EXPERIMENTAL
CELL
RESEARCH
195,
27-37
(1991)
Immunological and Ultrastructural Studies of the Nuclear Coiled Body with Autoimmune Antibodies IVANRASKA,~ Lu1sE.C. ANDRADE,ROBERT L. OCHS,EDWARD K.L. CHENG-MINGCHANG,G~RAN ROOS,’ ANDENG M. TANS W. M. Keck Autoimmune
Disease
Center,
Scripps
Clinic
and Research
Foundation,
10666
North
Torrey
Pines
CHAN,
Road,
La Jolla,
California
92037
storage, and degradation of various RNAs [4]. In addition, it seemslikely that the transport of material to and from the nucleolus occurs, at least in part, through the interchromatin space. However, information about the relationships between these different metabolic processes and structural organization in the interchromatin space is lacking. In order to gain deeper insight into structure-function relationships in the cell nucleus, probes defining specific structural domains within what may seem to be a homogeneous looking cell nucleus are of increasing importance. Specific antibodies have been employed very effectively using immunocytochemical approaches (e.g., [5]). However, it is often difficult to prepare specific sera or monoclonal antibodies against nuclear macromolecules since nuclear structures usually do not exhibit significant evolutionary differences and the relevant macromolecules are often present in low copy number [6]. On the other hand, high titer autoantibodies against a great variety of nuclear antigens can be found in sera of patients with autoimmune disorders, in particular systemic rheumatic diseases [7]. In this study, a novel autoantibody probe for an 80-kDa nuclear protein present in the coiled body, named p80coilin, is documented.
Studies with human autoimmune sera identified autoantibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function. (‘ 1991 Academic Press, Inc.
INTRODUCTION In the interior of the cell nucleus, only a limited number of morphologically well-defined structural components are found. Besides chromatin and nucleoli, four other categories of structures have been described in the remaining interchromatin space. They are perichromatin fibrils, perichromatin granules, interchromatin granules, and nuclear bodies [l, 21. In the category of nuclear bodies, only simple nuclear bodies and coiled bodies are regularly found [3]. Functionally, it has been assumed that all nuclear metabolism related to RNA polymerase II and III transcripts occurs in the interchromatin space by processes which include splicing, polyadenylation, transport,
MATERIALS
AND METHODS
Cells and tissues. Human HeLa S3, WI 38, MOLT-4, HEp-2, rat NRK, mouse 3T3, and marsupial PtK2 cell lines were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal calf serum and gentamicin. Commercial preparations of HEp-2 cells were obtained from Bion (F’ark Ridge, IL). Human lymphocytes were isolated from the blood of a healthy donor by means of Histopaque 1077 (Sigma). Mouse kidney and stomach sections were obtained from Kallestad Lab (Austin, TX). Fresh mouse liver imprints were made on 14.mm-round coverslips. Five-micrometer-thick cryosections of mouse and rat brain were prepared from paraformaldehyde (perfusion followed by immersion fixation)fixed tissue. Cell extracts and immunoblotting. Cultured cells were harvested by centrifugation, resuspended, and lysed in SDS-gel sample buffer
’ Present address: Institute of Experimental Medicine, Czechoslovak Academy of Sciences, Albertov 4,128 00, Prague, Czechoslovakia. ’ Present address: Clinical Cytology Laboratory, University Hospital, 90185 Umea. Sweden, ” TO whom reprint requests should be addressed.
25 Copyright All
rights
cc: 1991 of reproduction
hy Academic Press, Inc. in any form reserved.
28
RASKA
[8]. Whole cell lysates were separated on 12.5% SDS-PAGE gels according to Laemmli [8]. Separated proteins were transferred to nitrocellulose at 60 V for 150 min at 4OC. Nitrocellulose sheets were cut into strips and blocked in 3% nonfat milk in phosphate-bufIered saline (PBS) containing 0.05% Tween 20 (PBS-T buffer) for 30 min and then incubated for 1 h with different sera diluted loo-fold in the same blocking solution. ‘2”I-labeled protein A (ICN Biochemicals, Irvine, CA) was used as the secondary detection reagent. Protein standards (Bio-Rad, Richmond, CA) were phosphorylase B, BSA, ovalbu min, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme corresponding to 92.5, 66.2, 42.7, 31, 21.5, and 14.4 kDa, respectively. Antisera and antibodies. Sera from 20 patients with diverse autoimmune conditions exhibited a distinct fluorescent pattern consisting of brightly stained round bodies (usually less than six) in nuclei of HEp-2 cell preparations [Andrade et al., in preparation, 19911. All these sera were shown to contain anti-p80-coilin autoantibodies (see below). The titer of sera by immunofluorescence microscopy varied from 1:640 to 1:40,960. Other antisera employed in these studies included monoclonal antibody against bromodeoxyuridine (BrdU) (Becton-Dickinson, Basel, Switzerland), monoclonal antibodies against DNA of IgG class [9] and IgM class (Boehringer-Mannheim, Germany), anti-Sm monoclonal antibody and human autoantibody against 5s rRNP [lo, II] (kindly provided by Dr. 3. A. Steitz and Dr. T. Yario), anti-U1 RNP monoclonal antibody [la] (kindly provided by Dr. S. Hoch), rabbit antibodies and human autoantibodies against DNA topoisomerase I [13], mouse monoclonal antibodies [14] and human autoantibodies against fibrillarin [15], and human autoantibodies against RNA polymerase I (16,171, rabbit anti-trimethyl guanosine-capped (m,G) RNA antibodies 1181 (kindly provided by Dr. R. Liihrmann), rabbit antibodies against nucleolin [19] (kindly provided by Dr. M. Olson), monoclonal antibodies against protein B23 [20] (kindly provided by Dr. P. K. Ghan), monoclonal antibodies against L protein of heterogeneous nuclear RNP (hnRNP) 1211 (kindly provided by Dr. P. Pinol-Roma and Dr. G. Dreyfuss), monoclonal antibody 3C5 against a component of interchromatin granules [22] (kindly provided by Dr. B. Turner), and monoclonal antibody SC35 against a component of spliceosomes [23] (kindly provided by Dr. X.-D. Fu and Dr. T. Maniatis). Afinity purification of antibodies. Affinity purification of antip80-coilin antibodies from nitrocellulose filters was performed according to the method of Olmsted [24]. HeLa cell extract served as the antigen source. After blocking with 3% nonfat milk in PBS-T buffer for 30 min, filters were incubated for 1 h with serum diluted 1:50 in this same buffer and washed with PBS-T. Bound antibodies were eluted by a brief 30-s exposure to 0.1 M phosphate buffer (pH 2.5) containing 0.15 M NaCl and 0.1% BSA, and the solution was immediately neutralized by addition of 1 M Tris-HCI (pH 8.8). The antibodies were concentrated with Centricon 30 microconcentrators (Amicon, Danvers, MA), and stored at 4°C. Processing of cells for light microscopy. Cells grown in monolayers were cultured on 14-mm-round coverslips which enabled easy handling. Cells were washed in PBS and fixed 10 min in 2% paraformaldehyde in 0.12 M phosphate buffer (pH 7.2). After several washes in the same buffer, the cells were permeabilized 5 min with 0.5% Triton X-100 in phosphate buffer or in cold (-20°C) acetone for 1 min. Alternatively, cells were precipitated in cold (~20°C) methanol for 5 min and then for 20 s in cold acetone (~20°C). Cells grown in suspension were cytocentrifuged (Shandon, Astmoore, Cheshire, England) for 5 min at 1000 rpm onto round coverslips placed on microscope slides and processed in the same manner as cells grown on coverslips. Double-label immunofluorescence was performed as described previously [25]. Affinity-purified goat secondary antibodies conjugated to FITC and TRITC (Caltag, San Francisco, CA) were used. Incubation times were usually 30 min for primary antibodies and 25 min for the secondary antibodies. However, in order to obtain an
ET
AL.
