShyg/HA-Mcm10, ptetGFP-Mcm10 or ptetHA11-Mcm10 was digested with AgeI and DraI, filled with T4 DNA polymerase, and the resulting fragment containing the entire coding region for GFP-tagged Mcm10 or HA-tagged Mcm10 was cloned into the BamHI site of pIREShyg (Clontech). Cell Culture and Synchronization—HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. For synchronization at the mitotic phase, exponentially growing cells were treated with 50 ng/ml nocodazole (Aldrich) for 4 h and collected by mitotic shake-off. For synchronization in the G1 phase, the mitotic cells were collected as described above, washed twice with nocodazole-free medium, and cultured with fresh medium for 1.5 h. For synchronization at the G1/S boundary, the mitotic cells were collected as described above and replated in the presence of 15 μm aphidicolin (Sigma) for 12–14 h. The cells were released in aphidicolin-free medium and harvested at the indicated time points. Fractionation of cellular proteins and immunoblotting were performed as described (21Izumi M. Yatagai F. Hanaoka F. J. Biol. Chem. 2001; 276: 48526-48531Google Scholar). Transfection—The expression vector pIREShyg/GFP-Mcm10 or pIREShyg/HA-Mcm10 was linearized with SspI and transfected into HeLa cells by using LipofectAMINE reagent (Invitrogen) as described previously (23Izumi M. Gilbert D.M. J. Cell. Biochem. 1999; 76: 280-289Google Scholar). Transfected cells were selected with 400 μg/ml hygromycin B (Invitrogen) for 2 weeks. Individual colonies were isolated and screened for the expression of GFP-Mcm10 or HA-Mcm10 by immunoblotting. Stable transformant cell lines were maintained in the presence of hygromycin B. Flow Cytometry—To prepare cells for analysis by flow cytometry, mitotic cells were collected by mitotic shake-off and released into normal medium. The cells were harvested at the various time points and fixed in ice-cold 70% ethanol. The fixed cells were treated with RNaseA (0.25 mg/ml) at 37 °C for 30 min and then stained with propidium iodide (40 μg/ml). The DNA content of cells was analyzed with a FACSort (Becton Dickinson). Indirect Immunofluorescence Microscopy—Cells grown on coverslips were washed three times with ice-cold PBS, fixed with ice-cold 3.7% formaldehyde in PBS at 4 °C for 20 min, and permeabilized with 0.5% Nonidet P-40 in PBS at 4 °C for 5 min. In experiments where cells were first extracted, the cells were washed three times with ice-cold PBS and then incubated in cytoskeleton buffer (CSK buffer: 10 mm PIPES, pH 6.8, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, I mm dithiothreitol, 0.1 mm ATP, 0.2 mm Na3VO4, 10 mm NaF) containing 0.1% Triton X-100 and 2× Complete™ (Roche Applied Science) for 10 min at 4 °C before fixation. The coverslips were processed for immunofluorescence staining as described previously (24Izumi M. Vaughan O.A. Hutchison C.J. Gilbert D.M. Mol. Biol. Cell. 2000; 11: 4323-4337Google Scholar). Proliferating cell nuclear antigen (PCNA) (sc-56; Santa Cruz Biotechnology) and nucleolin (sc-8031; Santa Cruz Biotechnology) were detected with mouse monoclonal antibodies and a Texas Red-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). In double-staining experiments where proteins were colocalized with sites of bromodeoxyuridine (BrdU)-substituted DNA, the cells were labeled with 30 μg/ml BrdU for 10 min and fixed in ethanol:acetic acid (19:1) at –20 °C for 10 min. The cells were first stained for the HA tag using a rabbit anti-HA tag antibody (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) and an Alexa Fluor-conjugated antibody (Molecular Probes, Inc.) or stained for PCNA using a mouse monoclonal antibody and a fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). After the primary and secondary antibodies were fixed in ethanol:acetic acid (19:1) at –20 °C for 10 min, DNA was depurinated with 2 n HCl, 0.5% Nonidet P-40 at 37 °C for 30 min. BrdU was detected by using a rat anti-BrdU monoclonal antibody (Harlan Sera-Lab Ltd.) and a Texas Red-conjugated donkey anti-Rat antibody (Jackson ImmunoResearch Laboratories). The cells were visualized using an Olympus IX70 fluorescent microscope equipped with the 100 × 1.35NA UplanApo oil-immersion objective. The images were taken by a CCD camera and assembled using IP Lab software. In Vivo Labeling and Immunoprecipitation—To label proteins with [35S]methionine, 1.5 × 106 cells were incubated in Dulbecco's modified Eagle's medium containing 10-fold less methionine