ARTICLE |
Correspondence to: Umberto De Boni, Dept. of Physiology, Medical Sciences Bldg., U. of Toronto, Toronto, Ontario, Canada M5S 1A8.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interphase nuclei are organized into structural and functional domains. The coiled body, a nuclear organelle of unknown function, exhibits cell type-specific changes in number and morphology. Its association with nucleoli and with small nuclear ribonucleoproteins (snRNPs) indicates that it functions in RNA processing. In cycling cells, coiled bodies are round structures not associated with nucleoli. In contrast, in neurons, they frequently present as nucleolar "caps." To test the hypothesis that neuronal differentiation is accompanied by changes in the spatial association of coiled bodies with nucleoli and in their morphology, PC12 cells were differentiated into a neuronal phenotype with nerve growth factor (NGF) and coiled bodies detected by immunocytochemical localization of p80-coilin and snRNPs. The fraction of cells that showed coiled bodies as nucleolar caps increased from 1.6 ± 0.9% (mean ± SEM) in controls to 16.5 ± 1.6% in NGF-differentiated cultures. The fraction of cells with ring-like coiled bodies increased from 17.2 ± 5.0% in controls to 57.8 ± 4.4% in differentiated cells. This was accompanied by a decrease, from 81.2 ± 5.7% to 25.7 ± 3.1%, in the fraction of cells with small, round coiled bodies. SnRNPs remained associated with typical coiled bodies and with ring-like coiled bodies during NGF-induced recruitment of snRNPs to the nuclear periphery. Together with the observation that coiled bodies are also present as nucleolar caps in sensory neurons, the results indicate that coiled bodies alter their morphology and increase their association with nucleoli during NGF-induced neuronal differentiation. (J Histochem Cytochem 45:1523-1531, 1997)
Key Words: nuclear coiled bodies, snRNPs, p80-coilin, nucleolus, PC12 cells, neuronal differentiation, sensory neurons, immunocytochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The INTERPHASE NUCLEUS is organized into distinct structural and functional subnuclear domains (
Components of the interchromatin space are considered to contribute to this substructure, and include perichromatin fibrils, interchromatin granule clusters, and nuclear bodies. Among these, nuclear bodies have received considerable attention as dynamic structures (
Much of the recent interest in CBs has resulted from the identification of human autoimmune antibodies that recognize an 80-kD CB-specific protein named p80-coilin (
The identification of a subset of nucleolar antigens in CBs suggests the existence of a structural similarity between CBs and nucleoli. In fact, early electron microscopic studies in neurons revealed that CBs occur in close association with the nucleolus, sometimes appearing as a budding structure (
On the basis of their association with the nucleolus, CBs were believed to have a direct role in the processing of rRNA (
In actively cycling cells, CBs have been most often described as nucleoplasmic, uniformly staining round bodies, enriched in snRNPs and other splicing components (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
Rat pheochromocytoma cells (
Dorsal root ganglia (DRG) were excised aseptically from newborn CD-1 mice and suspended in a drop of cold Hanks' balanced salt solution (HBSS; GIBCO). Ganglia were sedimented (500 x g, 2 minutes) and the pellet incubated in trypsin (0.25%; 2 ml, 37C, 11 min; GIBCO) to allow dissociation of cells. Upon inactivation of trypsin with FBS (1 ml), the ganglia were mechanically disrupted by trituration with a Pasteur pipette and the dissociated cells sedimented (500 x g, 5 min). The pellet was resuspended in 0.2 ml of prewarmed (37C) DRG culture medium (90% MEM with Hanks' salts and L-glutamine, 10% FBS, supplemented with 6 mg/ml glucose and 7s NGF (100 ng/ml) and seeded onto calfskin collagen-coated (0.5 mg/ml, 1:1000 glacial acetic acid), air-dried glass coverslips and maintained by quantitative exchange of the culture medium every 3 days.
