Article |
Address correspondence to Christian Mielke, Dept. of Clinical Chemistry, Medizinische Poliklinik, University of Würzburg, Klinikstrasse 6-8, D-97070 Würzburg, Germany. Tel.: 49-931-201-7008. Fax: 49-931-201-7098. E-mail: christian.mielke{at}mail.uni-wuerzburg.de
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Abstract |
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Key Words: DNA topoisomerase II; cell cycle; nucleolus; nuclear matrix; chromosomal scaffold
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Introduction |
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Another open issue regards the biological roles of the two isoforms of mammalian topo II ( and ß), which are similar in primary structure, have almost identical catalytic properties in vitro (Austin and Marsh, 1998), and are both able to complement topo II function in
Top2 strains of Saccharomyces cerevisiae (Jensen et al., 1996a). In particular, the latter observation has argued against isoform-specific biological functions of topo II
and IIß. However, this might not be conclusive because yeast has only one form of topo II and might not utilize the mammalian enzymes in an isoform-specific manner.
Research on functions of mammalian topo II isoforms in their native environment has been impeded by various obstacles. For example, it has not been possible to unambiguously clarify their subnuclear localization. It has been established that topo II binds to condensed chromosomes and accumulates at centromeres in metaphase (Rattner et al., 1996; Sumner, 1996), but it is unclear whether the enzyme is distributed in the chromosome arms in a uniform (Boy de la Tour and Laemmli, 1988) or axial manner (Earnshaw and Heck, 1985; Gasser et al., 1986; Taagepera et al., 1993; Saitoh and Laemmli, 1994; Rattner et al., 1996; Sumner, 1996). The mitotic localization of topo IIß is even less clear. Several investigations indicate that it is cytoplasmic during cell division (Petrov et al., 1993; Chaly et al., 1996; Meyer et al., 1997), whereas others also describe its association with chromosomes (Taagepera et al., 1993; Sugimoto et al., 1998). All of these studies were restricted to fixed or fractionated specimen, and the disparate results have in part been blamed on the use of different staining techniques and antibodies (Chaly and Brown, 1996; Warburton and Earnshaw, 1997; Austin and Marsh, 1998). The most comprehensive study on topo II in living cells is based on microinjection of purified, fluorescently tagged topo II from Drosophila melanogaster into living embryos, and has revealed a dynamic but homogenous chromosomal distribution of the enzyme (Swedlow et al., 1993). Comparable in vivo studies of the mammalian topo II isoforms using stable heterologous expression of GFP chimera are missing, probably because overexpression of topo II
induces apoptotic cell death (McPherson and Goldenberg, 1998). On the other hand, transient expression of topo II
fused to the green fluorescent protein (GFP) has not yielded conclusive data from cells in interphase, although it allowed confirmation of the enzyme's association with mitotic chromosomes (Mo and Beck, 1999). Reports on the disposition of topo IIß in living cells are not available.
Here, we achieved constitutive expression of active GFP chimera of human topo II at physiological levels by a balanced coexpression with a selection marker from bicistronic transcripts. Thus, we could monitor dynamic relocations of both isozymes during mitosis and assess their localization and mobility during the whole cell cycle. Our data are suited to address most of the issues summarized above.
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Results |
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To compare expression levels of GFP-fused topo II isozymes with the endogenous enzymes, blots were probed with isoform-specific antibodies against topo II (Fig. 1 A, second panel) or ß (Fig. 1 A, third panel). The GFP chimera could clearly be discriminated from the corresponding endogenous enzymes as additional bands of slower migration (Fig. 1 A, arrows). From comparison within each lane, it became evident that they were expressed at much lower levels than the endogenous proteins. Moreover, the levels of untagged topo II
and IIß were the same in transfected and untransfected cells, indicating that the additional expression of GFP-tagged enzymes did not alter endogenous topo II expression.