acceptable fluorescent signal on formaldehyde-fixed cryosections of rat and mouse brain, overnight incubations of the primary and 2-h incubations of the secondary antibodies were used. Processing of cells for electron microscopy. For conventional electron microscopy, cultured cells were fixed in 2% glutaraldehyde in 0.1 A4 cacodylate buffer for 1 h at 4”C, postfixed in 1% osmium tetroxide for 1 h, and embedded in epon. Thin sections stained with uranyl acetate and lead citrate were observed in a Hitachi HU 12A electron microscope. For immunoelectron microscopy, cells were fixed in paraformaldehyde, infused with sucrose, frozen, thin sectioned, immunolabeled, and postembedded in methylcellulose as described [3, 2.51. Some of the secondary 5-, lo-, and 15nm gold probes (Janssen Pharmaceutica, Beerse, Belgium) were kindly donated by the manufacturer. Extraction and digestion procedures. Cells grown in monolayers were either extracted in 0.1 N HCI or digested with DNase I, RNase A, or trypsin according to the procedure of Fritzler et al. [26]. Acid extraction removed most of the histones leaving behind DNA and residual matrix-bound proteins, while enzyme digestions demonstrated whether antigenic determinants were associated with DNA or RNA. For the preparation of nuclear matrix, the protocol of Turner and Franchi 1221 was followed, which involved consecutive treatments with detergent, nuclease, and high molarity salt.
RESULTS
Human autoimmune sera were initially selected based on their reactivity with discrete round nuclear bodies in immunofluorescence microscopy. The size of the stained bodies usually ranged between 0.3 to 1.0 ym, seldom exceeding 1 pm. Immunoblotting of these selected sera, using extracts of cycling human cells (HeLa, Molt 4) yielded a common reactive band corresponding to 80,000 Daltons. Several of the sera also presented other unrelated reactivities (Fig. 1). The immunofluorescence pattern on cycling interphase mammalian cells consisted of small brightly stained round bodies distributed throughout the cell nucleus (Figs. 2a and 2b). This staining pattern was especially apparent for sera which were judged monospecific by immunoblotting. The overall weak homogeneous nucleoplasmic staining, fine speckled nuclear staining, and in some cases cytoplasmic staining were not observed at higher serum dilutions, The number of stained nuclear bodies was variable in cycling mammalian cells, being usually less than six per nucleus (see below). Affinity-purified antibodies from the 80-kDa band gave essentially the same staining pattern as monospecific autoimmune sera (Figs. 2c and 2d). By electron microscopic immunocytochemistry performed with monospecific autoimmune sera on thawed cryosections, gold particles were highly enriched over coiled bodies (Fig. 4a). This finding was further verified by the use of affinity-purified autoantibodies to the 80kDa antigen (Fig. 4b). Since autoantibodies which blotted only the 80-kDa antigen labeled the coiled body specifically, we have named the 80-kDa antigen of the coiled body “p80coilin”. Since the coiled body is a ubiquitous nuclear structure
AUTOANTIBODIES
AS
PROBES
FOR
FIG. 1. Immunoblotting of human autoantibodies recognizing discrete nuclear bodies. Whole HeLa cell lysate was separated by SDS-PAGE, transferred to nitrocellulose, and probed with different sera. Lane 1, positive control autoimmune serum known to contain anti-Ku antibody which blots bands at 70 and 82 kDa as well as unidentified lower molecular weight bands. Lanes 2 to 5, reactivity of four selected autoimmune sera which exhibit positivity at 80 kDa. Note that one serum (lane 3) gives a monospecific reaction. Lane 6, negative control serum (normal human serum). Lane 7, another human serum positive for the 80.kDa protein. Lane 8, affinity-purified autoantibodies from the 80.kDa band of lane 7. Relative mobilities for molecular weight markers designated by arrows are 92.5, 66.2, 42.7, 31, 21.5, and 14.4 kDa, respectively. The BO-kDa band is indicated by an arrowhead.
(Fig. 3) in both plant and animal cells [l, 3, 27, 281, the presence of p80-coilin was investigated in cells of various origins by light microscopic immunocytochemistry. Positivity could be established for mammalian cell lines of human (HeLa S3, MOLT 4, HEp-2, WI 38), mouse (3T3), rat (NRK-52E), or marsupial (PtK2) origin. P80-coilin was also shown in different tissues: mouse liver cells (Fig. 5), rat and mouse brain cells (Fig. 6), mouse kidney, stomach and muscle cells, and human resting peripheral blood lymphocytes (Fig. 7).
THE
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COILED
BODY
29
In frozen sections of rat and mouse brain, a frequent association between p80-coilin positive staining and nucleoli was noted (Fig. 6). Nucleolus-associated structures staining for p80-coilin usually formed one or several caps at the nucleolar periphery (Fig. 6b). This relationship was investigated in more detail in cycling cells by means of co-localization studies (see below). However, in HeLa cells less than 15% of coiled bodies were found in close association with the nucleolus. Coiled bodies were not evident at mitosis, but were present in the cell during the S phase as monitored by double label immunofluorescence of p80-coilin and incorporated bromodeoxyuridine (Fig. 9). In addition, we did not observe incorporated bromodeoxyuridine in p80-coilin positive bodies (e.g., Fig. 9). The number of coiled bodies found in any particular cell nucleus depended on the cell type. Most cycling cells of human origin exhibited 2 to 5 coiled bodies, with the number of negative cells being less than 10%. In the mouse cell line 3T3, the number of positive structures was not greater than two in most cells, and the percentage of negative cells approached 30%. In PtK2 cells, positive cells usually contained only one coiled body. Human peripheral blood lymphocytes usually possessed only one small weakly stained dot (Fig. 7). A number of additional antigens were localized to the coiled body (Table 1; Figs. 6,8, and 10 to 16). The most striking was the association with fibrillarin (Figs. 6, 8, and 15). In general, fibrillarin staining might be regarded as depicting “true” nucleoli and a number of bodies which, according to some investigators, represent prenucleolar bodies, micronucleoli, or satellite nucleoli (double arrowheads in Fig. 8) [ 15,29,30,31]. This study showed that p80-coilin positive structures are among those nuclear bodies which are stained for fibrillarin (single arrowhead in Fig. 8). P80-coilin positive bodies were also identified in conjunction with staining for Sm, Ul RNP, m,G-capped RNA, and DNA topoisomerase I (Figs. 10 to 12). The staining of these latter antigens gave rise to typical speckled nuclear patterns (e.g., Tan, 1989), and it was observed that some of the most brightly stained speckles costained for p80coilin (Figs. 10 to 12). For example, one of the brightly Sm-stained speckles in Fig. 1Oa was also positive for p80-coilin in Fig. lob. Antigens not detected in the coiled bodies included DNA, nucleolin, nucleolar protein B23, 5s rRNP, hnRNP protein L, a component of interchromatin granules detected by 3C5 antibody, and the 35-kDa spliceosome protein of Fu and Maniatis (Table 1; Fig. 13). Among these, an interesting feature was the staining pattern of the 35-kDa protein of Fu and Maniatis [23], since in many instances a SC35 positively stained body was located in proximity to a p80-coilin-stained body (Fig. 13), but as far as we could determine there was no co-localization.
