Article |
Address correspondence to Angus I. Lamond, Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK. Tel.: 44-1382-345473. Fax: 44-1382-345695. E-mail: a.i.lamond{at}dundee.ac.uk
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Abstract |
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Key Words: nucleolus; Cajal bodies; speckles; localization; nucleus
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Introduction |
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The best-studied subnuclear body is the nucleolus (for reviews see Pederson, 1998; Scheer and Hock, 1999; Carmo-Fonseca et al., 2000; Olson et al., 2000; Visintin and Amon, 2000). Whereas 10% of protein-coding genes are active at any time,
80% of the total cellular transcription activity is devoted to expression of the conserved ribosomal DNA (rDNA)* repeats in nucleoli by RNA polymerase I (Blobel and Potter, 1967). Multiple rDNA transcription units are joined together by nontranscribed intergenic spaces to form the so-called nucleolar organizing regions (NORs) located in human chromosomes 13, 14, 15, 21, and 22. Nucleoli are assembled on these chromosomal NOR regions after every round of mitosis. The rDNA-containing region is transcribed to yield a precursor, the 45S pre-rRNA, which is processed in a series of posttranscriptional cleavage and modification reactions to generate mature rRNA species that form the catalytic core of the ribosome (for review see Warner, 2001). Various small nucleolar RNAs (snoRNAs), for example U3 and U14, are needed for these separate rRNA cleavage steps, each snoRNA being required at specific steps. The snoRNAs can be categorized into two main classes, box C/D (i.e., U3, U8, U14) and box H/ACA (i.e., U17, U19, U64), according to conserved sequence elements and the way in which they are assumed to fold into defined secondary structures (for review see Weinstein and Steitz, 1999). These snoRNAs assemble together with protein factors to form snoRNPs that have roles in guiding and catalyzing posttranscriptional RNA modifications. For example, box C/D snoRNPs direct 2'-O-ribose methylation and box H/ACA snoRNPs direct pseudouridylations of specific rRNA nucleotides (for review see Lafontaine and Tollervey, 1998; Lewis and Tollervey, 2000).
Several nucleolar proteins, including the snoRNP factor fibrillarin, have been found in Cajal bodies (CBs) (for review see Gall, 2000). These subnuclear structures, originally called nucleolar accessory bodies, or coiled bodies, are often associated with the nucleolar periphery, or even located inside the nucleolus (Malatesta et al., 1994; Ochs et al., 1994; Lyon et al., 1997; Sleeman et al., 1998). In addition to snoRNPs, they also contain splicing snRNPs and some transcription factors; however, they do not contain non-snRNP protein splicing factors or nascent pre-mRNA. Their snRNP components are specifically newly assembled particles (Carvalho et al., 1999; Gall et al., 1999; Sleeman and Lamond, 1999a; Sleeman et al., 2001). Fluorescently labeled U3, U8, and U14 snoRNAs, when microinjected into Xenopus oocytes, accumulate transiently in CBs prior to nucleoli, suggesting that newly imported snoRNAs flow from the CBs to nucleoli (Narayanan et al., 1999b). Therefore, CBs might be involved in transport and maturation of both snRNPs and snoRNPs.
In higher eukaryotes, most pre-mRNAs must be spliced to generate functional mRNAs. Splicing is a nuclear process carried out by a complex RNA protein machine termed the spliceosome (for review see Staley and Guthrie, 1998). Although splicing often occurs cotranscriptionally, the majority of pre-mRNA splicing factors are not localized at active transcription sites; instead, they are enriched in domains called speckles (for reviews see Sleeman and Lamond, 1999b; Misteli, 2000). Recruitment of splicing factors to sites of transcription is believed to involve a cycle of phosphorylation and dephosphorylation (for review see Misteli, 1999).