Because protein functions could be disrupted by fusion to GFP, we wanted to ascertain that the topo IIGFP chimera had the same activity as the endogenous enzymes. We addressed this question by quantitative comparisons of the DNA decatenation activities of endogenous and GFP-fused enzymes isolated by immunoprecipitation. GFP-directed immunoprecipitates from cells expressing GFP chimera contained GFP-fused and unfused topo II in equal proportions (Fig. 1 B, inserts, lanes 2 and 4), suggesting heterodimers of GFP-fused and endogenous topo II. Upon comparing the DNA-decatenation activity of these mostly heterodimeric immunoprecipitates with similar amounts of endogenous enzymes obtained from untransfected 293 cells by topo IIdirected immunoprecipitation (Fig. 1 B, inserts, lane 1 and 2, respectively), it appeared that the GFP chimera of topo II (Fig. 1 B, second panel) and ß (Fig. 1 B, bottom) had the same activity as their endogenous counterparts (Fig. 1 B, top and third panel, respectively), and could likewise be inhibited by the ATPase-inhibitor orthovanadate (Fig. 1 B, outmost lanes on the right). Thus, fusion to GFP did not alter the catalytic properties of the enzymes. In addition, to ascertain that the GFP-fused enzymes were also active in the cells, we employed immunoband depletion (Fig. 1 C), which is based on the fact that the transient, covalent complex between topo II and genomic DNA is stabilized by specific topo II poisons (Liu, 1989). As a consequence, topo IIspecific signals are depleted in Western analyses due to a retention of topo IIDNA complexes in the gel slots. Untransfected and transfected 293 cells were treated with the topo II poison teniposide (VM 26), and subjected to immunoblotting using GFP antibodies (Fig. 1 C, top). Obviously, the treatment depleted topo IIfused GFP bands from the blots (Fig. 1 C, compare lanes 5 and 6, and lanes 7 and 8). The extent of depletion was similar to that of endogenous topo II (unpublished data). GFP alone or
-tubulin (Fig. 1 C, compare lanes 3 and 4, top and bottom, respectively) were not depleted by VM 26, attesting to the specificity of the assay. These results confirmed that topo IIGFP chimera were as active as endogenous topo II because they formed the same amount of catalytic intermediates in the genome of the transfected cells.
To judge the global functionality of topo IIGFP in complex cellular processes such as mitosis, we tested complementation of topo II function in S. cerevisiae topo II deletion strains (Jensen et al., 1996b). This assay (Fig. 1 D) demonstrated that GFP chimera of human topo II and IIß were fully capable of supporting the mitotic growth of
top2 yeast upon eradication of the endogenous salvage plasmid encoding Schizosaccharomyces pombe topo II (+5-fluoroorotic acid [FOA]), whereas cell growth was abolished in the absence of an additional topo II construct. Apparently, complementation by GFP chimera was as good as by untagged topo II. Thus, the functionality of human topo II is fully preserved in the corresponding GFP chimera.
Finally, we considered that fusion to GFP might disrupt cellular targeting of topo II. Therefore, we compared the distribution patterns of endogenous and GFP-tagged topo II by indirect immunofluorescence. Fig. 1 E shows examples of mitotic and of interphase cells labeled with isozyme-specific topo II antibodies. Untransfected cells (Fig. 1 E, top panels) gave rise to antibody-derived signals only, whereas cells expressing topo IIGFP (Fig. 1 D, bottom left) or topo IIßGFP (Fig. 1 E, bottom right) also emitted GFP fluorescence. The distribution patterns of GFP fluorescence (specific for GFP fusion proteins only) and immunofluorescence (specific for fused and endogenous enzymes) were virtually identical, attesting to the fact that the GFP chimera colocalized with their endogenous counterparts. In addition, distribution patterns of topo II in nontransfected and in topo IIGFP-expressing cells were identical (Fig. 1 D, compare top and bottom portion), making it unlikely that the GFP chimera disrupted localization of the endogenous enzymes. It should be noted that the patterns shown in Fig. 1 E do not represent the true localization of topo II
or IIß in living cells, because these cells were fixed and permeabilized which is known to alter the cellular distribution of topo II in an unpredictable fashion (Chaly and Brown, 1996). In fact, when we applied different protocols for permeabilization and/or fixation, we also obtained patterns different from those shown in Fig. 1 E, but in each case antibody staining and GFP fluorescence were identical. The colocalization of topo II and GFP epitopes in fixed cells shows that the cellular targeting of topo IIGFP chimera is similar to that of the endogenous enzymes. Thus, the GFP-tagged enzymes should also reflect faithfully the localization of topo II in living cells.