KASKA
ET
AL.
FIG. 2. (a, h) Phase (a) and immunofluorescence (b) of interphase HEp-2 cells by the same serum as used for Western blots in lane 7 of Fig. 1. Nuclear bodies are brightly stained (arrowheads). The number of stained nuclear bodies per cell ranges from two to four in this figure. Note the heterogeneity in their size. Additional weak nuclear fluorescence in some HEp-2 cells is also observed. x1370. (c, d) Phase (c) and immunofiuorescence (d) of HEp-2 cells stained with autoantibody eluted from the 80-kDa band in Western blottings. Nuclear bodies (arrowheads) are specifically labeled. X1370. FIG. 3. Conventional epon section of a HeLa cell. Coiled body (large arrow) and simple nuclear body (small arrow) are present in the nucleoplasm. Nucleolus, Nu; cytoplasm, Cy. ~20,100. FIG. 4. (a) Immunolocalization of p80-coilin in a cryosection of a HeLa cell with monospecific autoimmune serum. Ten-nanometer gold particles (arrowheads) are enriched in the coiled body (arrow). X24,900. (b) Localization of p80-coilin with affinity-purified autoantibody. Ten-nanometer gold particles (arrowheads) are concentrated over the coiled body. ~65,800.
Immunoelectron microscopy demonstrated that coiled bodies were enriched for m,G-capped RNA (Fig. 14). On the other hand, RNA polymerase I (Fig. 14) and DNA were not detected in coiled bodies. Positivity of coiled bodies for fibrillarin (Fig. 15), Sm, Ul RNP, and DNA topoisomerase I (Fig. 16) was also demonstrated by electron microscopic immunocytochemistry. In order to further characterize p80-coilin, a number of digestion and extraction experiments were per-
formed. Staining for p80-coilin positive bodies was not affected by DNase digestion, but it was diminished by RNase treatment to some extent, and the fluorescence of p80-coilin positive bodies was completely abolished by proteinase K (data not shown). P80-coilin was not substantially extracted by high molar salt, acid, or detergent treatment. In nuclear matrix preparations, the fluorescence of p80-coilin positive bodies was still very prominent (data not shown).
AUTOANTIBODIES
FIG. heads). FIG. and one FIG. indicate
AS PROBES
FOR
THE
NUCLEAR
COILED
BODY
31
5. Mouse hepatocytes stained for p80-coilin with the same serum as in Fig. 2. Fluorescence of coiled bodies is prominent (arrow~1230. 6. Frozen section of rat brain double-stained for tibrillarin (a) and for p80-coilin (b). PSO-coilin-positive structures form one strongly weakly stained cap (arrowheads) associated with the nucleolus. X1840. 7. Isolated human lymphocytes stained for p80-coilin. Coiled bodies (arrowheads) are small and weakly stained. Double arrowheads nonspecific staining of B cell cytoplasm, presumably due to the presence of endogenous human immunoglobulins. ~1230.
DISCUSSION
In the present study we have identified a novel protein which appears to be localized predominantly to the nuclear coiled body. The target antigen is an 80-kDa protein (named p80-coilin) recognized by antibodies in the sera of patients with various autoimmune diseases (Andrade et al., manuscript in preparation). By means of immunoelectron microscopy of interphase mammalian cells, the antigen was shown to be highly enriched in the coiled bodies. Thus, p80-coilin represents a macromolecule which may serve as a marker for these intranuclear bodies, and autoantibodies to p80-coilin may eventually contribute to the understanding of the nature and function of t.his structure. Within the limits of the immunoelectron microscopic method employed, no significant amount of p80-coilin was identified at sites other than the coiled body. However, two points should be kept in mind. First, there is a possibility that p80coilin may be found in other cellular sites at very low levels, beyond the threshold of detection of the techniques employed. Second, the present association between p80-coilin and the coiled body was derived from studies in a limited number of physiological cell states. It is certainly worthwhile to determine if this association holds true under experimental or pathological conditions, such as in hormone-stimulated or virus-infected cells in which other types of nuclear bodies are known to arise [l, 2, 28, 32-401. Historically, the coiled body has been described at the ultrastructural level as a round-to-oval corpuscle 0.5 to 1.0 pm in diameter, consisting of coiled fibrillar strands. Coiled bodies are reported to be located randomly in the nucleoplasm, sometimes in close association with the
nucleolus [l, 2, 3,411. Coiled bodies were first described at the light microscopic level in 1903 as the “accessory body” by the Spanish cytologist Ramon y Cajal, using special silver staining techniques [42]. The relationship of the light microscopic structure designated as the “accessory body” with the ultrastructurally defined coiled body was first suggested by Hardin et al. [41] and definitively demonstrated by Seite et al. [43] and Lafarga et al. [38]. It has been shown to be present in both mammalian and plant eukaryotic cells [27]. By means of electron microscopic cytochemistry, coiled bodies have been shown to contain ribonucleoproteins [1,381, orthophosphate ions, and acid phosphatase activity [27], but were negative for DNA [l] or newly transcribed RNA [44]. Some electron micrographs depict the coiled body in close association with the nucleolus, and the fact that both structures are argyrophilic gave rise to suggestions of a relationship between the nucleolus and the coiled body [I, 41,431. Aside from the above-mentioned observations not much else is known about this ubiquitous structure. Images similar to the brightly stained nuclear bodies observed in this study may have been previously described in the autoimmune literature as “dots,” “granules,” and “speckles” [45-491. These descriptions were essentially morphological, and since the antigens were not further defined, it is not possible to discuss their relationship to p80-coilin or to the coiled bodies. The number of coiled bodies observed in any one electron microscopic thin section is rarely greater than one. Indirect immunofluorescence observations with antip80-coilin, however, have allowed for an initial estimate of the number of coiled bodies per cell. The frequency of coiled bodies was shown to vary according to the species
32
RASKA
ET
AL.
FIG. 8. Mouse 3T3 cells were double-stained for fibrillarin (a) and p80-coilin (b). Note that in (a) true (large) nucleoli are stained as well as two small bodies (single and double arrowheads). One of these bodies is also positive for p80-coilin in (b) (single arrowheads). ~1650. FIG. 9. A HeLa cell in S phase was double-stained for incorporated bromodeoxyuridine (a) and p80coilin (b). Coiled bodies (arrowheads) are not co-localized with bromodeoxyuridine incorporation. X1650. FIG. 10. Mouse 3T3 cell double-stained for Sm (a) and p8O-coilin (bl. Note that the coiled body (arrowhead) is relatively enriched in Sm antigen. X1650. FIG. 11. Mouse 3T3 cell double-stained for m,G-capped RNA (a) and p80-coilin (b), with a similar result as in Fig. 10 (arrowhead indicates a stained coiled body). X1650. FIG. 12. HeLa cells double-stained for DNA topoisomerase I (a) and p80-coilin (h) show DNA topoisomerase I in the coiled body (arrowheads). X1230. FIG. 13. HeLa cell double-stained for a protein recognized by SC35 antibody (a) and p80-coilin (b). For some coiled bodies, SC35 positive speckles appear to be juxtapositioned, but not identically superimposed. Coiled bodies, arrowheads. X1230
AUTOANTIBODIES
TABLE
AS
PROBES
FOR
1
Immunofluorescence Analysis for Antigens in the Coiled Body Antigens detected p80-coilin m,G capped RNA DNA topoisomerase Sm, Ul snRNP fitxillarin
Antigens not detected
I
DNA 5s rRNP hnRNP protein L nucleolin
nucleolar protein B23 interchromatin granule 3C5 antigen SC35 spliceosome protein RNA polymerase I”
’ Also refer to Fig. 14.