and 3% dialyzed calf serum for 1 h and then in the same medium containing 100 μCi/ml [35S]methionine (Amersham Biosciences) for 1 h. For the pulse-chase experiment, the cells were chased with aphidicolin-free Dulbecco's modified Eagle's medium supplemented with normal 10% calf serum and 150 μg/ml methionine. For immunoprecipitation, the cells were lysed in 200 μl of buffer containing 20 mm Hepes, pH 7.5, 1% Nonidet P-40, 0.3 m NaCl, 10 mm NaF, 0.25 mm Na3VO4, and 2× Complete (Roche Applied Science) at 4 °C for 10 min and centrifuged to obtain the lysates. The lysates were incubated with 1 μl of anti-Mcm10 rabbit antiserum at 4 °C for 1 h, after which 10 μl of protein A-Sepharose Fast Flow (Amersham Biosciences) were added and incubated for an additional 1 h. The beads were washed five times with lysis buffer, and the precipitates were dissolved in 40 μl of 2× SDS-PAGE sample buffer. Expression of His6-tagged Mcm10 Protein in Escherichia coli—His6-tagged Mcm10 was constructed by cloning the PCR-amplified human Mcm10 into the SalI-NotI sites of pET24a (Novagen). Expression of His6-tagged Mcm10 was induced in the E. coli strain BL21(DE3) (Stratagene) by the addition of 1 mm isopropyl-1-thio-β-d-galactopyranoside at 30 °C for 3 h. After centrifugation, the cell pellets were suspended in 1/20 culture volume of lysis buffer (20 mm sodium phosphate buffer, pH 7.4, 0.15 m NaCl, 0.5% Triton X-100) and lysed on ice by mild sonication. The insoluble material containing His6-tagged Mcm10 was collected by centrifugation and solubilized in 8 m urea buffer (20 mm sodium phosphate buffer, pH 7.4, 0.15 m NaCl, 0.5% Triton X-100, 8 m urea). His6-tagged Mcm10 was purified over a TALON column (Clontech) according to the manufacturer's instructions. Establishment of HeLa Cell Lines Expressing GFP-tagged Mcm10 —To better understand the dynamics of human Mcm10 localization during the cell cycle, we first tried to detect endogenous Mcm10 protein by indirect immunofluorescence. This was unsuccessful because of the poor reactivity of the anti-Mcm10 antibody in immunohistochemistry. Therefore, we established stable HeLa cell lines that express a GFP-tagged Mcm10 protein. The expression of GFP-Mcm10 was maintained by the bicistronic message encoding the hygromycin B-resistant gene (hyg) because the expression of transfected genes in mammalian cells is usually repressed by epigenetic mechanisms. Forty individual clonal cell lines were expanded in the presence of hygromycin B and screened for GFP-Mcm10 expression by immunoblotting. Fig. 1a shows the immunoblot of the whole cell extracts made from 11 cell lines that were probed with the anti-Mcm10 antibody, which recognizes both the endogenous Mcm10 protein that migrates at 100 kDa and the GFP-tagged Mcm10 protein that migrates at 130 kDa. Each of these cell lines expressed GFP-Mcm10 at different levels relative to endogenous Mcm10 expression. G10-24 and G10-30 cells were initially considered to be the most desirable of the cell lines for further studies because they expressed the exogenous GFP-Mcm10 protein at significantly lower levels compared with endogenous Mcm10 expression, which minimizes the possibility of artifacts caused by overexpression. However, GFP-Mcm10 fluorescence in these cell lines was below the level of detection by fluorescence microscopy. Therefore, for further experiments, we used G10-1 cells, which express GFP-Mcm10 at twice the level of the endogenous Mcm10 protein. In nearly all of the cells in the G10-1 population, GFP-Mcm10 was detectable and expressed at relatively homogenous levels. We also found that the exogenous GFP-Mcm10 in the G10-1 cells did not interfere with normal cell cycle progression (Fig. 1b). GFP-Mcm10 Is Functionally Equivalent to the Endogenous Mcm10 Protein—We first examined whether GFP-Mcm10 showed the same affinity for chromatin as endogenous Mcm10. Thus, G10-1 cells were lysed in a Triton X-100-containing CSK buffer with 0.1 m NaCl or 0.3 m NaCl, and soluble and insoluble proteins were separated by low speed centrifugation and analyzed by immunoblotting. The GFP-Mcm10 protein as well as the endogenous Mcm10 remained in the insoluble chromatin fraction at 0.1 m NaCl but increasing the salt concentration to 0.3 m removed both proteins from the chromatin (Fig. 1c). We have previously reported that human Mcm10 is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner (21Izumi M. Yatagai F. Hanaoka F. J. Biol. Chem. 2001; 276: 48526-48531Google