Distribution of topo II and IIß in living cells
Monitoring of topo IIGFP and topo IIßGFP in living cells by GFP fluorescence revealed a very similar distribution of the isozymes in interphase nuclei (Fig. 2): both were exclusively in the nucleus, where they resided in the nucleoplasm and the nucleoli. The minor, nucleoplasmic subpopulation had a slightly uneven distribution. The major fraction of both isoforms accumulated in the nucleoli. It had a cloudy but otherwise unstructured distribution in the entire nucleolar space which distinctively excluded small globular compartments (not yet identified), imposing as dark holes within the brightly fluorescent nucleolus (Fig. 2, insert, arrows).
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Further evidence for this hypothesis was obtained from metaphase cells treated with the topo IIspecific drug VM 26 (Fig. 4 C). This caused a rapid redistribution of both topo IIGFP (Fig. 4 C, top) and topo IIßGFP (bottom) from the cytosol to the chromosomes, indicating that the drug efficiently trapped both isozymes on chromosomal DNA. Of interest, the VM 26induced chromosomal accumulation of topo II
GFP was faster and more complete than that of topo IIßGFP. In summary, these data indicate that during mitosis topo IIß has a propensity to engage in DNA turnover, but the enzyme is clearly biased to the cytosolic state. In addition, the rapid redistribution of both isoforms from the cytosol to the chromosomes after treatment with VM 26 suggests a dynamic interchange between the two populations thus identifying these enzymes as highly mobile constituents of the mitotic cell.
Mobility of topo II and IIß and the effects of topo II poisons
To follow up on the mobility of topo II and topo IIß in living cells, we employed photobleaching techniques. Fig. 5 summarizes a series of experiments, determining the mobility of topo II
and IIß in interphase nuclei via kinetics of FRAP (White and Stelzer, 1999). Topo IIlinked GFP fluorescence was bleached irreversibly in circular areas (Ø = 1 µm) by high-powered laser pulses and fluorescence recovery in the bleached spots as a consequence of topo IIGFP molecules moving in from unbleached areas was recorded over time. It is readily apparent from time-lapsed fluorescent images (Fig. 5, top) and quantitative plots of recovery kinetics (Fig. 5, bottom) that FRAP of a nucleoplasmic or a nucleolar area was in each compartment fast and complete for both topo II isoforms. This suggests that immobile molecules are virtually absent. Recovery kinetics of topo IIGFP were much faster than those of GFP-histone H3, a member of nucleosomal core proteins (Fig. 5, Ctrls.) known to be firmly immobilized on chromatin (Phair and Misteli, 2000). On the other hand, both topo II isozymes were by no means as freely diffusible as unfused GFP which exhibited recovery kinetics even too fast to be recorded with our experimental settings (Fig. 5, Ctrls.). Moreover, recovery of topo II
GFP and topo IIßGFP was notably slower in nucleoli (t1/2 = 6.2 and 10 s, respectively) than in the nucleoplasm (t1/2 = 1.8 and 3 s, respectively), suggesting less mobile enzyme subpopulations in the nucleoli. This, and the slight difference in mobility between the isozymes (topo IIß was somewhat slower), raised the question of what controls topo II mobility and which role DNA interactions play in this respect. We could address this issue experimentally, as covalent topo II·DNA intermediates are stabilized by specific agents such as VM 26 (compare Figs. 1 C and 4 C). VM 26 treatment induced a profound redistribution of topo II in the nucleus which was similar for both isoforms (Fig. 5, top panels, bottom rows). Nucleoli became largely depleted, and almost the entire enzyme pool concentrated at distinct sites in the nucleoplasm, forming there a granular pattern. FRAP analysis revealed that topo II was clearly much less mobile at these granular nucleoplasmic sites. Notably, immobilization was less pronounced for topo IIß which still exhibited a slight ascension of the recovery curve, whereas topo II