and also among members of the same cell population. It could be argued that such variation is due to the absence of p80-coilin, or to the masking of the p80coilin epitope(s) recognized by the antisera in some coiled bodies. Our immunoelectron microscopic observations argue against these possibilities, since each coiled body identified by electron microscopy was labeled with anti-p80-coilin antibodies. Of special interest is the intraspecies variation in the frequency of coiled bodies, which may be dependent on the cell cycle phase or metabolic state of the cell examined, or on both factors. The elucidation of the factors which influence the expression of coiled bodies may bring some insights into the nature and function of this distinctive nuclear structure. Other available information about the nature of the coiled body concerns its relationship with the nuclear matrix. Extractions and digestions designed to create in situ nuclear matrix preparations still showed bright coiled bodies stained by anti-p80-coilin antibodies. These results suggest that the coiled body is a structure tightly bound to the nuclear matrix, as observed previously by Chaly et al. [36]. The spatial distribution of highly specialized macromolecules in the cell nucleus is apparently related to their function. For example, recent studies on the localization of small nuclear RNPs have suggested a highly organized and reciprocal relationship between structure and function in the cell nucleus [23,4,50]. Thus, immunofluorescence images obtained with antinuclear antibodies may provide morphological “maps” of functional nuclear domains. Accordingly, an attempt was made to define the macromolecules present in the coiled body, and thereby gain some insight into its function. This was accomplished by means of immunoelectron microscopy or double label immunofluorescence with antip80-coilin as a reference marker and other specific antinuclear antibodies as detecting agents. Three distinct
examined
THE
NUCLEAR
COILED
BODY
33
groups of macromolecules were localized to the coiled body: snRNPs (recognized by anti-Sm, anti-U1 RNP, and anti-m,G-capped RNA), DNA topoisomerase I, and fibrillarin. The Sm antigen has been previously demonstrated in the coiled body by immunoelectron microscopy, using either monoclonal anti-Sm IgG [51] or mouse monoclonal and human autoimmune antibodies to Sm [52]. The snRNPs are known to be involved in the splicing of newly transcribed mRNA, which would suggest that the coiled body might be implicated in processing of pre-mRNA. However, we could not detect the presence of the L protein of hnRNA nor SC35, a recently described protein believed to be required for splicing activity [23]. Previous electron microscopic studies demonstrated that the coiled body does not incorporate [“Hluridine after short pulses [44]. These observations might suggest, that active transcription and splicing of pre-mRNA takes place outside the coiled body. If this is the case, the coiled body might play some role in other aspects of RNA metabolism such as the transport, storage, mat,uration, or degradation of snRNPs. Fibrillarin is a component of nucleolar U3 snRNP [ 15, 531, and it was recently shown that this RNP is involved in early processing of pre-rRNA [54]. The presence of fibrillarin in the coiled body might indicate some relationship to t.he nucleolus. At the electron microscopic level, we were able to confirm the spatial association of coiled bodies with the nucleolus in specialized cells such as neurons [41, 38, 551 (see also [56] for an example of other kinds of specialized cells). In our hands, this association was less impressive in cycling cells. Another interesting piece of evidence is the apparent interspecies correlation between the number of nucleolar organizing regions (NORs) and coiled bodies. We observed a maximum of 8,3, and 1 coiled bodies per cell in human, murine, and PtK2 cell lines, respectively, as compared to 10,6, and 2 nucleolar organizing regions in diploid G, nuclei of t,hese same cells. At the cytochemical level, Seite et al. [43] and Raska et al. (551 have shown that coiled bodies are stained by the NOR-silver staining technique in a way similar to the fibrillar regions of the nucleolus. On the other hand, the major nucleolar proteins nucleolin and protein B23 [57] were not found in the coiled bodies, and neither was 5s rRNP [II]. In order to determine the function of the coiled body, several lines of investigation should be pursued, including the study of these structures in various physiological, pharmacological, and pathological states, as well as its isolation and biochemical analysis. The characterization of a protein, p80-coilin, highly enriched in the coiled body and the availability of cognate autoantibodies should be of help in the implementation of further investigations.
34
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36
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I.R. expresses his gratitude to Dr. E. M. Tan for the opportunity to work at the W. M. Keck Autoimmune Disease Center. L.E.C.A. is a recipient of Grant 204776/88-O from the Brazilian National Council for Development of Science and Technology. E.K.L.C. is a recipient of an Arthritis Foundation Investigator Award. The authors would like to thank all their colleagues in the laboratory, in particular Ms. Carol Peebles, Dr. Rufus Burlingame, and Dr. Michael Pollard for their support and help. This work was supported by NIH Grants AR32063 and A110386. This is publication 6516.MEM from the Research Institute of Scripps Clinic. REFERENCES A., and Bernhard,
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Steitz, J. A., Berg, C., Hendrick, J. P., La Branche-Chabot, H., Metspalu, A., Rinke, J., and Yario, T. (1988) J. Cell Biol. 106, 545-556. Billings, P. B., Allen, R. W., Jensen, F. C., and Hoch, S. 0. (1982) J. Immunol. 128,1176~1181.
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Raska, I., Reimer, G., Jarnik, M., Kostrouch, Z., and Raska, Jr. (1989) Biol. Cell 85, 79-82. Liihrmann, R., Appel, B., Bringmann, P., Rinke, J., Roethe, and Bald, R. (1982) Nucleic Acids Res. 10, 7103-7113.
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Padykula, H. A., and Clark, J. H. (1981) in The Cell Nucleus Busch, Ed.) Vol 9, pp. 309-339, Academic Press, New York.
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Leser, G. P., Fakan, 50, 376-389.
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A. M., Mauchamp, J., Stahl, J. Ultrustruct. Res. 70, 37-51.
A., and
R., and Franke,
J. H., Spicer,
H. G. (1989)
T. E. (1989) Proc. N&l.
S. S., and Greene,
(H.
W. W. (1984)
M., Hervas, J. P., Santa-Cruz, M. C., Villegas, D. (1983) Anut. Embriol. 166, 19-30. S., and Martin,
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43.
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Moreno
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A., Risueno,
FIG. 14. A cryosection of a HeLa cell labeled for RNA polymerase I with 10.nm gold particles (large arrowheads) and m,G-capped RNAs with 5.nm particles (small arrowheads). The coiled body (arrows) is heavily labeled with 5-nm gold particles, while only one 10.nm gold particle is observed. This may reflect either low RNA polymerase I concentration in the coiled body or its complete absence. Label is also present over the nucleolus (Nu). Ten-nanometer particles are enriched over the fibrillar centers (F), while 5-nm particles are found more frequently over dense fibrillar components (D). Condensed chromatin, Ch. X70,800. FIG. 15. A cryosection of a HeLa cell labeled for fibrillarin. Ten-nanometer gold particles (arrowheads) are found over nucleolar dense fibrillar components and over a coiled body (large arrows). Nucleolus, Nu; simple nuclear body, small arrow. X45,700. FIG. 16. A cryosection of a HeLa cell labeled for DNA topoisomerase I. Ten-nanometer gold particles are enriched over the nucleolus, in particular over dense fibrillar components (D), and over a fibrillar center (F), as well as over a coiled body (arrows). Nucleolar interstitia, NI; cytoplasm, Cy. X71,200.
