Article |
Address correspondence to Joseph G. Gall, Dept. of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210. Tel.: (410) 554-1217. Fax: (410) 243-6311. E-mail: gall{at}ciwemb.edu
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Abstract |
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Key Words: coilin; diffusion; FRAP; TATA-binding protein; U7 snRNA
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Introduction |
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Targeting of epitope-tagged molecules to CBs provides evidence that some components may transit through CBs on their way to sites of transcription in the nucleus (Gall et al., 1999; Narayanan et al., 1999; Gall, 2000). Similarly, in vivo visualization of CB components tagged with GFP suggests that some molecules localize to CBs as a primary step in RNP maturation (Sleeman et al., 1998; Sleeman and Lamond, 1999; Verheggen et al., 2001, 2002). Such studies imply that CBs play an active role in RNA biogenesis, but leave open the question of how individual CB components behave at steady state.
The application of photophysical techniques to live-cell imaging has transformed our understanding of nuclear dynamics. In particular, FRAP and related techniques (Axelrod et al., 1976; Dewey, 1991) have made it clear that many proteins rapidly and continuously enter and exit nuclear organelles, even though their steady-state distributions give the impression that they might be stable components of these organelles (Misteli et al., 1997; Phair and Misteli, 2000, 2001; Snaar et al., 2000; Chen et al., 2002; Dundr et al., 2002).
In this work, we extend the analysis of CB structure and function by observing the steady-state behavior of three fluorescently labeled CB components: U7 small nuclear RNA (snRNA), coilin, and TATA-binding protein (TBP). First, we demonstrate that exchange of CB components between the nucleoplasm and CBs is slow compared with their mobility in the nucleoplasm. Second, we find that the rate of fluorescence recovery in CBs is independent of the bleach spot diameter and is multi-phasic. Finally, we show that CBs are not appreciably more viscous or dense than the surrounding nucleoplasm. These data allow us to rule out diffusion as the rate-limiting factor in the movement of these three components in and out of CBs. Computer analyses of our FRAP data are most consistent with models in which each molecule exists in three distinct kinetic states within CBs. We conclude that CBs maintain their steady-state concentration of specific factors by selective, multi-state binding.
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Results |
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The U7 snRNP is involved in the maturation of histone pre-mRNAs. After being transcribed and exported to the cytoplasm, U7 snRNA receives a trimethylguanosine (TMG) cap and forms a complex with Sm- and snRNP-specific proteins before it is reimported into the nucleus (Birnstiel and Schaufele, 1988; Mattaj, 1988; Smith et al., 1991; Pillai et al., 2001). In the Xenopus GV, endogenous U7 snRNA is localized primarily in the CBs (Wu and Gall, 1993; Wu et al., 1996). Here, we show that fluorescein-U7 that has been injected into the oocyte cytoplasm, like endogenous U7, can be immunoprecipitated from GV extracts by mAb Y12 (Fig. 1 A). Y12 recognizes a symmetrical dimethylarginine epitope found on several Sm proteins and on coilin (Brahms et al., 2001; Hebert et al., 2002). Fluorescein-U7 that has been recovered from injected oocytes migrates at the expected molecular weight on a Northern blot, and shows little sign of degradation after prolonged incubation inside cells (Fig. 1 B). Finally, when fluorescein-U7 snRNA is injected into the oocyte cytoplasm, and GVs are subsequently isolated for cytological analysis, one sees brightly fluorescent CBs against a low level of nucleoplasmic staining (Fig. 1 C). In contrast, when fluorescein-U7 is injected directly into oil-isolated GVs, fluorescence is barely detectable in CBs, even after overnight incubation (Fig. 1 C). These results suggest that under standard conditions of cytoplasmic injection, fluorescein-U7 transcripts associate properly with Sm proteins in the cytoplasm before they enter the nucleus, where they are stable. Fluorescein-U7 snRNPs are then concentrated in CBs in a pattern that is indistinguishable from that of the endogenous snRNP.
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The slow fluorescence recovery inside CBs cannot be explained by high CB viscosity
The slow rate at which photobleached U7, coilin, and TBP recovered inside CBs relative to the nucleoplasm is consistent with the idea that these molecules bind transiently to other molecules within CBs. However, it remains a formal possibility that fluorescence recovery within CBs might be impeded by high internal viscosity.