did not. This is in agreement with previous pharmacological studies showing that topo IIß is less sensitive to VM 26 (Drake et al., 1989). In comparison, the small amount of topo II
and ß remaining inside nucleoli after VM 26 treatment retained a much higher mobility and, here again, topo IIß was more mobile (less affected by VM 26) than topo II
. These data demonstrate for the first time directly that the vast majority of topo II
and IIß molecules present in a living cell becomes indeed trapped by VM 26 in its covalently DNA-bound intermediate state which must be relieving for oncologists, relying on this mechanism of action for tumor therapy. These findings also suggest that nuclear sites where topo II normally accumulates (Fig. 2) do not necessarily represent sites where the enzymes are most actively engaged in DNA catalysis. This is most evident in nucleoli where topo II concentration is highest (Fig. 2), enzyme mobility is lowest (Fig. 5, untreated cells), but entrapment by VM 26 is least efficient (Fig. 5, VM 26-treated cells). Thus, interaction of topo II with genomic DNA seems not to constitute the major determinant of topo II localization or mobility.
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Mobility of mitotic topo II
The high degree of mobility of topo II and IIß in interphase nuclei makes it unlikely that the enzymes play a structural role in building a karyoskeleton. However, it is still possible that they participate in building the mitotic chromosome scaffold which is the major structural role proposed for these enzymes (Adachi et al., 1991). To address this issue, we determined the mobility of topo II
in chromosomes (Fig. 8). These experiments were restricted to the
-isozyme, because chromosomal and cytosolic locations of topo IIßGFP could not be reliably discriminated (compare Figs. 8 and 4 B). Unexpectedly, fluorescence recovery of topo II
GFP (Fig. 8 A, upper panel) after photobleaching of a chromosomal area in a metaphase plate, was fast (t1/2 = 3.5 s) and complete. We obtained similar recovery kinetics with chromosomes of cells in pro-, ana- or telophase (unpublished data). Moreover, the FLIP experiment shown in Fig. 8 B demonstrates that all topo II
GFP fluorescence was lost from chromosomes and the cytoplasm of a given cell when repeated bleach pulses were applied to a distinct chromosomal area. Comparable results were obtained when the cytosol was repeatedly bleached (unpublished data). In summary, these data indicate that topo II
is mobile within the chromosome. An immobile fraction is virtually absent. The FLIP experiment also demonstrates unrestricted movements of the enzyme between chromosomes and the cytoplasm. To corroborate these controversial findings, we needed to prove that we would have detected an immobile fraction, if present. This was achieved in two ways. First, we demonstrated immobility of GFP-histone H3 in mitotic chromosomes (Fig. 8 A, bottom). Second, we demonstrated immobility of topo II
GFP and topo IIßGFP trapped on chromosomal DNA by VM 26 (Fig. 8 A, middle panels). Both control experiments attest to the validity of our data regarding the unexpected degree of mobility of chromosomal topo II
in the absence of VM 26.
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Fig. 9 A shows high-resolution confocal microscopy images of topo IIGFP and GFP-histone H3 in single chromosomes of living cells. The quantitative analysis of fluorescence intensity distribution in representative cross sections of the chromosomes (boxes) shows that both proteins covered a similar chromosome diameter of 0.81 µm, and had a similar distribution. Because histone H3 is an integral part of the chromatin and hence can be assumed to cover the entire chromosome, these data suggest that, in living cells, topo II
is not confined to chromosome axes.