AUTOANTIBODIES
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M. C., Medina, F. J., and Fernandez-Gomez, tron Microsc. 2, 240-241.
M. E. (1980)
Bernstein, R. M., Neuberger, J. M., M. E., Hughes, G. R. V., and Williams, munol. 55, 5533560.
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49.
Freundlich, B., Makover, D., and Maul, tern.Med. 109,295-297. Elicieri, G. L.. and Ryerse, J. S. (1984) 449-451.
50.
Received Revised
M. A. H., and Bernstein,
December 6, 1990 version received February
M.,
28, 1991
THE
NUCLEAR
COILED
37
BODY
51.
Fakan, S., Leser, 358-363.
Ar-
52.
Nyman, U., Hallman, H., Hadlaczky, G., Pettersson, I., Sharp, G., and Ringertz, N. R. (1986) J. Cell Biol. 102, 137-144.
Bunn, C. C., Callender, R. (1984) Clin. Exp. Im-
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54.
Kass, S., Tyc, K., Steitz, J. A., and Sollner-Webb, B. (1990) Cell 60,897-908. Raska, I., Ochs, R. L., Andrade, L. E. C., Chan, E. K. L., Burlingame, R., Peebles, C., Gruol, D., and Tan, E. M. (1990) J. Strut. Biol. 104, 120-127.
R. M. (1987)
F. B., Lenzi, 38, 801-805.
FOR
Elec-
Fritzler, M. J., Valencia, D. W., and McCarty, thritis Rheum. 27, 92-96. 46.
PROBES
Volta,
G. A. (1984)
Ann.
Rheum.
U., and
G. G. (1988)
46,
Pisi, Ann.
J. Cell. Physiol.
E.
55.
In-
121,
56.
Schultz,
57.
Borer, (19891
G., and Martin,
M. C. (1989)
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R. A., Lehner, C. F., Eppenberger, Call 56, 379-390.
J. Cell Biol.
98,
21--25. E., and Nigg,
H. M.
CELL
RESEARCH
195,
27-37
(1991)
Immunological and Ultrastructural Studies of the Nuclear Coiled Body with Autoimmune Antibodies IVANRASKA,~ Lu1sE.C. ANDRADE,ROBERT L. OCHS,EDWARD K.L. CHENG-MINGCHANG,G~RAN ROOS,’ ANDENG M. TANS W. M. Keck Autoimmune
Disease
Center,
Scripps
Clinic
and Research
Foundation,
10666
North
Torrey
Pines
CHAN,
Road,
La Jolla,
California
92037
storage, and degradation of various RNAs [4]. In addition, it seemslikely that the transport of material to and from the nucleolus occurs, at least in part, through the interchromatin space. However, information about the relationships between these different metabolic processes and structural organization in the interchromatin space is lacking. In order to gain deeper insight into structure-function relationships in the cell nucleus, probes defining specific structural domains within what may seem to be a homogeneous looking cell nucleus are of increasing importance. Specific antibodies have been employed very effectively using immunocytochemical approaches (e.g., [5]). However, it is often difficult to prepare specific sera or monoclonal antibodies against nuclear macromolecules since nuclear structures usually do not exhibit significant evolutionary differences and the relevant macromolecules are often present in low copy number [6]. On the other hand, high titer autoantibodies against a great variety of nuclear antigens can be found in sera of patients with autoimmune disorders, in particular systemic rheumatic diseases [7]. In this study, a novel autoantibody probe for an 80-kDa nuclear protein present in the coiled body, named p80coilin, is documented.
Studies with human autoimmune sera identified autoantibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function. (‘ 1991 Academic Press, Inc.
INTRODUCTION In the interior of the cell nucleus, only a limited number of morphologically well-defined structural components are found. Besides chromatin and nucleoli, four other categories of structures have been described in the remaining interchromatin space. They are perichromatin fibrils, perichromatin granules, interchromatin granules, and nuclear bodies [l, 21. In the category of nuclear bodies, only simple nuclear bodies and coiled bodies are regularly found [3]. Functionally, it has been assumed that all nuclear metabolism related to RNA polymerase II and III transcripts occurs in the interchromatin space by processes which include splicing, polyadenylation, transport,
MATERIALS
AND METHODS
Cells and tissues. Human HeLa S3, WI 38, MOLT-4, HEp-2, rat NRK, mouse 3T3, and marsupial PtK2 cell lines were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal calf serum and gentamicin. Commercial preparations of HEp-2 cells were obtained from Bion (F’ark Ridge, IL). Human lymphocytes were isolated from the blood of a healthy donor by means of Histopaque 1077 (Sigma). Mouse kidney and stomach sections were obtained from Kallestad Lab (Austin, TX). Fresh mouse liver imprints were made on 14.mm-round coverslips. Five-micrometer-thick cryosections of mouse and rat brain were prepared from paraformaldehyde (perfusion followed by immersion fixation)fixed tissue. Cell extracts and immunoblotting. Cultured cells were harvested by centrifugation, resuspended, and lysed in SDS-gel sample buffer
’ Present address: Institute of Experimental Medicine, Czechoslovak Academy of Sciences, Albertov 4,128 00, Prague, Czechoslovakia. ’ Present address: Clinical Cytology Laboratory, University Hospital, 90185 Umea. Sweden, ” TO whom reprint requests should be addressed.
25 Copyright All
rights
cc: 1991 of reproduction
hy Academic Press, Inc. in any form reserved.