The advent of green fluorescent protein (GFP) technology allows the visualization of tagged proteins and their movement in living cells (for review see Swedlow and Lamond, 2001). The use of photobleaching techniques with the GFP fusion proteins also allows analysis of protein dynamics (Phair and Misteli, 2001; Reits and Neefjes, 2001). Here we describe a novel nuclear pathway shown by the nucleolar factor NHPX. The NHPX protein was recently identified as a putative human homologue of yeast NHP2p (Saito et al., 1996), and later, independently, as a 15.5-kD RNA-binding protein (Nottrott et al., 1999). Subsequent analyses indicated that NHPX is the human orthologue of the Saccharomyces cerevisiae Snu13p (Chang et al., 1999; Nottrott et al., 1999). The human NHPX protein was also independently identified in a directed proteomic analysis of nucleoli isolated from cultured HeLa cells (Andersen et al., 2002); it shares a common RNP structure that binds to both box C/D snoRNAs and spliceosomal U4 snRNA (Nottrott et al., 1999; Vidovic et al., 2000; Watkins et al., 2000). However, NHPX localizes in nucleoli by immunofluorescence (Chang et al., 1999). Here we analyze the in vivo pathway by which NHPX accumulates in nucleoli, and show that it involves an unexpected, transient interaction with splicing speckles. The movement of NHPX from speckles to nucleoli is dependent on pre-mRNA transcription. These data suggest that NHPX may be involved in, and possibly link, several parallel RNA metabolic pathways that occur in distinct nuclear domains.
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Results |
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To confirm that the EYFPNHPX fusion protein expressed in the HeLaEYFPNHPX cell line behaves biochemically as the endogenous protein, its in vivo RNA-binding specificity was investigated. An extract from HeLaEYFPNHPX cells was immunoprecipitated using an anti-GFP antibody. RNAs in the immunoprecipitate were separated by Urea-polyacrylamide gel electrophoresis, transferred to nylon membrane, and hybridized with probes for U1, U2, U4, U5, and U6 snRNAs and U3 snoRNA (Fig. 1 E). This showed that U3 snoRNA and U4 snRNA, but not U1, U2, U5, or U6 snRNAs, were preferentially isolated with the anti-GFP antibody (Fig. 1 E, lane 3). Control experiments, i.e., bead control and an HeLaEGFPH2B cell extract, showed that although the anti-GFP antibody still pulled down the fluorescent protein fusion protein (unpublished data), it did not pull down any of the RNAs tested from these extracts (Fig. 1 E, lane 1 and 2), whereas the same anti-GFP antibody pulled down each of the U1, U2, U4, U5, and U6 snRNAs, but not U3 snoRNA, from a HeLaECFPSmB cell extract (Fig. 1 E, lane 4). These data show that the EYFPNHPX fusion protein in vivo is specifically complexed with the same RNA targets that NHPX was shown to bind directly in vitro (Nottrott et al., 1999; Watkins et al., 2000). Given that U4 and U6 snRNAs usually exist as a duplex inside the nucleus, the immunoprecipitation of U4 snRNA alone is surprising. The disruption of the U4/U6 interaction in this assay is unlikely, because a non-Sm RNA, i.e., U6 snRNA, can be coimmunoprecipitated with U4 from the ECFPSmB cell extract under the same experimental conditions and using the same anti-GFP antibody (Fig. 1 E, lane 4). Another interesting observation is that only a small amount of the total U4 snRNA was pulled down in these experiments (Fig. 1 E, lane 3), even though U4 snRNA is present in similar abundance to U3 snoRNA inside HeLa cells (Reddy and Bush, 1988). This suggests that only a subset of U4, which is likely not bound to U6, may be interacting with NHPX in vivo. Alternatively, the fraction of NHPX bound to the U4/U6 duplex could be inaccessible to the anti-GFP antibodies.
NHPX is colocalized with U3-containing snoRNPs, rather than U4-enriched splicing speckles, during interphase
The ability of NHPX to bind both U3 snoRNA and U4 snRNA, which normally are located in different subnuclear structures, prompted us to investigate further the localization of NHPX under different conditions (Fig. 2). The binding of NHPX to the spliceosomal U4 snRNA suggests that it should colocalize, at least in part, with other splicing factors. However, EYFPNHPX in the HeLaEYFPNHPX cell line does not show a speckled nuclear pattern similar to other human splicing factors such as SC35 and U1A (Fig. 2 A, arrowhead indicates CB, arrow indicates nucleolus, broken arrow indicates speckles). The anti-NHPX antiserum also does not label speckles (Fig. 1 B, Chang et al., 1999). Instead, EYFPNHPX is accumulated in nucleoli and CBs and colocalizes with the snoRNP protein FIB (Figs. 1 A and 2 A ; unpublished data). Also, nucleolar segregation, caused by treating the cells with the transcription inhibitor actinomycin D, results in the relocation of NHPX to the nucleolar periphery along with FIB, but does not cause it to colocalize with either SC35, or other splicing factors (unpublished data).