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In summary, these observations demonstrate that the localization of topo II in chromosome spreads differs notably from the situation in living cells. Apparently, hypotonic treatment of cells triggers a series of events eventually generating an axial pattern of topo II. First, the whole complement of the enzymes relocates rapidly from the cytosol to the chromosomes. Then, a loosely bound fraction of topo II is gradually lost over time from the outer region of the chromosome arms. This unveils another fraction bound to the centromere and the chromosomal core in a more stable manner. We envisage that during immunohistochemical staining procedures topo II can easily be lost from the chromosomal periphery, thus leading to the impression that topo II is confined to the chromosome axis. This would also explain why in some cases both patternsaxial and even distribution over chromosome armshave been detected within the same sample (Sumner, 1996).
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Discussion |
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On the other hand, topo II is obviously restricted in its mobility, because it moves slower than GFP which is believed to be freely diffusible. It has been proposed that such an attenuation in mobility is caused by interactions with less mobile nuclear components (Misteli, 2001). However, the nature of such componentsthe chromatin, a putative karyoskeleton, or bothis an unsolved issue (Shopland and Lawrence, 2000; Misteli, 2001). In this respect, our observation that VM 26 treatment depletes topo II from nucleoli is of interest, because it suggests that it is not the direct interaction with chromatin that slows down topo II and causes its accumulation in this compartment. Moreover, nucleoli recruit most of the topo II complement of the nucleus and it is not likely that such a large fraction is required for the topological organization of the nucleolar chromatin which is small in comparison to the entire genome. Thus, the decreased mobility of topo II and IIß in nucleoli is likely due to interactions with other proteins and not with DNA. On the other hand, considering the dense packing of topo II
and IIß in the nucleoli of living cells (Fig. 2), and considering that among numerous topo IIinteracting proteins described in the literature, none is a nucleolar protein, it could also be imagined that polymerization of topo II (Vassetzky et al., 1994) might create a transient, less mobile structure thus contributing to accumulation of the enzymes in nucleoli.
Our observations also shed some light on ongoing discussions about isoform-specific topo II functions in mammalian cells. It is clear that topo II function in general is required for maintenance of the nucleolar structure and the folding of rDNA into functionally organized nucleolar genes in yeast and mammals (Christman et al., 1988; Hirano et al., 1989; Iarovaia et al., 1995). We find that both isoforms accumulate in nucleoli and exhibit the same subnucleolar distribution, indicating that they are both engaged in these functions. Thus, topo II and IIß have probably many similar functions in the interphase nucleus. However, our data clearly indicate that topo IIß differs significantly from topo II
in its contribution to the mitotic cycle. We find that in metaphase topo IIß is scarcely chromosome-associated. This fits with the recent observation that disruption of the TOP2ß gene has little effect on early embryonal development in mouse (Yang et al., 2000), and with our previous finding that in a mammalian cell, topo IIß cannot substitute topo II
during mitosis (Grue et al., 1998). It now seems quite clear that topo IIß is not required for mitotic cell division; however, in a living cell, topo IIß is not fully excluded from metaphase chromosomes (Fig. 4 B), and to some extent it retains the ability to engage in catalytic DNA turnover in the chromosome (Fig. 4 C). This might be sufficient to support mitotic topo II functions in yeast (Jensen et al., 1996a; Fig. 1 C), but not in mammals (Grue et al., 1998).
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Materials and methods |
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Cell culture and transfection
The human embryonal kidney cell line 293 (DSMZ) was grown at 37°C in a humidified atmosphere of 5% CO2 in DME with Glutamax-I (GIBCO BRL) supplemented with 10% fetal bovine serum, 100 U penicillin/ml, and 100 µg ml-1 streptomycin. Cells were transfected using Lipofectamine (GIBCO BRL) according to the manufacturer's instructions. After 2 d, stable cell lines were selected and maintained thereafter in medium containing 0.35 µg ml-1 puromycin.
Complementation assay
Yeast complementation was carried out as described by Jensen and colleagues (1996a). Briefly, LEU2 expression plasmids for human topo II isoforms and the respective GFP chimera were transformed into the haploid top2 strain BJ201 (pHT173 [S.pombe TOP2/URA-ARS/CEN] MAT
ura3 trp1 leu2 his3 pep4::HIS3 prb1Dcan1 top2::TRP1 GAL) which contains a URA3 plasmid carrying the S. pombe TOP2 gene to substitute for the essential topo II activity. Colonies from selection plates (without Leu) were then streaked onto media containing 1 mg ml1 5-FOA to counterselect against the plasmid carrying the S. pombe TOP2 gene.