28
RASKA
[8]. Whole cell lysates were separated on 12.5% SDS-PAGE gels according to Laemmli [8]. Separated proteins were transferred to nitrocellulose at 60 V for 150 min at 4OC. Nitrocellulose sheets were cut into strips and blocked in 3% nonfat milk in phosphate-bufIered saline (PBS) containing 0.05% Tween 20 (PBS-T buffer) for 30 min and then incubated for 1 h with different sera diluted loo-fold in the same blocking solution. ‘2”I-labeled protein A (ICN Biochemicals, Irvine, CA) was used as the secondary detection reagent. Protein standards (Bio-Rad, Richmond, CA) were phosphorylase B, BSA, ovalbu min, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme corresponding to 92.5, 66.2, 42.7, 31, 21.5, and 14.4 kDa, respectively. Antisera and antibodies. Sera from 20 patients with diverse autoimmune conditions exhibited a distinct fluorescent pattern consisting of brightly stained round bodies (usually less than six) in nuclei of HEp-2 cell preparations [Andrade et al., in preparation, 19911. All these sera were shown to contain anti-p80-coilin autoantibodies (see below). The titer of sera by immunofluorescence microscopy varied from 1:640 to 1:40,960. Other antisera employed in these studies included monoclonal antibody against bromodeoxyuridine (BrdU) (Becton-Dickinson, Basel, Switzerland), monoclonal antibodies against DNA of IgG class [9] and IgM class (Boehringer-Mannheim, Germany), anti-Sm monoclonal antibody and human autoantibody against 5s rRNP [lo, II] (kindly provided by Dr. 3. A. Steitz and Dr. T. Yario), anti-U1 RNP monoclonal antibody [la] (kindly provided by Dr. S. Hoch), rabbit antibodies and human autoantibodies against DNA topoisomerase I [13], mouse monoclonal antibodies [14] and human autoantibodies against fibrillarin [15], and human autoantibodies against RNA polymerase I (16,171, rabbit anti-trimethyl guanosine-capped (m,G) RNA antibodies 1181 (kindly provided by Dr. R. Liihrmann), rabbit antibodies against nucleolin [19] (kindly provided by Dr. M. Olson), monoclonal antibodies against protein B23 [20] (kindly provided by Dr. P. K. Ghan), monoclonal antibodies against L protein of heterogeneous nuclear RNP (hnRNP) 1211 (kindly provided by Dr. P. Pinol-Roma and Dr. G. Dreyfuss), monoclonal antibody 3C5 against a component of interchromatin granules [22] (kindly provided by Dr. B. Turner), and monoclonal antibody SC35 against a component of spliceosomes [23] (kindly provided by Dr. X.-D. Fu and Dr. T. Maniatis). Afinity purification of antibodies. Affinity purification of antip80-coilin antibodies from nitrocellulose filters was performed according to the method of Olmsted [24]. HeLa cell extract served as the antigen source. After blocking with 3% nonfat milk in PBS-T buffer for 30 min, filters were incubated for 1 h with serum diluted 1:50 in this same buffer and washed with PBS-T. Bound antibodies were eluted by a brief 30-s exposure to 0.1 M phosphate buffer (pH 2.5) containing 0.15 M NaCl and 0.1% BSA, and the solution was immediately neutralized by addition of 1 M Tris-HCI (pH 8.8). The antibodies were concentrated with Centricon 30 microconcentrators (Amicon, Danvers, MA), and stored at 4°C. Processing of cells for light microscopy. Cells grown in monolayers were cultured on 14-mm-round coverslips which enabled easy handling. Cells were washed in PBS and fixed 10 min in 2% paraformaldehyde in 0.12 M phosphate buffer (pH 7.2). After several washes in the same buffer, the cells were permeabilized 5 min with 0.5% Triton X-100 in phosphate buffer or in cold (-20°C) acetone for 1 min. Alternatively, cells were precipitated in cold (~20°C) methanol for 5 min and then for 20 s in cold acetone (~20°C). Cells grown in suspension were cytocentrifuged (Shandon, Astmoore, Cheshire, England) for 5 min at 1000 rpm onto round coverslips placed on microscope slides and processed in the same manner as cells grown on coverslips. Double-label immunofluorescence was performed as described previously [25]. Affinity-purified goat secondary antibodies conjugated to FITC and TRITC (Caltag, San Francisco, CA) were used. Incubation times were usually 30 min for primary antibodies and 25 min for the secondary antibodies. However, in order to obtain an
ET
AL.
acceptable fluorescent signal on formaldehyde-fixed cryosections of rat and mouse brain, overnight incubations of the primary and 2-h incubations of the secondary antibodies were used. Processing of cells for electron microscopy. For conventional electron microscopy, cultured cells were fixed in 2% glutaraldehyde in 0.1 A4 cacodylate buffer for 1 h at 4”C, postfixed in 1% osmium tetroxide for 1 h, and embedded in epon. Thin sections stained with uranyl acetate and lead citrate were observed in a Hitachi HU 12A electron microscope. For immunoelectron microscopy, cells were fixed in paraformaldehyde, infused with sucrose, frozen, thin sectioned, immunolabeled, and postembedded in methylcellulose as described [3, 2.51. Some of the secondary 5-, lo-, and 15nm gold probes (Janssen Pharmaceutica, Beerse, Belgium) were kindly donated by the manufacturer. Extraction and digestion procedures. Cells grown in monolayers were either extracted in 0.1 N HCI or digested with DNase I, RNase A, or trypsin according to the procedure of Fritzler et al. [26]. Acid extraction removed most of the histones leaving behind DNA and residual matrix-bound proteins, while enzyme digestions demonstrated whether antigenic determinants were associated with DNA or RNA. For the preparation of nuclear matrix, the protocol of Turner and Franchi 1221 was followed, which involved consecutive treatments with detergent, nuclease, and high molarity salt.
RESULTS
Human autoimmune sera were initially selected based on their reactivity with discrete round nuclear bodies in immunofluorescence microscopy. The size of the stained bodies usually ranged between 0.3 to 1.0 ym, seldom exceeding 1 pm. Immunoblotting of these selected sera, using extracts of cycling human cells (HeLa, Molt 4) yielded a common reactive band corresponding to 80,000 Daltons. Several of the sera also presented other unrelated reactivities (Fig. 1). The immunofluorescence pattern on cycling interphase mammalian cells consisted of small brightly stained round bodies distributed throughout the cell nucleus (Figs. 2a and 2b). This staining pattern was especially apparent for sera which were judged monospecific by immunoblotting. The overall weak homogeneous nucleoplasmic staining, fine speckled nuclear staining, and in some cases cytoplasmic staining were not observed at higher serum dilutions, The number of stained nuclear bodies was variable in cycling mammalian cells, being usually less than six per nucleus (see below). Affinity-purified antibodies from the 80-kDa band gave essentially the same staining pattern as monospecific autoimmune sera (Figs. 2c and 2d). By electron microscopic immunocytochemistry performed with monospecific autoimmune sera on thawed cryosections, gold particles were highly enriched over coiled bodies (Fig. 4a). This finding was further verified by the use of affinity-purified autoantibodies to the 80kDa antigen (Fig. 4b). Since autoantibodies which blotted only the 80-kDa antigen labeled the coiled body specifically, we have named the 80-kDa antigen of the coiled body “p80coilin”. Since the coiled body is a ubiquitous nuclear structure
AUTOANTIBODIES
AS
PROBES
FOR
FIG. 1. Immunoblotting of human autoantibodies recognizing discrete nuclear bodies. Whole HeLa cell lysate was separated by SDS-PAGE, transferred to nitrocellulose, and probed with different sera. Lane 1, positive control autoimmune serum known to contain anti-Ku antibody which blots bands at 70 and 82 kDa as well as unidentified lower molecular weight bands. Lanes 2 to 5, reactivity of four selected autoimmune sera which exhibit positivity at 80 kDa. Note that one serum (lane 3) gives a monospecific reaction. Lane 6, negative control serum (normal human serum). Lane 7, another human serum positive for the 80.kDa protein. Lane 8, affinity-purified autoantibodies from the 80.kDa band of lane 7. Relative mobilities for molecular weight markers designated by arrows are 92.5, 66.2, 42.7, 31, 21.5, and 14.4 kDa, respectively. The BO-kDa band is indicated by an arrowhead.
(Fig. 3) in both plant and animal cells [l, 3, 27, 281, the presence of p80-coilin was investigated in cells of various origins by light microscopic immunocytochemistry. Positivity could be established for mammalian cell lines of human (HeLa S3, MOLT 4, HEp-2, WI 38), mouse (3T3), rat (NRK-52E), or marsupial (PtK2) origin. P80-coilin was also shown in different tissues: mouse liver cells (Fig. 5), rat and mouse brain cells (Fig. 6), mouse kidney, stomach and muscle cells, and human resting peripheral blood lymphocytes (Fig. 7).