Immunocytochemistry
Cultures were fixed (12 min) with formaldehyde freshly prepared from paraformaldehyde (4% w/v) in PBS (130 mM NaCl, 7 mM Na2PO4, 3 mM NaH2PO4, pH 7.2), washed in PBS (three times for 5 min), and permeabilized [Triton X-100, 0.1% (v/v), PBS, 30 min]. To reduce nonspecific binding of antibodies, cultures were blocked with bovine serum albumin (BSA) [4% (w/v), PBS, 30 min].
CBs were labeled using rabbit R288 antibody to p80-coilin (
snRNPs were labeled using human anti-Sm autoimmune serum (1:200, 4% BSA, 2 hr; ANA Human Reference Serum no. 5, Centers for Disease Control, Atlanta, GA) and detected with TRITC-conjugated goat anti-human IgG (H+L, 1:100, 4%BSA, 1 hr; Jackson ImmunoResearch). For double labeling of p80-coilin and snRNPs, sequential incubation in both anti-Sm and anti-p80-coilin with an intervening wash was carried out before sequential incubation with the appropriate secondary antibodies. For visualization of nuclear and nucleolar outlines, when required, cells were counterstained with ethidium bromide (1 µg/ml, PBS, 10 min; Sigma).
After labeling, coverslips were inverted on glass slides into a drop of freshly prepared p-phenylenediamine solution (1 mg/ml, 50% glycerol, PBS) to reduce photobleaching (
For analysis, cells exposed to NGF for 10-14 days were categorized as neuronal or non-neuronal on the basis of cell morphology. Specifically, only those cells that exhibited large (diameter >20 µm) rounded cell bodies and extensive neurite outgrowth (minimal length >2 x cell body diameter) were designated as neuronal (Figure 1). Changes in the morphology and in the number of CBs per cell nucleus were compared among controls, NGF-treated cells of neuronal morphology, and NGF treated cells that failed to exhibit morphological evidence of differentiation. For quantification of the number of CBs per nucleus, nuclei were optically sectioned at 0.4-µm steps.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NGF-induced Neuronal Differentiation of PC12 Cells Is Accompanied by a Change in CB Morphology Without a Change in CB Number
In PC12 cells, CBs, detected by immunocytochemistry of associated p80-coilin, varied in morphology and were classified into three categories. As described in previous studies (
|
|
Although all three of these CB subtypes were observed in both untreated and treated PC12 cells, the frequency with which they occurred differed between cells with neuronal and non-neuronal morphologies (Figure 4). Specifically, in PC12 cells that were not exposed to NGF, CBs most commonly appeared as nucleoplasmic TCBs and less commonly as RLCBs. Only in a small fraction of untreated cells were CBs observed in association with the nucleolus, as nucleolar caps. In cells treated with NGF but that failed to exhibit a neuronal morphology, the frequencies with which these CB subtypes occurred did not differ from those observed in untreated cells. In contrast, however, in cells that responded to NGF by differentiation into cells of neuronal morphology, the fraction of cells with TCBs was significantly lower than that observed in non-neuronal control cells and in non-neuronal NGF-treated cells within the same culture. This decrease was paralleled by a significant increase in the fraction of cells that exhibited RLCBs and nucleolar caps, compared to untreated and NGF-nonresponsive cells (Figure 4).
|
To determine whether increases in the number of cells exhibiting RLCBs and nucleolar caps were attributable to the formation of new coiled bodies, the number of CBs per nucleus were quantified in untreated and NGF-treated PC12 cells. In cells exposed to NGF, the mean number of CBs per nucleus in both those cells responding to NGF with a neuronal morphology and in NGF-nonresponsive cells did not differ from that observed in untreated (control) cultures (ANOVA; p=0.05). In addition, the fraction of cells that exhibited either none, one, two, or multiple (range 3-7) CBs did not differ (ANOVA; p=0.05) between controls, NGF-responsive, and NGF-unresponsive cells. In all these cases, nuclei most commonly contained one CB (Figure 5).