Immunoblotting and band depletion assay
5 x 105 cells were harvested, resuspended in PBS, and lysed by addition of an equal volume of twofold lysis buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 8 M urea, 20% glycerol, 0.04% bromophenol blue, 10 mM 4-[2-Aminoethyl]-benzenesulfonyl fluoride, 1 mM PMSF, 20 µg ml-1 Aprotinin, 10 µg ml-1 Pepstatin A). Whole cell lysates were separated by SDS-PAGE, and blotted onto PVDF membranes (Immobilon P; Millipore). Blots were blocked in 2% BSA, 0.05% Tween 20 in CMF-PBS (167 mM NaCl, 25.5 mM Na2HPO4, 2 mM KH2PO4, pH 7.5) and then incubated for 1 h in the same buffer with diluted primary antibodies (mouse monoclonal topo II antibody KiS1 [Boege et al., 1995]; mouse monoclonal peptide topo IIß antibody 3H10 [Kimura et al., 1996]; mouse monoclonal GFP-antibody [CLONTECH Laboratories, Inc.]; and mouse monoclonal
-tubulin antibody [Sigma-Aldrich]). After washing, the filters were incubated with HRP-conjugated goat antirabbit or goat antimouse antibodies in 2% BSA, 0.1% Tween 20 in CMF-PBS for 1 h. After extensive washing with the same buffer, specific protein bands were detected with the ECL Plus system (Amersham Pharmacia Biotech). For immunoband depletion, cells were incubated with 200 µM VM 26 for 1 h prior to harvesting.
Immunoprecipitation and kDNA decatenation
Magnetic particles (Dynabeads M 500; Dynal) were covalently coated (according to the producers manual) with rabbit antibodies against topoisomerase II (Genosys) and mouse monoclonal antibodies against topo IIß (Meyer et al., 1997) or GFP (Roche). 2 x 107 coated beads were saturated for 2 h at 4°C with nuclear extracts obtained from 109 cells (Meyer et al., 1997). Subsequently, the beads were collected with a magnet, washed with binding buffer (175 mM NaCl, 6 mM Hepes, pH 7.5, 2 mM EDTA, 14% glycerol, 0.2 mM DTT, 0.5 mM 4-[2-Aminoethyl]-benzenesulfonyl fluoride, 10 µg ml-1 aprotinin, 5% FBS), and then with assay buffer (120 mM KCl, 50 mM Tris HCl, pH 7.9, 10 mM MgCl2, 1 mM ATP, 0.5 mM DTT, 0.5 mM EDTA, 30 µg ml-1 BSA). Finally, the beads were incubated for 2 h at 37°C with 1 µg crithidia fasciculata kinetoplast DNA (kDNA; TopoGen, Inc.) in a final volume of 40 µl assay buffer. Negative controls included addition of 300 µM Na3VO4 and beads not incubated with nuclear extracts. Alternatively, the beads were eluted with 100 mM Na3PO4, 100 mM NaCitrate, pH 3 and 3 M urea. Eluates were subjected to SDS-PAGE (6% gels) and visualized by silver staining.
Immunocytochemistry
Cells were grown on microscopic coverslips, washed in PBS, fixed in 2% paraformaldehyde in PBS for 15 min on ice, and permeabilized with 0.25% Triton X-100 in the presence of 1% paraformaldehyde in PBS for 10 min on ice. All subsequent steps were carried out at ambient temperature. After washing with PBS, cells were blocked for 1 h in PBS, 2% BSA, 5% goat serum and then incubated for 1 h with topo II antibody KiS1 or topo IIß antibody 3H10 diluted 1:500 in blocking solution. After washing, bound antibodies were visualized by incubation for 1 h with Cy3TM-conjugated goat antirabbit or antimouse F(ab')2 fragments (Dianova) diluted 1:1,000. Coverslips were then washed three times for 5 min in PBS. The first wash contained 80 ng ml-1 DAPI to stain genomic DNA.