According to the Stokes-Einstein equation, the diffusion coefficient of a molecule is inversely proportional to the viscosity of the medium in which it diffuses. Therefore, other experimental parameters being equal, high internal viscosity could contribute to the slow FRAP inside CBs relative to the nucleoplasm. To address this issue, we performed FRAP on fluorescein-UTP, which is neither excluded from CBs nor concentrated in them relative to the nucleoplasm (Fig. 9 A). The uniform distribution of fluorescein-UTP presumably results from its ability to penetrate CBs without appreciably interacting with CB components. We reasoned that if the internal milieu of CBs is more viscous than the nucleoplasm, then after photobleaching, fluorescein-UTP within CBs should recover more slowly than fluorescein-UTP in the nucleoplasm.
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For purposes of illustration, we performed the same experiment with the confocal microscope. The high intensity pulse of 488-nm light also bleaches Alexa 546U7, even though 488 nm is well below the absorption peak of the Alexa dye. Recovery of fluorescein-UTP in both the nucleoplasm and the CB occurred before the first postbleach image was taken (Fig. 9 A). Because the recovery of photobleached U7 takes minutes, we could confirm that the CB had been photobleached by observing the red fluorescence of U7 (Fig. 9 B). We could also confirm by focusing that the bleached area lay completely within the CB.
The slow fluorescence recovery inside CBs cannot be explained by high CB density
The diffusion of molecules in subcellular compartments is influenced by additional parameters not considered in the Stokes-Einstein equation (Kao et al., 1993; Seksek et al., 1997). One such parameter is molecular crowding, reflected in the physical density of the compartment under consideration. For example, CBs might consist of a dense meshwork of molecules whose effective pore size limits the free movement of incoming macromolecules. Such a network could, in theory, explain the slow FRAP we observe for fluorescein-U7, GFP-coilin, and GFP-TBP inside CBs. The density of CBs and other nuclear organelles can be estimated by measuring their refractive index with an interferometer microscope (Davies, 1958; Hale, 1958). Such measurements are underway in our laboratory and will be reported in a separate publication. However, simple phase contrast and differential interference contrast observations on oil-isolated nuclei show that CBs are barely visible when surrounded by nucleoplasm, whereas nucleoli and B-snurposomes display higher contrast (Fig. 4, A and B). Because phase contrast and differential interference contrast both depend on differences in refractive index between the specimen and the medium in which it is observed, we can conclude that the refractive index of CBs, and hence their protein concentration, does not differ by more than a few percent from that of the nucleoplasm. A small difference in protein concentration could not account for the >100-fold difference in mobility that we observe for CB components in CBs relative to the same components in the nucleoplasm.
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Discussion |
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In contrast, a mature oocyte nucleus of Xenopus is 400 µm in diameter. The vast majority of its volume lacks resolvable structure at the light microscope level, and thus can be characterized as nucleoplasm. The remaining volume contains the 18 lampbrush chromosomes plus thousands of nuclear organelles that are readily discernible with transmitted light. A single GV can contain up to 100 CBs with diameters in the range of 210 µm. Because of its large size, a GV can be isolated by hand, either in an appropriate saline solution or in oil. Oil-isolated Xenopus GVs maintain energy-dependent functions for many hours after removal from the cytoplasm (Lund and Paine, 1990; Paine et al., 1992; Yu et al., 1998). Therefore, this system is useful for studying various aspects of nuclear biochemistry, such as RNA splicing and modification. Here, we have used oil-isolated GVs to follow the in vivo steady-state dynamics of CB components in both CBs and nucleoplasm by FRAP. The same system should be equally valuable for studying other nuclear organelles, such as nucleoli, B-snurposomes, and lampbrush chromosomes.
CB components are freely diffusible in the oocyte nucleoplasm
The translational mobility of a solute in a complex macromolecular mixture is influenced by the fluid-phase viscosity, collision between the solute and macromolecular obstacles, and binding of the solute to other macromolecules (Kao et al., 1993). The contribution of each of these parameters to the mobility of molecules in bulk cytoplasm, cytoplasmic organelles, and membranes has been analyzed extensively because the physical structure of these compartments is thought to impose significant restraint on the rates of biochemical reactions within and between compartments (Kao et al., 1993; Seksek et al., 1997; Dayel et al., 1999). These same parameters undoubtedly influence translational mobility in the nucleus. Here, we have addressed the contribution of these parameters to the mobility of CB components in CBs and the nucleoplasm.