|
CBs Remain Enriched in snRNPs During NGF-induced Redistribution of Sm Antigens to the Nuclear Periphery
In PC12 cells, snRNPs are recruited to the nuclear periphery in response to NGF (
CBs Appear as Paranucleolar Caps in Nuclei of DRG Neurons
To compare the distribution of CBs in PC12 cells that exhibit an NGF-induced neuronal morphology to that typically observed within nuclei of normal neurons, CBs were detected by immunocytochemical labeling of associated p80-coilin in nuclei of murine DRG neurons in vitro. In such neurons (Figure 6), CBs most commonly occurred as nucleolar caps (1-5 per nucleolus), with a spatial distribution similar to that observed in NGF-differentiated PC12 cells as described above and as described previously (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the present study, which show increases in the frequency of RLCBs and nucleolar caps in nuclei of NGF-responsive PC12 cells of neuronal morphology, indicate that NGF-induced neuronal differentiation is accompanied by, in addition to changes in the morphology of CBs, an increased association of CBs with nucleoli (Figure 2 and Figure 4). It is noteworthy that the changes in frequency of occurrence of RLCBs and nucleolar caps were restricted to those cells that were induced to exhibit a neuronal morphology, and were not detected in NGF-exposed cells within the same cultures that failed to differentiate. The mode of action of NGF that results in the above changes in morphology and spatial position of CBs therefore remains unclear. However, in DRG neurons, CBs similarly occur in association with nucleoli, as nucleolar caps. An association between CBs and nucleoli, therefore, appears to be a feature common to neuronal cells and may represent a nuclear organization associated with neuronal differentiation, as previously reported for hippocampal neurons (
It was also observed that changes in the frequency of occurrence of the three morphological subtypes of CBs on NGF-induced neuronal differentiation of PC12 cells were not accompanied by a change in the number of CBs per nucleus. This suggests that increases in the occurrence of ring-like CBs and nucleolar caps are not associated with the formation of new CBs, as has been proposed to occur through assembly and disassembly of CBs at the periphery of nucleoli in rat hippocampal neurons (
It must also be considered that the increase in the incidence of ring-like CBs and nucleolar caps may have resulted from changes in the morphology and distribution of existing CBs, in which ring-like CBs may represent intermediate structures before their association with nucleoli as caps. It is interesting to note that, similar to RLCBs, nucleolar caps also exhibit peripheral p80-coilin with an unlabeled core, as demonstrated in the present study in differentiated PC12 cells. Such peripheral labeling of nucleolar caps was demonstrated in previous studies in which p80-coilin was described as labeling a cap-like patch at the nucleolar periphery (
The association of CBs with nucleoli during NGF-induced neuronal differentiation may be a consequence of changes in nucleolar organization associated with nucleolar fusion, a process that has been shown to accompany neuronal differentiation (
Given that CBs contain snRNPs (
The results of the present study, which show that CBs alter their morphology and exhibit an increased association with the nucleolus during NGF-induced neuronal differentiation, support the view that CBs are dynamic structures, undergoing cell state-dependent changes in morphology and in their intranuclear spatial distribution.
![]() |
Acknowledgments |
---|
Supported by the National Science and Engineering Research Council of Canada.
The authors acknowledge the gift of R288 anti-coilin antibody from Dr E.K.L. Chan.