Microscopy
Epifluorescent images were acquired with appropriate filter sets (AHF Analysentechnik) at 630x magnification using an inverted microscope (Axiovert 100; Carl Zeiss) equipped with a cooled charge-coupled device camera (Sensys; Photometrics Ltd.) and an additional 4x magnification lens. Living cells were grown and inspected in CO2-independent medium (GIBCO BRL) in poly-L-lysinecoated live-cell chambers (Bioptechs, Inc.) at 37°C.
Confocal imaging, FRAP and FLIP analyses of living cells were performed at 37°C with a Zeiss LSM 510 inverted confocal laser scanning microscope equipped with a CO2-controlled on-stage heating chamber using a heated 63x/1.4 NA oil-immersion objective and the FITC filter setting (488 nm/ 515 nm). It should be emphasized that strict temperature control was crucial for topo II localization and mobility. For FRAP experiments, a single optical section was acquired with 5.6x zoom. Images were taken before and at 1.577 s time intervals after bleaching of a circular area at 20 mW nominal laser power with three iterations. The imaging scans were aquired with a laser power attenuated to 0.11% of the bleach intensity. The same settings were used for FLIP experiments where cells were repeatedly bleached and imaged at intervals of 15 s. For quantitative FRAP analysis, fluorescence intensities of the bleached region and the entire cell nucleus were measured at each time point. Data were corrected for extracellular background intensity and for the overall loss in total intensity as a result of the bleach pulse itself and the imaging scans. Unless stated otherwise, FRAP recovery curves were generated by calculating the relative intensity of the bleached area Irel as described (Phair and Misteli, 2000). FLIP depletion curves of selected areas outside the bleach spot were just corrected for the loss of fluorescence intensity caused by the imaging scans by comparison with the fluorescence intensity of a neighboring cell nucleus. Bleaching of mitotic cells (Fig. 8) was performed in the presence of 0.1 µM paclitaxel to reduce movements of the chromosomes.
Spreading of native chromosomes
107 exponentially growing cells were harvested, collected by centrifugation for 5 min at 160 g, and carefully resuspended in 6 ml hypotonic solution (0.075 M KCl). Cells were pelleted as above, resuspended in 400 µl hypotonic solution and drops of about 50 µl were then spotted on a slightly tilted glass slide. Prior to this, slides were carefully cleaned in methanol and then soaked in double distilled water. After spreading, unattached cells were removed by letting 200 µl isotonic cytoskeleton CSK buffer (100 mM KCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 1 mM EGTA, 10 mM 4-[2-Aminoethyl]-benzenesulfonyl fluoride, 1 mM PMSF, 20 µg/ml Aprotinin, 10 µg/ml Pepstatin A) containing 80 ng/ml DAPI run down the slightly tilted slide. Spread chromosomes were then carefully mounted in the same buffer.
Online supplemental material
Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200112023/DC1)(10 images per second) shows topo IIGFP- and topo IIßGFP-expressing cells proceeding through a mitotic cycle from prophase to early G1 phase. Time-lapsed images (one image every 30 s) were acquired by confocal microscopy, and corresponding images of transmitted light (left) and green fluorescence (right) are mounted side by side.
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Footnotes |
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* Abbreviations used in this paper: FLIP, fluorescence loss in photobleaching; FOA, fluoroorotic acid; GFP, green fluorescent protein; topo, DNA topoisomerase; TPI, triose phosphate isomerase.
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bo 910/3-1, Bo 910/4-1, GRK 639, HA 1434/13-1), the Danish Cancer Society (97-100-32, 97-143-09-9132, 97-143-10-9132), the Danish Research Council, the Danish Center for Molecular Gerontology, and the Thaysen Foundation.
Submitted: 6 December 2001
Revised: 20 February 2002
Accepted: 21 February 2002
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