Recent FRAP measurements on GFP-constructs now suggest that various proteins move freely within the nucleoplasm and also exchange between the nucleoplasm and subnuclear compartments, such as the nucleoli, CBs, and splicing factor compartments (speckles; Misteli et al., 1997; Snaar et al., 2000; Carmo-Fonseca et al., 2002; Chen et al., 2002; Dundr et al., 2002). In the nucleoplasm of mammalian cultured cells, the apparent diffusion coefficient for three GFP-labeled nuclear proteins (HMG-17, SF2/ASF, and fibrillarin) ranged from 0.24 to 0.53 µm2 sec-1 (Phair and Misteli, 2000). Likewise, Politz and colleagues (1999) used a caged fluorescent oligonucleotide hybridized to poly(A) to show that bulk poly(A) RNA had a diffusion coefficient of 0.6 µm2 sec-1 in rat myoblast nuclei. Our values for fluorescein-U7, GFP-coilin, and GFP-TBP in the GV nucleoplasm are similar, 0.260.40 µm2 sec-1.
Earlier measurements of FITC-dextran in the nucleoplasm of cultured mammalian cells showed more rapid diffusion with D values ranging from 2.5 to 10 µm2 sec-1 for molecular weights in the range of 70 kD2 MD (Seksek et al., 1997). It will be interesting to measure FITC-dextran in the GV to determine whether these differences are related to the type of nucleus or to the nature of the molecules.
Reaction limited mobility of CB components in CBs
Fluorescein-U7, GFP-coilin, and GFP-TBP within CBs differ in at least two important respects from the same molecules in the nucleoplasm. First, the steady-state concentration in each case is much higher within the CB than outside. Second, the recovery of fluorescence after bleaching is much slower and shows at least three kinetic components. Slow recovery was also found for GFP-fibrillarin in CBs from human osteosarcoma cells in culture (Snaar et al., 2000), suggesting that CBs in oocytes and somatic tissues may have similar kinetic properties.
CBs and other nuclear organelles lack membranes like those that surround many cytoplasmic organelles or the nucleus itself. Thus, the accumulation of CB components against a concentration gradient is not likely to involve specific pumps or channels like those found in cytoplasmic membranes or the nuclear envelope. In the absence of such mechanisms, specific binding is the most probable factor involved in the accumulation of CB components.
In the simplest binding scenario, components might associate with one or more classes of binding sites within CBs and remain there for a time period well beyond what we have measured experimentally. This type of binding might involve storage of macromolecular complexes to be used days or weeks later by the developing embryo, as is the case of yolk granules in the oocyte cytoplasm. This is clearly not the case for U7 snRNA, coilin, and TBP, where fluorescence recovery occurs on a time scale of minutes. Furthermore, we have shown experimentally that when exogenous U7 is introduced into the GV, endogenous U7 exits the CBs (Fig. 7), whereas the concentration of U7 remains the same (Fig. 6). Thus, U7 in CBs is clearly in equilibrium with U7 in the nucleoplasm on a time scale of minutes, and the same is probably true for coilin and TBP.
Given that reversible binding best explains the data for U7, coilin, and TBP, we can turn to a more detailed quantitative analysis of the FRAP curves. As discussed in the supplemental material, a good fit of the data for each of these molecules requires at least three exponential terms. That is, the data resolve three states for each protein or RNP within the CB; one that exchanges with t1/2 15 s, a slower one with t1/2
37 min, and a very slow component with t1/2
35 min.
We used computer modeling to determine how these three RNP/protein states might be arranged and interconnected in a functional pathway (Phair and Misteli, 2001). A simple hypothesis is that each state represents one stage in the sequential assembly of a macromolecular complex (Fig. 10 A). In any sequential model, the state with the slowest turnover must occupy the largest fraction of the total population of molecules. Our computer analyses indicated that this is not true for any of the three molecules tested in this study. Therefore, we can rule out the linear assembly model for these molecules.
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Further biochemical experiments will be required to test the model in Fig. 10 B. However, we feel confident that the slow compartments exist and are significant features of CB biology. Because enzymes that perform post-transcriptional and post-translational modifications occur in CBs, and because particles comparable in size to ribosomes are present in CBs (Gall et al., 1999), it is tempting to speculate that the slow states we observe in the FRAP experiments represent modification and/or assembly events that occur inside CBs.