Scholar). To verify that GFP-Mcm10 is regulated in the same manner as endogenous Mcm10, the cells were synchronized in metaphase with nocodazole followed by mitotic shake-off. The cells were either collected as metaphase populations or released into the cell cycle and collected 1.5 h after release (G1 phase), or accumulated at the G1/S boundary with aphidicolin. The cells were then fractionated into Triton X-100-soluble and -insoluble fractions, and the subcellular localization of GFP-Mcm10 was investigated by immunoblotting (Fig. 1d). In cells that had been synchronized to be in the G1 phase or at the G1/S boundary, most of the GFP-Mcm10 proteins were mainly found in the insoluble fraction. GFP-Mcm10 appeared to be more soluble at the G1/S boundary, which may be due to the effect of GFP-tag. At metaphase, however, the GFP-Mcm10 protein dissociated from the chromatin and was found in the soluble fraction. This pattern reflects that of endogenous Mcm10. Moreover, the expression of GFP-Mcm10 decreased in the early G1 phase, as did the expression of endogenous Mcm10. The down-regulation of GFP-Mcm10 could be explained by its proteasome-mediated proteolysis because the presence of proteasome inhibitors stabilized GFP-Mcm10 levels (data not shown). In addition, like endogenous Mcm10, GFP-Mcm10 in metaphase showed slower electrophoretic mobility, which could be due to its phosphorylation (21Izumi M. Yatagai F. Hanaoka F. J. Biol. Chem. 2001; 276: 48526-48531Google Scholar). Thus, the GFP-tagged protein appears to be regulated in the same fashion as its endogenous counterpart throughout the cell cycle. This suggests that GFP-Mcm10 is functional and equivalent to endogenous Mcm10 in living cells and that GFP-Mcm10 does not impair the function of endogenous Mcm10 as a dominant negative mutant. The Intracellular Distribution of GFP-Mcm10 Changes throughout the Cell Cycle—We next examined the localization of GFP-Mcm10 in G10-1 cells. In an asynchronous population of G10-1 cells, GFP-Mcm10 was found to be concentrated in the nucleus, but weak fluorescence was also detected in the cytoplasm (Fig. 2a, left panels). The distribution of GFP-Mcm10 in the nucleus was relatively homogeneous (Fig. 2a, right panels). However, a different distribution of GFP-Mcm10 was observed when the cells were subjected to Triton X-100 extraction to remove soluble proteins (data not shown). In these cells, GFP-Mcm10 was observed as discrete foci in the nucleus. This distribution has also been observed for many DNA replication proteins, including PCNA (25Leonhardt H. Rahn H.-P. Weinzierl P. Sporbert A. Cremer T. Zink D. Cardoso M.C. J. Cell Biol. 2000; 149: 271-279Google Scholar), and replication foci (26O'Keefe R.T. Henderson S.C. Spector D.L. J. Cell Biol. 1992; 116: 1095-1110Google Scholar). The fluorescence of GFP-Mcm10 in the cytoplasm disappeared after extraction. The distribution, number, and size of the GFP-Mcm10 foci in the individual cells varied. This is probably due to the fact that the cell population was asynchronous. Thus, to determine more precisely how the various staining patterns correlated with particular cell cycle phases, the cells were arrested at the G1/S boundary by aphidicolin treatment following mitotic shake-off and then released in aphidicolin-free medium. Three kinds of localization patterns were observed, each of which was observed at specific time points during the S phase (Fig. 2, b and c). The first pattern (type I) appeared in the first 3 h from the G1/S boundary. This pattern was characterized by hundreds of small foci distributed throughout the nuclei with the exception of the nucleoli. The second pattern (type II) appeared 3 h after the release into S phase. This pattern was characterized by foci concentrated around the nucleoli and in the nuclear periphery. At 3 h after release, weak fluorescence was still detected in the nucleoplasm. This disappeared 4.5 h after release. At 6 h after release, the number of foci decreased. The third pattern (type III) was observed toward the end of the S phase (6–7.5 h after release). This pattern was characterized by the appearance of GFP-Mcm10 in nucleoli. That GFP-Mcm10 was localized in the nucleoli during this phase was confirmed by the colocalization of nucleolin and GFP-Mcm10 (Fig. 2d). The physiological function of Mcm10 in nucleoli in late S phase is unknown. It may be that localization in the nucleoli serves to sequestrate Mcm10, thereby preventing