Analysis of the HeLaEYFPNHPX cells at different stages of mitosis also showed no evidence for the association of NHPX with splicing factors (Fig. 2 B). Time-lapse analysis of live HeLaEYFPNHPX cells showed that NHPX associated with snoRNP-containing nucleoli immediately after the nuclear envelope reforms (Fig. 2 C; unpublished data). Combined with other immunofluorescence data of fixed cells counterstained with anti-FIB (unpublished data), we conclude that EYFPNHPX does not accumulate with snRNPs in speckles, but instead colocalizes with the snoRNP protein FIB at all cell cycle stages and metabolic conditions tested. The only colocalization of NHPX with splicing snRNPs detected in vivo was specifically in CBs, structures known to accumulate newly imported snRNPs and snoRNPs when they first enter the nucleus.
Newly imported NHPX transiently colocalizes with splicing factors
Next, we investigated the localization of newly synthesized NHPX because recent data have shown a temporal pathway for snRNP and snoRNP localization in nuclei (Fig. 3; Carvalho et al., 1999; Narayanan et al., 1999a, 1999b; Sleeman and Lamond, 1999a; Sleeman et al., 2001). To our surprise, microinjection of the expression vector pAL107EYFPNHPX into the cultured cell lines, followed by examination in the fluorescence microscope, revealed that 1 h after microinjection, EYFPNHPX is accumulated in splicing speckles and CBs (Fig. 3). 26 h postmicroinjection, EYFPNHPX is also detected in nucleoli, whereas the level of EYFPNHPX in speckles shows a concomitant decrease. At later time points, the signal in speckles can no longer be detected and EYFPNHPX accumulates specifically in nucleoli and CBs, resembling the pattern observed in the HeLaEYFPNHPX cell line during interphase.
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Newly synthesized NHPX does not colocalize with U3 snoRNP in speckles
Next, we examined whether the speckle localization of newly synthesized NHPX is a consequence of a previously unknown behavior of U3 snoRNP. To test this, an expression vector, pAL118ECFPFIB, encoding ECFP-tagged FIB, a known U3 snoRNP component, was cotransfected with pAL107EYFPNHPX into the parental HeLa cell line and analyzed at various time points posttransfection (Fig. 4). At 1 h posttransfection, ECFPFIB had already accumulated in nucleoli and CBs, whereas in the same cells, EYFPNHPX accumulated in speckles and CBs, but not in nucleoli (Fig. 4 A, arrow indicates nucleolus, broken arrow indicates speckles). Several hours later, EYFPNHPX began to accumulate in nucleoli and the signal in speckles decreased, whereas the ECFPFIB remained specifically in nucleoli, and both proteins were detected in CBs (unpublished data). After one cell cycle, EYFPNHPX and ECFPFIB both quantitatively colocalized in the dense fibrillar component inside nucleoli and in CBs (Figs. 1 A and 4 B, arrowhead indicates CB, arrow indicates nucleolus, broken arrow indicates speckles; unpublished data). Therefore, the transient presence of newly expressed NHPX in nuclear speckles is not explained by its association with U3 snoRNP.
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The expression of exogenous NHPX raises the possibility that the target RNAs it binds to may be upregulated, and thereby results in the observed temporal sequence of nuclear localization. Therefore, we investigated the in vivo localizations of U3 and U4 RNAs in the HeLa cells that were microinjected with pAL107EYFPNHPX using complementary 2'-O-methyl antisense RNAs (Fig. 5; for review see Lamond, 1993). The U3 in microinjected cells remained localized in the dense fibrillar component of nucleoli and CBs at both early and late time points, similar to control, nonmicroinjected cells (Fig. 5 A and B, arrowhead indicates CB, arrow indicates nucleolus, broken arrow indicates speckles; unpublished data). The U4 snRNA was detected in speckles and CBs in both the microinjected cells at different time points and in control, nonmicroinjected cells (Fig. 5, C and D). Because the localization of U3 and U4 remained unaltered, the differential localization of NHPX at different time points is likely not due to the movement or relocalization of either of these NHPX target RNAs. These data are consistent with NHPX binding U4 snRNA in speckles and U3 snoRNA in the nucleolus. However, we cannot exclude that NHPX also binds to different and/or unknown target RNAs at the different nuclear structures.