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Materials and methods |
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Plasmids and transcripts
Human TBP cDNA was amplified by PCR from a vector containing the full-length coding sequence with the following primers: upstream, 5'-GCGACGCCGATCGATATGGATCAGAACAACAGCCTGCC-3'; downstream, 5'-GCGACGCCGATCGATGCGTCGTCTTCCTGAATCCC-3'. The underlined sequences are ClaI restriction sites. Amplified products were cloned into the pCRII vector (Invitrogen), grown in Dam- Escherichia coli SCS110, purified by mini-prep (QIAGEN), and then digested with ClaI. The fragment containing the TBP cDNA was gel purified and ligated to ClaI-digested pCS2(GFP) vector (Huang et al., 1999) upstream of the GFP open reading frame. Xenopus wild-type U7 snRNA construct no. 401 (Wu et al., 1996) was linearized with SalI and transcribed with T7 polymerase to generate sense-strand molecules for injection. The Xenopus GFP-coilin construct (Handwerger et al., 2002) was provided by Z. Wu (Carnegie Institution). Transcripts of fluorescein-U7 snRNA, GFP-coilin mRNA, and GFP-TBP mRNA were synthesized in vitro as described previously (Handwerger et al., 2002).
Microinjections and incubations
Needles were pulled from capillary tubing (0.5-mm inner diameter, 1.2-mm outer diameter) with a vertical pipette puller (David Kopf Instruments). Approximately 1 ng of fluorescein-U7 snRNA, GFP-coilin mRNA, or GFP-TBP mRNA was microinjected in a volume of 9.223 nl into the cytoplasm of 1-mm diam oocytes, using a dissecting microscope in combination with a Nanoject microinjection apparatus (Drummond Scientific). Cells were held for a maximum of 48 h at 18°C in OR2 medium before GV preparations were made.
RNA isolation and Northern blots
GVs were isolated from oocytes and stored for up to 1 h on ice in 100 µl isolation medium (83 mM KCl, 17 mM NaCl, 6.5 mM Na2HPO4, 3.5 mM KH2PO4, 1 mM MgCl2, and 1 mM dithiothreitol, pH 7.0). RNA was extracted and precipitated as described previously (Gall et al., 1999). Samples were electrophoresed on a 12% polyacrylamide/8M urea/TBE gel at 35 mA for 1.5 h. Northern blots and UV cross-linking were performed as described previously (Bellini and Gall, 1998) with probes of antisense U7 snRNA (106 cpm/ml; Wu and Gall, 1993) and U6 snRNA (2.5 x 103 cpm/ml; Wu et al., 1991). Blots were exposed to a PhosphorImager screen, and the resultant images were scanned with a Storm 860 detector and software (Molecular Dynamics, Inc.).
Protein isolation, protein gels, immunoprecipitation, and Western blots
GVs were isolated from oocytes and stored for up to 1 h on ice in isolation medium. GVs were mixed with an equal volume of 2x gel buffer (Laemmli, 1970) and loaded onto a 10% SDS-PAGE mini-gel (Bio-Rad Laboratories). Gels were run for 1 h at 30 mA and blotted onto Immobilon-P nylon filter (Millipore) at 50 V for 4 h at 4°C. Blots were briefly immersed in methanol, rinsed in transfer buffer, and blocked overnight at 4°C in 5% nonfat dry milk in PBS. Membranes were washed three times for a total of 30 min in 0.05% Tween 20 in PBS and incubated for 1 h at RT in primary antibody. Blots were washed and incubated as above with secondary antibody that was either alkaline phosphataselinked goat antirabbit IgG (1:1,000) in 0.025% Tween 20 in PBS, or goat antimouse IgG (1:10,000; Amersham Biosciences). After washing, the membrane was exposed to developer according to the manufacturer's protocol (ECF kit; Amersham Biosciences). An image of the chemifluorescence was obtained with the Storm 860 detector and software (Molecular Dynamics, Inc.).