THE
NUCLEAR
COILED
BODY
29
In frozen sections of rat and mouse brain, a frequent association between p80-coilin positive staining and nucleoli was noted (Fig. 6). Nucleolus-associated structures staining for p80-coilin usually formed one or several caps at the nucleolar periphery (Fig. 6b). This relationship was investigated in more detail in cycling cells by means of co-localization studies (see below). However, in HeLa cells less than 15% of coiled bodies were found in close association with the nucleolus. Coiled bodies were not evident at mitosis, but were present in the cell during the S phase as monitored by double label immunofluorescence of p80-coilin and incorporated bromodeoxyuridine (Fig. 9). In addition, we did not observe incorporated bromodeoxyuridine in p80-coilin positive bodies (e.g., Fig. 9). The number of coiled bodies found in any particular cell nucleus depended on the cell type. Most cycling cells of human origin exhibited 2 to 5 coiled bodies, with the number of negative cells being less than 10%. In the mouse cell line 3T3, the number of positive structures was not greater than two in most cells, and the percentage of negative cells approached 30%. In PtK2 cells, positive cells usually contained only one coiled body. Human peripheral blood lymphocytes usually possessed only one small weakly stained dot (Fig. 7). A number of additional antigens were localized to the coiled body (Table 1; Figs. 6,8, and 10 to 16). The most striking was the association with fibrillarin (Figs. 6, 8, and 15). In general, fibrillarin staining might be regarded as depicting “true” nucleoli and a number of bodies which, according to some investigators, represent prenucleolar bodies, micronucleoli, or satellite nucleoli (double arrowheads in Fig. 8) [ 15,29,30,31]. This study showed that p80-coilin positive structures are among those nuclear bodies which are stained for fibrillarin (single arrowhead in Fig. 8). P80-coilin positive bodies were also identified in conjunction with staining for Sm, Ul RNP, m,G-capped RNA, and DNA topoisomerase I (Figs. 10 to 12). The staining of these latter antigens gave rise to typical speckled nuclear patterns (e.g., Tan, 1989), and it was observed that some of the most brightly stained speckles costained for p80coilin (Figs. 10 to 12). For example, one of the brightly Sm-stained speckles in Fig. 1Oa was also positive for p80-coilin in Fig. lob. Antigens not detected in the coiled bodies included DNA, nucleolin, nucleolar protein B23, 5s rRNP, hnRNP protein L, a component of interchromatin granules detected by 3C5 antibody, and the 35-kDa spliceosome protein of Fu and Maniatis (Table 1; Fig. 13). Among these, an interesting feature was the staining pattern of the 35-kDa protein of Fu and Maniatis [23], since in many instances a SC35 positively stained body was located in proximity to a p80-coilin-stained body (Fig. 13), but as far as we could determine there was no co-localization.
KASKA
ET
AL.
FIG. 2. (a, h) Phase (a) and immunofluorescence (b) of interphase HEp-2 cells by the same serum as used for Western blots in lane 7 of Fig. 1. Nuclear bodies are brightly stained (arrowheads). The number of stained nuclear bodies per cell ranges from two to four in this figure. Note the heterogeneity in their size. Additional weak nuclear fluorescence in some HEp-2 cells is also observed. x1370. (c, d) Phase (c) and immunofiuorescence (d) of HEp-2 cells stained with autoantibody eluted from the 80-kDa band in Western blottings. Nuclear bodies (arrowheads) are specifically labeled. X1370. FIG. 3. Conventional epon section of a HeLa cell. Coiled body (large arrow) and simple nuclear body (small arrow) are present in the nucleoplasm. Nucleolus, Nu; cytoplasm, Cy. ~20,100. FIG. 4. (a) Immunolocalization of p80-coilin in a cryosection of a HeLa cell with monospecific autoimmune serum. Ten-nanometer gold particles (arrowheads) are enriched in the coiled body (arrow). X24,900. (b) Localization of p80-coilin with affinity-purified autoantibody. Ten-nanometer gold particles (arrowheads) are concentrated over the coiled body. ~65,800.
Immunoelectron microscopy demonstrated that coiled bodies were enriched for m,G-capped RNA (Fig. 14). On the other hand, RNA polymerase I (Fig. 14) and DNA were not detected in coiled bodies. Positivity of coiled bodies for fibrillarin (Fig. 15), Sm, Ul RNP, and DNA topoisomerase I (Fig. 16) was also demonstrated by electron microscopic immunocytochemistry. In order to further characterize p80-coilin, a number of digestion and extraction experiments were per-
formed. Staining for p80-coilin positive bodies was not affected by DNase digestion, but it was diminished by RNase treatment to some extent, and the fluorescence of p80-coilin positive bodies was completely abolished by proteinase K (data not shown). P80-coilin was not substantially extracted by high molar salt, acid, or detergent treatment. In nuclear matrix preparations, the fluorescence of p80-coilin positive bodies was still very prominent (data not shown).
AUTOANTIBODIES
FIG. heads). FIG. and one FIG. indicate
AS PROBES
FOR
THE
NUCLEAR
COILED
BODY
31
5. Mouse hepatocytes stained for p80-coilin with the same serum as in Fig. 2. Fluorescence of coiled bodies is prominent (arrow~1230. 6. Frozen section of rat brain double-stained for tibrillarin (a) and for p80-coilin (b). PSO-coilin-positive structures form one strongly weakly stained cap (arrowheads) associated with the nucleolus. X1840. 7. Isolated human lymphocytes stained for p80-coilin. Coiled bodies (arrowheads) are small and weakly stained. Double arrowheads nonspecific staining of B cell cytoplasm, presumably due to the presence of endogenous human immunoglobulins. ~1230.
DISCUSSION
In the present study we have identified a novel protein which appears to be localized predominantly to the nuclear coiled body. The target antigen is an 80-kDa protein (named p80-coilin) recognized by antibodies in the sera of patients with various autoimmune diseases (Andrade et al., manuscript in preparation). By means of immunoelectron microscopy of interphase mammalian cells, the antigen was shown to be highly enriched in the coiled bodies. Thus, p80-coilin represents a macromolecule which may serve as a marker for these intranuclear bodies, and autoantibodies to p80-coilin may eventually contribute to the understanding of the nature and function of t.his structure. Within the limits of the immunoelectron microscopic method employed, no significant amount of p80-coilin was identified at sites other than the coiled body. However, two points should be kept in mind. First, there is a possibility that p80coilin may be found in other cellular sites at very low levels, beyond the threshold of detection of the techniques employed. Second, the present association between p80-coilin and the coiled body was derived from studies in a limited number of physiological cell states. It is certainly worthwhile to determine if this association holds true under experimental or pathological conditions, such as in hormone-stimulated or virus-infected cells in which other types of nuclear bodies are known to arise [l, 2, 28, 32-401. Historically, the coiled body has been described at the ultrastructural level as a round-to-oval corpuscle 0.5 to 1.0 pm in diameter, consisting of coiled fibrillar strands. Coiled bodies are reported to be located randomly in the nucleoplasm, sometimes in close association with the
nucleolus [l, 2, 3,411. Coiled bodies were first described at the light microscopic level in 1903 as the “accessory body” by the Spanish cytologist Ramon y Cajal, using special silver staining techniques [42]. The relationship of the light microscopic structure designated as the “accessory body” with the ultrastructurally defined coiled body was first suggested by Hardin et al. [41] and definitively demonstrated by Seite et al. [43] and Lafarga et al. [38]. It has been shown to be present in both mammalian and plant eukaryotic cells [27]. By means of electron microscopic cytochemistry, coiled bodies have been shown to contain ribonucleoproteins [1,381, orthophosphate ions, and acid phosphatase activity [27], but were negative for DNA [l] or newly transcribed RNA [44]. Some electron micrographs depict the coiled body in close association with the nucleolus, and the fact that both structures are argyrophilic gave rise to suggestions of a relationship between the nucleolus and the coiled body [I, 41,431. Aside from the above-mentioned observations not much else is known about this ubiquitous structure. Images similar to the brightly stained nuclear bodies observed in this study may have been previously described in the autoimmune literature as “dots,” “granules,” and “speckles” [45-491. These descriptions were essentially morphological, and since the antigens were not further defined, it is not possible to discuss their relationship to p80-coilin or to the coiled bodies. The number of coiled bodies observed in any one electron microscopic thin section is rarely greater than one. Indirect immunofluorescence observations with antip80-coilin, however, have allowed for an initial estimate of the number of coiled bodies per cell. The frequency of coiled bodies was shown to vary according to the species
32
RASKA
ET
AL.