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The progression of NHPX from speckles to nucleoli is dependent on Pol II, but not Pol I transcription
Next, we tested whether the progression of NHPX from speckles to nucleoli requires gene expression, including both Pol I and Pol II transcription (Fig. 10). We again employed the heterokaryon approach between HeLaECFPFIB and HeLaEYFPNHPX cells. At 4 h postfusion, only a small amount of EYFPNHPX was in speckles, whereas most accumulated in nucleoli (Figs. 6 D and 10 A, arrowheads indicate CBs, arrows indicate nucleoli, broken arrows indicate speckles). The heterokaryons formed between HeLaEYFPNHPX and HeLaECFPFIB cells were subjected to transcription inhibitors targeted to specific polymerases. Low levels of actinomycin D cause the segregation of nucleoli and inhibit rRNA transcription, but not pre-mRNA transcription. Newly synthesized EYFPNHPX moved to the speckles of recipient HeLaECFPFIB nuclei, prior to accumulating in the segregated nucleoli (Fig. 10 B, arrows indicate segregated nucleoli, broken arrows indicate speckles), suggesting that pol I transcription and/or ribosome biogenesis is not a prerequisite for the NHPX pathway. Similarly, the immunosuppressant rapamycin, which inhibits transcription of a subset of ribosomal protein genes and hence ribosome assembly, gave the same results (unpublished data). However, when RNA pol II transcription was inhibited, either by -amanitin or DRB, progression of the newly synthesized NHPX from speckles to the nucleolus was blocked (Fig. 10 C, arrows indicate nucleoli, broken arrows indicate speckles; unpublished data). This suggests that one or more factors must be continually synthesized to allow the newly imported NHPX to move from speckles to nucleoli.
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Discussion |
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Our recent proteomic analysis of nucleoli isolated from cultured HeLa cells shows that they contain >270 different proteins (Andersen et al., 2002). So far there has been no nucleolar targeting motif identified common to all of these factors and it seems likely that multiple, parallel nucleolar localization pathways can operate. Nonetheless, analyses of proteins that show a steady-state accumulation in nucleoli have shown that they usually move rapidly into the nucleolus when they enter the nucleus. This is illustrated here by the rapid nucleolar accumulation of EYFPFIB when it is transiently expressed in vivo (Fig. 4). The finding that newly expressed NHPX accumulates in nuclear speckles transiently before accumulating specifically in the nucleolus defines a new localization pathway for nucleolar proteins. It is interesting to compare this with the recently reported pathway for nucleolar localization of snoRNAs, which showed that multiple snoRNAs accumulate in CBs prior to nucleoli upon initial entry into the nucleus (Narayanan et al., 1999a, 1999b; Verheggen et al., 2001). However, none of the snoRNAs showed a transient accumulation in speckles, consistent with our finding that the snoRNP protein FIB also does not transiently accumulate in speckles prior to nucleoli (Fig. 4). We also observed that NHPX localizes to CBs as well as speckles upon its initial entry into the nucleus, but unlike its transient association with speckles, NHPX is also detected in CBs at later stages of expression when the bulk of the protein is concentrated in nucleoli. At present, we cannot distinguish whether NHPX accumulates in CBs prior to speckles, or in both structures at the same time. However, the CB association does not appear to be obligatory for the NHPX localization pathway because a similar transient association with speckles prior to nucleolar accumulation is observed in cell lines lacking prominent CBs (Fig. 3 B). However, similar molecular events may occur either within the nucleoplasm or in CBs that are too small to detect.