Immunofluorescence and quantitation
Nuclear spreads were prepared as described previously (Gall et al., 1999). The resulting slides were blocked for 10 min in PBS containing 0.2% cold water fish gelatin (Sigma-Aldrich), 5% BSA, and 0.02% sodium azide. GV spreads were incubated with 10 µl of 1 µg/ml mAb K121 (Oncogene Research Products) for 1 h at RT. Slides were washed 35 min with PBS and incubated for 1 h with 10 µl of 1 µg/ml Alexa 488labeled goat antimouse IgG (Molecular Probes, Inc.). The slides were again washed with PBS and mounted in 50% glycerol containing 1 mg/ml 1,4-diaminobenzene to retard fading. Images of fluorescent CBs were captured on a CCD camera (MicroMax; Princeton Instruments) and stored as IP Lab Spectrum files (Scanalytics) or as TIFF files in Adobe Photoshop®. mAb Y12 was provided by J. Steitz (Yale University, New Haven, CT), and mAb H1 was provided by R. Tuma and M. Roth (Fred Hutchinson Cancer Research Center, Seattle, WA).
Preparation of oil-isolated GVs
Single oocytes that had been injected with fluorescein-U7, GFP-coilin mRNA, or GFP-TBP mRNA were transferred from OR2 saline to a piece of Whatman #1 filter paper (Whatman, Ltd.). Most of the aqueous medium surrounding each oocyte was absorbed by the filter paper. The oocyte was then transferred to a 35-mm Petri dish containing mineral oil (EC No. 2324558; Sigma-Aldrich) and the GV was removed manually with jeweler's forceps (Lund and Paine, 1990; Paine et al., 1992). Individual GVs were transferred to a standard 3" x 1" microscope slide in 5 µl of oil and were gently squashed under a 22-mm2 glass coverslip. After the oil had spread to the edges of the coverslip by capillary action, the sample was observed in the microscope.
FRAP of CBs
For confocal FRAP, oil-isolated GVs were prepared as above. Imaging and bleaching were conducted on a laser scanning confocal microscope (TCS SF2; Leica) using a 100x, NA 1.4 oil immersion objective. Pre- and postbleach images were collected with the 488-nm line of an argon laser set at minimal output. Each consisted of a single optical section through the center of a CB (512 x 512 pixels; 2x frame-averaged; 25% gain of the acousto-optical tunable filter). A diffraction-limited spot was photobleached inside the CBs by applying full intensity of the beam for 500 ms without scanning. After bleaching, images were scanned every 15 s for 45 s, then every minute until there was no visible difference in fluorescence between the bleached and nonbleached areas (typically 2030 min). The average pixel intensity within bleached and unbleached areas was quantified with MetaMorph® software (Universal Imaging Corp.). All bleach spot values were corrected for the amount of bleaching that occurred during image acquisition and were then normalized to the prebleach value.
FRAP measurements in the nucleoplasm
Oil-isolated GVs from injected oocytes were prepared for microscopy as above. A fluorescence microscope (Axioplan; Carl Zeiss MicroImaging, Inc.) fitted with a Plan-Neofluar objective (100x oil, NA 1.30) was used to select an area of the GV that was devoid of nucleoli, B-snurposomes, and CBs. An attenuated beam from a 488-nm argon laser was focused on an 1.0-µm spot of nucleoplasm, and the baseline fluorescence intensity was measured with a photomultiplier tube. The sample was then bleached with full laser intensity for 20 ms. Recovery of fluorescence was recorded every 50 ms with the attenuated beam over a period of 1520 s and analyzed with custom software (M. Edidin, Johns Hopkins University, Baltimore, MD). To display the recovery series, raw data points were exported to Microsoft Excel software.
Online supplemental material
The online supplemental material gives details on the compartment models and on fitting the FRAP curves to a sum of exponentials. The procedure for wide-field FRAP is also given. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200212024/DC1.
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Footnotes |
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K.E. Handwerger's present address is Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142.
* Abbreviations used in this paper: CB, Cajal body; GV, germinal vesicle; snRNA, small nuclear RNA; TBP, TATA-binding protein; TMG, trimethylguanosine.
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Acknowledgments |
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This work was supported in part by Research Grant GM33397 from the National Institute of General Medical Sciences of the National Institutes of Health (to J.G. Gall). J.G. Gall is American Cancer Society Professor of Developmental Genetics.
Submitted: 3 December 2002
Revised: 7 January 2003
Accepted: 8 January 2003
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