Received for publication February 20, 1997; accepted June 9, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrade LEC, Chan EKL, Raska I, Pebbles CL, Roos G, Tan EM (1991) Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J Exp Med 173:1407-1419 [Abstract]
Andrade LEC, Tan EM, Chan EKL (1993) Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation. Proc Natl Acad Sci USA 90:1947-1951 [Abstract]
Berciano MT, Calle E, Andres MA, Berciano J, Lafarga M (1996) Schwann cell nuclear remodelling and formation of nuclear and coiled bodies in Guillain-Barré syndrome. Acta Neuropathol (Berl) 92:386-394 [Medline]
Billia F, Baskys A, Carlen PL, De Boni U (1992) Rearrangement of centromeric satellite DNA in hippocampal neurons exhibiting long-term potentiation. Mol Brain Res 14:101-108 [Medline]
Billia F, De Boni U (1991) Localization of centromeric satellite and telomeric DNA sequences in dorsal root ganglia neurons, in vitro. J Cell Sci 100:219-226 [Abstract]
Bohmann K, Ferreira JA, Lamond AI (1995) Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus. J Cell Biol 131:817-831 [Abstract]
Borden J, Manuelidis L (1988) Movement of the X chromosome in epilepsy. Science 242:1687-1691 [Medline]
Brasch K, Harrington S, Blake H (1989) Isolation and analysis of nuclear bodies from estrogen-stimulated chick liver. Exp Cell Res 182:425-435 [Medline]
Brasch K, Ochs RL (1992) Nuclear bodies (Nbs): a newly "rediscovered" organelle. Exp Cell Res 202:211-223 [Medline]
Brodsky VY, Marshak TL, Karavanov AA, Zatsepina OV, Nosikov VV, Korochkin LI, Braga EA (1988) Cell differentiation as assayed by topography and number of ribosomal genes. Cell Differ 24:201-208 [Medline]
Buschmann MB, LaVelle A (1981) Morphological changes of the pyramidal cell nucleolus and nucleus in hamster frontal cortex during development and aging. Mech Ageing Dev 15:385-397 [Medline]
Carmo-Fonseca M, Ferreira J, Lamond AI (1993) Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosisevidence that the coiled body is a kinetic nuclear structure. J Cell Biol 120:841-852
[Abstract]
Cremer T, Cremer C, Baumann H, Leudtke EK, Sperling K (1982) Rabl's model of the interphase chromosome arrangement tested in Chinese hamster cells by premature chromosome condensation and laser-UV-microbeam experiments. Hum Genet 60:46-56 [Medline]
Crespo D, Viadero CF, Villegas J, Lafarga M (1988) Nucleoli numbers and neuronal growth in supraoptic nucleus neurons during postnatal development in the rat. Dev Brain Res 44:151-155 [Medline]
De Boni U, Mintz AH (1986) Curvilinear, three-dimensional motion of chromatin domains and nucleoli in neuronal interphase nuclei. Science 234:863-866 [Medline]
Fakan S, Leser G, Martin TE (1984) Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections. J Cell Biol 98:358-363 [Abstract]
Gall JG, Tsvetkov A, Wu ZA, Murphy C (1995) Is the sphere organelle/coiled body a universal nuclear component? Dev Genet 16:25-35[Medline]
Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:2424-2428 [Abstract]
Hardin JH, Spicer SS, Greene WB (1969) The para-nucleolar structure, accessory body of Cajal, sex chromatin, and related structures in nuclei of rat trigeminal neurons: a cytochemical and ultrastructural study. Anat Rec 164:403-432 [Medline]
Hochstrasser M, Sedat JW (1987) Three-dimensional organization of Drosophila melanogaster interphase nuclei. II. Chromosomal spatial organization and gene regulation. J Cell Biol 104:1471-1483 [Abstract]
Jackson DA (1991) Structure-function relationships in eukaryotic nuclei. Bioessays 13:1-10 [Medline]
Janevski J, Park PC, De Boni U (1995) Organization of centromeric domains in hepatocyte nuclei: rearrangement associated with de novo activation of the vitellogenin gene family in Xenopus laevis. Exp Cell Res 217:227-239 [Medline]
Jimenez-Garcia LF, Segura-Valdez ML, Ochs RL, Rothblum LI, Hannan R, Spector DL (1994) Nucleogenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis. Mol Biol Cell 9:955-966
Johnson GD, Nogueiro-Aguaro GMC (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43:349-350 [Medline]