It is also interesting to compare the NHPX pathway with the recently identified nuclear pathway for splicing snRNPs where FP-tagged snRNP Sm proteins accumulate in CBs, and nucleoli, prior to speckles, upon their initial nuclear entry (Sleeman and Lamond, 1999a; Sleeman et al., 2001). Therefore, this pathway appears to be complementary to that of NHPX. By analyzing heterokaryons formed between separate stable HeLa cell lines expressing EYFPNHPX and ECFPSmB, we could show that both these complementary pathways can operate simultaneously within the same nuclei (Fig. 6). These data confirm the specificity of the pathways and highlight the dynamic mechanisms operating to organize the distribution of proteins and RNPs in the nucleus. The results also point to the localization specificity of the separate subnuclear bodies, including nucleoli, CBs, and speckles, although they are not enclosed by membranes.
In order to answer why NHPX shows the observed transient accumulation in speckles prior to nucleoli, it may be important to consider that it is specifically newly expressed and imported NHPX protein that is detected in speckles. Several experiments showed that NHPX does not localize to speckles by default, and that the nucleolar pool of NHPX does not cycle continually to and from speckles. For example, in micronuclei that lack NOR-containing chromosomes (and hence do not have nucleoli), NHPX does not accumulate back in speckles or colocalize with splicing factors (Fig. 8). FLIP photobleaching experiments also showed that whereas nucleolar EYFPNHPX can exchange rapidly between separate nucleoli within the same nucleus, little or no exchange occurs with the pool of NHPX in speckles (Fig. 9). This contrasts with the behavior of the nucleolar protein PSP1, which was recently shown to cycle continually between nucleoli and paraspeckles (Fox et al., 2002).
Our data strongly indicate that the association of NHPX with speckles is a temporal phenomenon linked to the entry of newly expressed NHPX into the nucleus. For example, transient expression of EYFPNHPX in HeLa cells expressing ECFPNHPX, which already accumulated in nucleoli, shows a transient accumulation of the EYFPNHPX in speckles before it later colocalizes quantitatively with the existing nucleolar ECFPNHPX (Fig. 7). Also, when the HeLaEYFPNHPX cells undergo mitosis, EYFPNHPX immediately relocalizes to the reforming nucleoli during telophase and does not accumulate in speckles in the postmitotic nuclei (Fig. 2). Therefore, the speckles association is not a result of nuclear import of NHPX per se, but rather relates to an effect specific for newly expressed protein. We propose that a likely explanation for this behavior of NHPX could be related to it having a function required for the assembly or maturation of some form of nuclear protein or RNP complex, prior to its subsequent stable association with U3 and/or other nucleolar snoRNPs. This could imply either that the affinity of NHPX for different target RNAs changes after it enters the nucleus for the first time or that its access to bind snoRNA targets is initially restricted.
Based upon the results of previous biochemical studies on the structure and binding specificity of NHPX, the U4 snRNA is a possible candidate target for NHPX in speckles. NHPX binds U4 snRNA in vitro via the 5' stem loop sequence (Nottrott et al., 1999; Vidovic et al., 2000). Consistent with this idea, U4 snRNA has been localized to speckles in HeLa cells by hybridization experiments (Carmo-Fonseca et al., 1992; Fig. 5). The fact that we show here that EYFP-NHPX likely interacts in vivo with a form of U4 snRNA that is not stably associated with U6 snRNA suggests that NHPX may transiently interact in speckles with an immature form of U4 snRNP (Fig. 1 E). The observed requirement for gene expression in order for NHPX to move from speckles to nucleoli might reflect a requirement for other factors to be expressed to allow NHPX to complete its transient role in speckles (Fig. 10). Whether this is connected to U4 snRNP assembly and/or some other events remains to be established. Future studies will aim to analyze further the molecular mechanism involved in the novel nucleolar localization pathway detected for NHPX and to establish what biological role this may play.
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Materials and methods |
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Cell culture, transfection, and establishment of stable cell line
HeLa, MCF7, HEK293, and primary fibroblast htert1787 were grown in DME supplemented with 10% FCS and 100 U/ml penicillin and streptomycin (Invitrogen). 2 µg plasmid construct per 100-mm dish was used for transfection using Effectene (QIAGEN) according to the manufacturer's instruction. EYFPNHPX and EYFPFIB stable cell lines were generated essentially as described in Sleeman et al. (2001). ECFPSmB stable cell line (CFPSmBE8.8) was previously described (Sleeman et al., 2001). EGFPH2B stable cell line was a gift from T. Kanda (The Salk Institute for Biological Studies, La Jolla, CA) (Kanda et al., 1998). Drug treatments were carried out as follows: Actinomycin D (0.04 µg/ml, 3 h; Sigma-Aldrich); DRB (100 µM, 4 h; Sigma-Aldrich); -amanitin (40 µM, 4 h; Sigma-Aldrich); and colcemid (0.5 µg/ml, 31 h; Sigma-Aldrich).