it from reaching its target, which may help to ensure that a replicated chromosome does not reinitiate replication.Fig. 2Subcellular localization of GFP-Mcm10 during the cell cycle.a, asynchronous populations of G10-1 cells were fixed with 3.7% formaldehyde and permeabilized with 0.5% Nonidet P-40. DNA was stained with DAPI, and the cells were observed by fluorescence microscopy. b, G10-1 cells were synchronized at the G1/S boundary and released into the S phase. To monitor DNA synthesis after release from the G1/S boundary, cells at each time point were pulse-labeled with BrdU, and the percentage of BrdU-positive cells was scored. c, at various intervals from the G1/S boundary, the cells were extracted with CSK buffer containing 0.1% Triton X-100 for 10 min on ice prior to fixation and then observed by fluorescence microscopy. d, cells were extracted with CSK buffer containing 0.1% Triton X-100 and fixed at 4.5 or 7.5 h from the G1/S boundary. Nucleolin was detected by indirect immunofluorescence with an anti-nucleolin antibody and a Texas Red-conjugated anti-mouse secondary antibody. e, whole cell extracts (wce) and Triton X-100-soluble fractions (sup) and insoluble fractions (ppt) were prepared at each time point after release from the G1/S boundary and subjected to immunoblotting as described in the legend to Fig. 1b. f, cells undergoing mitosis were fixed with 3.7% formaldehyde and permeabilized with 0.5% Nonidet P-40. DNA was stained with DAPI, and the cells were observed by fluorescence microscopy.View Large Image Figure ViewerDownload (PPT) It is known that some fixation protocols alter the localization of proteins. To test the veracity of our confocal studies, we fixed cells with either 3.7% formaldehyde or ethanol:acetic acid (19: 1). The same results were obtained. We also found that the same patterns were obtained when a different cell line was tested (data not shown), which indicates that these patterns are also not specific to one cell clone. Moreover, we examined the subcellular localization of GFP-Mcm10 and endogenous Mcm10 during S phase progression by immunoblotting after fractionating the cells into Triton X-100-soluble and -insoluble samples. Both GFP-Mcm10 and endogenous Mcm10 bound chromatin during the S phase and dissociated from chromatin in the G2 phase (Fig. 2e). This is consistent with our earlier observations (Fig. 1c). Also consistent with this is our observation that during mitosis, GFP-Mcm10 was absent from condensed chromosomes and had diffused into the cytoplasm (Fig. 2f). GFP-Mcm10 Is Recruited to Replication Sites before They Replicate—We next investigated the spatial relationship between the GFP-Mcm10 foci and PCNA foci. The nuclear foci of PCNA have been well characterized and are known to colocalize with sites of newly synthesized DNA (24Izumi M. Vaughan O.A. Hutchison C.J. Gilbert D.M. Mol. Biol. Cell. 2000; 11: 4323-4337Google Scholar). Thus, synchronized cells were extracted prior to fixation, and PCNA was detected by indirect immunofluorescence (Fig. 3a). The cells arrested at the G1/S boundary by aphidicolin did not display any PCNA fluorescent labeling, whereas GFP-Mcm10 bound chromatin and appeared to be distributed throughout the nucleoplasm. Between 10 min and 1.5 h after release from the G1/S boundary, both the GFP-Mcm10 foci and the PCNA foci showed the type I pattern. However, the GFP-Mcm10 foci and the PCNA foci did not colocalize in most cases. At 3 h after release, the GFP-Mcm10 foci started to accumulate around the nucleoli and the nuclear periphery, whereas the PCNA foci were still distributed throughout the nuclei. Between 4.5 and 6 h, both the GFP-Mcm10 foci and the PCNA foci were concentrated around the nucleoli and in the nuclear periphery. At 7.5 h, GFP-Mcm10 was mainly concentrated in the nucleoli, whereas PCNA formed a few discrete foci around the nucleoli and in the nuclear periphery. Each of the patterns were enriched at specific time points, and the transition in the GFP-Mcm10 distributions preceded that of the PCNA distributions by 30–60 min (Fig. 3b). To confirm the above results, we examined the correlation of the appearance of Mcm10 foci with that of the DNA replication foci. The DNA replication foci were detected by pulse labeling with the nucleotide analog BrdU and staining with an antibody against BrdU. Because the acid treatment denatures GFP and results in the loss of fluoresce
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