Lafarga M, Andres MA, Berciano MT, Maquiera E (1991) Organization of nucleoli and nuclear bodies in osmotically stimulated supraoptic neurons of the rat. J Comp Neurol 308:329-339 [Medline]
Lafarga M, Andres MA, Fernandez-Viadero C, Villegas J, Berciano MT (1995) Number of nucleoli and coiled bodies and distribution of fibrillar centers in differentiating rat Purkinje neurons of chick and rat cerebellum. Anat Embryol 191:359-367 [Medline]
Lafarga M, Hervas JP, Santa-Cruz MC, Villegas J, Crespo D (1983) The "accesory body" of Cajal in the neuronal nucleus. A light and electron microscopic approach. Anat Embryol 166:19-30 [Medline]
LaSalle JM, Lalande M (1996) Homologous association of oppositely imprinted chromosomal domains. Science 272:725-728 [Abstract]
Malatesta M, Zancanaro C, Martin TE, Chan EKL, Amalric F, Lührmann R, Vogel P, Fakan S (1994) Is the coiled body involved in nucleolar functions? Exp Cell Res 211:415-419[Medline]
Meier UT, Blobel G (1992) Nopp 140 shuttles on tracks between nucleolus and cytoplasm. Cell 70:127-138 [Medline]
Monneron A, Bernhard W (1969) Fine structural organization of the interphase nucleus in some mammalian cells. J Ultrastruct Res 27:266-288 [Medline]
Münkel C, Eils R, Imhoff J, Dietzel S, Cremer C, Cremer T (1995) Simulation of the distribution of chromosome targets in cell nuclei under topological constraints. Bioimaging 3:108-120
Ochs RL, Stein TW, Jr, Andrade LEC, Gallo D, Chan EKL, Tan EM, Brasch K (1995) Formation of nuclear bodies in hepatocytes of estrogen-treated roosters. Mol Biol Cell 6:345-356 [Abstract]
Park PC, De Boni U (1991) Dynamics of nucleolar fusion in neuronal interphase nuclei in vitro: association with nuclear rotation. Exp Cell Res 197:213-221 [Medline]
Park PC, De Boni U (1996) Transposition of DNAse hypersensitive chromatin to the nuclear periphery coincides temporally with NGF-induced upregulation of gene expression in PC12 cells. Proc Natl Acad Sci USA 93:11646-11651
Pienta KJ, Getzenberg RH, Coffey DS (1991) Cell structure and DNA organization. Crit Rev Eukaryotic Gene Express 355-385
Ramon y Cajal S (1903) Un sencillo metodo de coloracion selectiva del reticulo protoplasmico y sus efectos en los diversos organos nerviosos de vertebrados e invertebrados. Trab Lab Invest Biol 2:129-221
Raska I, Andrade LEC, Ochs RL, Chan EKL, Chang C, Roos G, Tan EM (1991) Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp Cell Res 195:27-37 [Medline]
Raska I, Ochs RL, Andrade LEC, Chan EKL, Burlingame R, Peebles C, Gruol D, Tan EM (1990) Association between the nucleolus and the coiled body. J Struct Biol 104:120-127 [Medline]
Rebelo L, Almeida F, Ramos C, Bohmann K, Lamond AI, Carmo-Fonseca M (1996) The dynamics of coiled bodies in the nucleus of adenovirus-infected cells. Mol Biol Cell 7:1137-1151 [Abstract]
Sahlas DJ, Milankov K, Park PC, De Boni U (1993) Distribution of small snRNPs, splicing factor SC-35 and actin in interphase nuclei: immunocytochemical evidence for differential distribution during changes in functional states. J Cell Sci 105:347-357
Santama N, Dotti CG, Lamond AI (1996) Neuronal differentiation in the rat hippocampus involves a stage-specific reorganization of subnuclear structure both in vivo and in vitro. Eur J Neurosci 8:892-905 [Medline]
Seite R, Pebusque MJ, Bio-Cigna M (1982) Argyrophylic proteins on coiled bodies in sympathetic neurons identified by Ag-NOR procedure. Biol Cell 46:97-100
Spector DL (1993) Macromolecular domains within the cell nucleus. Annu Rev Cell Biol 9:265-315
Thiry M (1995) Nucleic acid compartmentalization within the cell nucleus by in situ transferase-immunogold techniques. Microsc Res Tech 31:4-21 [Medline]
van Driel R, Wansink DG, van Steensel B, Grande MA, Schul W, de Jong L (1995) Nuclear domains and the nuclear matrix. Int Rev Cytol 162A:151-189
Xing Y, Lawrence JB (1991) Preservation of specific RNA distribution with the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol 112:1055-1063 [Abstract]