FACS analysis
Parental HeLa and HeLaEYFPNHPX cells were harvested by trypsinization, and fixed in 70% ethanol for 3 h at 4°C. Cells were stained with PI (25 µg/ml) containing RNase A (100 µg/ml). Fluorescence was measured using a FACScan (Becton Dickinson). Cell debris and fixation artifacts were gated out. Data analysis was done using Cell Quest software (Becton Dickinson).
Immunoprecipitation and immunoblotting
For immunoprecipitation, 100 µg protein G Sepharose (Amersham Pharmacia Biotech) was preincubated with 10 µg anti-GFP antibodies (Roche). Extracts were prepared using nuclear lysis buffer (Sleeman et al., 2001). Extracts were precleared with 100 µg protein G Sepharose and then incubated with antibody-bound protein G Sepharose for 16 h. Beads were then washed three times with the lysis buffer, and bound RNAs were released by Proteinase K (2.24 mg/ml) in extraction buffer (0.63% SDS, 26.25 mM EDTA, 26.25 mM Tris-HCl, pH 8.0, 0.28 mg/ml yeast tRNA) for 45 min at 65°C. RNAs were precipitated by adding 7 vol of EtOH/NH4OAc (86% EtOH, 0.57 M NH4OAc), and washed once with 70% EtOH. Northern hybridizations were done by standard procedures (Lamond et al., 1989).
For immunoblotting, protein samples were separated by 412% Bis-Tris gels (Novex), and subsequently transferred onto nitrocellulose membrane using a submarine system (Novex). After blocking with 5% milk powder in PBS+0.05% Tween-20, the membranes were incubated with either rabbit anti-NHPX R86 (1:100; Chang et al., 1999) or mouse monoclonal anti-GFP (1:1,000; Roche), and the bound antibody was then probed using antirabbit HRP (1:2,000; Pierce Chemical Co.) and antimouse HRP conjugate, respectively (1:5,000; Pierce Chemical Co.) in PBS containing 5% milk powder and 0.05% Tween-20, and detected via chemiluminescence with ECL Plus (Amersham Pharmacia Biotech).
Immunostaining and 2'-O-methyl RNA hybridization
Cells grown on coverslips were washed in PBS and fixed for 10 min in 3.7% (wt/vol) paraformaldehyde in CSK buffer (10 mM Pipes, pH 6.8, 10 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 2 mM EDTA) at RT, permeabilized with 1% Triton X-100 in PBS for 10 min at room temperature, mounted onto glass slides using VectorShield (Vector Lab), and imaged as described below. Antibodies used were anti-FIB monoclonal 72b9 (1:10; Turley et al., 1993); anti-NHPX peptide antibody R86 (1:100; Chang et al., 1999); anti-SC35 monoclonal (1:500; Sigma-Aldrich); anti-coilin 204/10 (1:300; Bohmann et al., 1995); anti-B23 (1:75; Santa Cruz Biotechnology); and TRITC-, Texas red, and Cy5-conjugated secondary antibodies (Jackson Laboratories). Before being mounted on slides, coverslips were incubated with 1 µM DAPI (Sigma-Aldrich) for 30 s to stain DNA. Fluorescence microscopy of fixed cells was carried out using a 100x NA 1.4 Plan-Apochromat objective. Three-dimensional images and sections were recorded either on a LSM410 Confocal microscope (ZEISS) or on a Zeiss DeltaVision Restoration microscope (Applied Precision, Inc.). Images presented here are maximal intensity projections of the entire nuclear fluorescence.