FIG. 8. Mouse 3T3 cells were double-stained for fibrillarin (a) and p80-coilin (b). Note that in (a) true (large) nucleoli are stained as well as two small bodies (single and double arrowheads). One of these bodies is also positive for p80-coilin in (b) (single arrowheads). ~1650. FIG. 9. A HeLa cell in S phase was double-stained for incorporated bromodeoxyuridine (a) and p80coilin (b). Coiled bodies (arrowheads) are not co-localized with bromodeoxyuridine incorporation. X1650. FIG. 10. Mouse 3T3 cell double-stained for Sm (a) and p8O-coilin (bl. Note that the coiled body (arrowhead) is relatively enriched in Sm antigen. X1650. FIG. 11. Mouse 3T3 cell double-stained for m,G-capped RNA (a) and p80-coilin (b), with a similar result as in Fig. 10 (arrowhead indicates a stained coiled body). X1650. FIG. 12. HeLa cells double-stained for DNA topoisomerase I (a) and p80-coilin (h) show DNA topoisomerase I in the coiled body (arrowheads). X1230. FIG. 13. HeLa cell double-stained for a protein recognized by SC35 antibody (a) and p80-coilin (b). For some coiled bodies, SC35 positive speckles appear to be juxtapositioned, but not identically superimposed. Coiled bodies, arrowheads. X1230
AUTOANTIBODIES
TABLE
AS
PROBES
FOR
1
Immunofluorescence Analysis for Antigens in the Coiled Body Antigens detected p80-coilin m,G capped RNA DNA topoisomerase Sm, Ul snRNP fitxillarin
Antigens not detected
I
DNA 5s rRNP hnRNP protein L nucleolin
nucleolar protein B23 interchromatin granule 3C5 antigen SC35 spliceosome protein RNA polymerase I”
’ Also refer to Fig. 14.
and also among members of the same cell population. It could be argued that such variation is due to the absence of p80-coilin, or to the masking of the p80coilin epitope(s) recognized by the antisera in some coiled bodies. Our immunoelectron microscopic observations argue against these possibilities, since each coiled body identified by electron microscopy was labeled with anti-p80-coilin antibodies. Of special interest is the intraspecies variation in the frequency of coiled bodies, which may be dependent on the cell cycle phase or metabolic state of the cell examined, or on both factors. The elucidation of the factors which influence the expression of coiled bodies may bring some insights into the nature and function of this distinctive nuclear structure. Other available information about the nature of the coiled body concerns its relationship with the nuclear matrix. Extractions and digestions designed to create in situ nuclear matrix preparations still showed bright coiled bodies stained by anti-p80-coilin antibodies. These results suggest that the coiled body is a structure tightly bound to the nuclear matrix, as observed previously by Chaly et al. [36]. The spatial distribution of highly specialized macromolecules in the cell nucleus is apparently related to their function. For example, recent studies on the localization of small nuclear RNPs have suggested a highly organized and reciprocal relationship between structure and function in the cell nucleus [23,4,50]. Thus, immunofluorescence images obtained with antinuclear antibodies may provide morphological “maps” of functional nuclear domains. Accordingly, an attempt was made to define the macromolecules present in the coiled body, and thereby gain some insight into its function. This was accomplished by means of immunoelectron microscopy or double label immunofluorescence with antip80-coilin as a reference marker and other specific antinuclear antibodies as detecting agents. Three distinct
examined
THE
NUCLEAR
COILED
BODY
33
groups of macromolecules were localized to the coiled body: snRNPs (recognized by anti-Sm, anti-U1 RNP, and anti-m,G-capped RNA), DNA topoisomerase I, and fibrillarin. The Sm antigen has been previously demonstrated in the coiled body by immunoelectron microscopy, using either monoclonal anti-Sm IgG [51] or mouse monoclonal and human autoimmune antibodies to Sm [52]. The snRNPs are known to be involved in the splicing of newly transcribed mRNA, which would suggest that the coiled body might be implicated in processing of pre-mRNA. However, we could not detect the presence of the L protein of hnRNA nor SC35, a recently described protein believed to be required for splicing activity [23]. Previous electron microscopic studies demonstrated that the coiled body does not incorporate [“Hluridine after short pulses [44]. These observations might suggest, that active transcription and splicing of pre-mRNA takes place outside the coiled body. If this is the case, the coiled body might play some role in other aspects of RNA metabolism such as the transport, storage, mat,uration, or degradation of snRNPs. Fibrillarin is a component of nucleolar U3 snRNP [ 15, 531, and it was recently shown that this RNP is involved in early processing of pre-rRNA [54]. The presence of fibrillarin in the coiled body might indicate some relationship to t.he nucleolus. At the electron microscopic level, we were able to confirm the spatial association of coiled bodies with the nucleolus in specialized cells such as neurons [41, 38, 551 (see also [56] for an example of other kinds of specialized cells). In our hands, this association was less impressive in cycling cells. Another interesting piece of evidence is the apparent interspecies correlation between the number of nucleolar organizing regions (NORs) and coiled bodies. We observed a maximum of 8,3, and 1 coiled bodies per cell in human, murine, and PtK2 cell lines, respectively, as compared to 10,6, and 2 nucleolar organizing regions in diploid G, nuclei of t,hese same cells. At the cytochemical level, Seite et al. [43] and Raska et al. (551 have shown that coiled bodies are stained by the NOR-silver staining technique in a way similar to the fibrillar regions of the nucleolus. On the other hand, the major nucleolar proteins nucleolin and protein B23 [57] were not found in the coiled bodies, and neither was 5s rRNP [II]. In order to determine the function of the coiled body, several lines of investigation should be pursued, including the study of these structures in various physiological, pharmacological, and pathological states, as well as its isolation and biochemical analysis. The characterization of a protein, p80-coilin, highly enriched in the coiled body and the availability of cognate autoantibodies should be of help in the implementation of further investigations.
34
RASKA
ET
AI,
AUTOANTIBODIES
AS PROBES
FOR
THE
NUCLEAR
COILED
BODY
35
36
RASKA
I.R. expresses his gratitude to Dr. E. M. Tan for the opportunity to work at the W. M. Keck Autoimmune Disease Center. L.E.C.A. is a recipient of Grant 204776/88-O from the Brazilian National Council for Development of Science and Technology. E.K.L.C. is a recipient of an Arthritis Foundation Investigator Award. The authors would like to thank all their colleagues in the laboratory, in particular Ms. Carol Peebles, Dr. Rufus Burlingame, and Dr. Michael Pollard for their support and help. This work was supported by NIH Grants AR32063 and A110386. This is publication 6516.MEM from the Research Institute of Scripps Clinic. REFERENCES A., and Bernhard,
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