For 2'-O-Methyl RNA hybridization (Carmo-Fonseca et al., 1992), cells were permeabilized with 0.5% Triton X-100 in CSK buffer containing Complete (Roche) on ice for 3 min, and were then fixed in freshly prepared 3.7% paraformaldehyde in CSK buffer for 10 min at room temperature. Cells were washed three times in PBS, one time with 6 x SSPE, and prehybridized with 6 x SSPE/5 x Denhardt's solution containing yeast tRNA (0.5 mg/ml) for 15 min. Cells were then hybridized with the same buffer with biotinylated 2'-O-methyl antisense oligonucleotide probe (2 µM) for 30 min, and then were washed three times with 6 x SSPE and rinsed with avidin wash buffer (0.03 M Hepes, pH 7.9, 0.15 M KCl, 0.05% Tween-20, 1% donkey serum) before incubating with Texas redconjugated avidin DCS (Vector Labs) at 2 µg/ml for 30 min. They were then washed and mounted on slides for microscopic studies as above. (U3 probe 1011: 5' *C*CUUUCGGUGCUC*C*C 3'; U4 probe 1012: 5' *C*CUGCCACUGCGCAAAGCU*C*C 3'; * denotes biotinylated sites).
Mitotic studies of living cells
Cells were grown on 40-mm glass coverslips (Intracel) in medium containing 200 µg/ml G418. Cells were maintained at 37°C by use of a closed-system perfusion chamber (Bioptech) in DME media (20 mM Hepes, without Phenol red; Invitrogen). Images were collected using the 100x NA 1.4 Plan-Apochromat objective on the Zeiss-DeltaVision Restoration microscope. For each nucleus, 2030 optical sections in the z-axis were recorded. The Hg lamp was attenuated with a 0.5-OD neutral density filter, and images were recorded every 3 min over a time period of 2 h (3 x 3 binning). Time-lapse images were viewed as maximal intensity projections of each time point (SoftWoRx; Applied Precision, Inc.).
Microinjection and heterokaryon formation
For microinjection, pAL107 was diluted to 20 µg/ml with injection buffer (100 mM glutamic acid, pH 7.2 [with citric acid], 140 mM KOH, 1 mM MgSO4, and 1mM DTT) prior to injection into living cells using an Eppendorf 5242 microinjector. For heterokaryon formation, two different cell lines expressing fluorescent proteins were mixed in a ratio of 1:1 in 100-mm diameter petri dishes containing coverslips and cultured until 8090% confluent. The culture medium was drained, and 1.5 ml of 50% Polyethylene Glycol (PEG hybrimax; Sigma-Aldrich) was added. The dishes were rocked gently for 90 s and washed thoroughly by several changes of fresh culture medium (a modification of Sleeman et al. 2001).
Photobleaching analysis
Cells were grown on 42-mm glass coverslips (no. 1; Helmut Sauer) in medium containing 200 µg/ml G418. Cells were maintained at 37°C by use of a closed perfusion chamber (Bachofer) in DME media (20 mM Hepes, no Phenol red; Invitrogen). Photobleaching experiments were carried out on a Zeiss 510 confocal laser scanning microscope equipped with an argon-krypton laser (ZEISS). The 488-nm laser and a 63x plan Apolens with a 1.4 NA and a laser power of 2.5% was used for image acquisition, and 25% was used for photobleaching. An area of 16 x 16 pixels was bleached with an iteration of 250 (duration of bleach was 3 s). An image was collected after every bleaching event, with 20-s intervals between each bleaching event over a period of 15 min. To locate splicing speckles in vivo, plasmid pDsRED-U1A was transfected into the cells 2436 h before imaging. Speckles were defined by red fluorescence, whereas nucleoli were defined by both phase contrast and the absence of U1A.
Online supplemental material
Fig. S1 (available at http://www.jcb.org/cgi/content/full/200201120/DC1) depicts immunofluorescence labeling of HeLa cells using anti-NHPX antibodies either with (A) or without (B) transient transfection with pAL107EYFPNHPX. Panel A is identical to Fig. 1 B in the text.
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Footnotes |
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Acknowledgments |
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This work was supported by the Wellcome Trust. A.I. Lamond is a Wellcome Trust Principal Research Fellow. A.K.L. Leung is a Croucher Foundation Scholar (HK) and ORS awardee (UK).
Submitted: 28 January 2002
Revised: 20 March 2002
Accepted: 22 March 2002
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References |
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