Benavente R et al. (1984), Karyoskeletal proteins and the organization of ...

XB-ART-29919

J Cell Sci Suppl 1984 Jan 01;1:161-86.

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Karyoskeletal proteins and the organization of the amphibian oocyte nucleus.

Benavente R , Krohne G , Schmidt-Zachmann MS , Hügle B , Franke WW .

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We have investigated the existence of structural components in the nucleus of the oocyte of Xenopus laevis and other amphibia that are insoluble in non-denaturing detergents and buffers of low and high ionic strength. These cells are particularly suitable for such studies as they have a high frequency of extrachromosomal amplified nucleoli and pore complexes of the nuclear envelope. Using biochemical and immunological techniques, we have shown these structures to contain only two major proteins. These are a polypeptide of Mr 145000, which is located in a meshwork of filaments specific to the nucleolar cortex, and certain nucleoplasmic bodies probably derived therefrom, and a polypeptide of Mr 68000, which is the predominant constituent of the lamina-pore complex structure. We show that the latter protein is related to, but not identical to, lamina proteins ('lamins') of somatic cells, indicating cell type-specificity of the expression of polypeptides of the lamin family. In addition, we describe a protein of Mr 180000, which is the major constituent of the dense fibrillar component of the nucleolus. This can be partially solubilized in buffers of moderately high ionic strength. We interpret proteins of this category as karyoskeletal components involved in the architectural organization of specific functional topology within the nucleus. In contrast to previous reports for other cell types we have found no other prominent high-salt-insoluble structures in the nuclear interior, indicating the absence of an extended internal nuclear matrix in this kind of nucleus.

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Species referenced: Xenopus laevis
Genes referenced: alb lxn rs1 tsc1

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	Fig. 1. Biochemical characterization of the major karyoskeletal protein of the nuclear contents of X. laevis oocytes (see also Figs 16, 17). Polypeptides after separated by SDS/PAGE (10% acrylamide) were visualized by Coomassie Blue staining (lanes a, b) and autoradiography (lanes c-e). Lane a, reference proteins: myosin heavy chain (M, 200000), phosphorylase a (M, 94000), bovine serum albumin (BSA; M, 68000), actin (M, 42000) and chymotrypsinogen (M, 25 000). Lanes b, c, in manually isolated and sedimented nuclear contents theM, 145 000 polypeptide is present in significant amounts (b, arrowhead) and can be identified by the immunoblotting test (c) using specific antibodies (for details see Krohne et al. 1982). Lanes d, e, polypeptides of karyoskeletal residues (insoluble in HS-T-buffer; Ps fraction, see scheme 1) of nuclear contents prepared from oocytes incubated with e5S]methionine (d; 300f-tCi/ml, 24h) or [32P]orthophosphate (e; 500 f-tCi/ml, 24 h). TheM, 145 000 polypeptide is the most intensely labelled protein (arrowheads in d and e) of this fraction. Fig. 2. Immunofluorescence microscopy of culturedX.laevis kidney epithelial cells (line A6) with affinity-purified antibodies against theM, 145 000 polypeptide. This antibody shows punctate reaction in the periphery of nucleoli and in distinct granules free in the nucleoplasm. A. Phase contrast; B, epifluorescence. Bar, 20f-tm. Fig. 3. A. Two-dimensional gel electrophoresis (first dimension non-equilibrium pH gradient electrophoresis, NEPHGE; second dimension: SDS/PAGE (10%); silver staining) of residual polypeptides (insoluble in HS-T buffer) from X. laevis oocyte nuclear contents.TheM, 145 000 polypeptide (arrow) is the major protein of the fraction. Co-electrophoresed reference proteins are BSA (B), phosphoglycerokinase (P) and actin (A). B. An enlarged part of A, showing that theM, 145 000 polypeptide can be resolved into two isoelectric variants (arrows); B, BSA.
	Fig. 4. Immunofluorescence microscopy of a frozen section through aX. laevis ovary after incubation with affinity-purified antibodies against the J\11, 145 000 polypeptide. Part of an oocyte nucleus is shown. These antibodies show intense reaction with dot-like areas in the cortices of the amplified nucleoli (arrows) and MFBs free in the nucleoplasm (some are denoted by arrowheads). The position of the nuclear envelope is marked by a broken line. Bar, SO ~-tm. Fig. 5. Electron micrograph of an ultrathin section through an extracted nucleolus of X. laevis oocytes obtained after treatment with HS-T buffer (Ps fraction; for details see Franke et a/ . 1981a). Nucleolar filaments that tend to form dense aggregates in the nucleolar cortex (arrows) are characte ristic of this structure (see Figs 16, lane e and 17, lane a, for biochemical data). Bar, 1~-tm. Fig. 6. Electron microscopic immunolocalization of theM, 145 000 polypeptide in the nucleolar skeletal structure obtained as described in Fig. 5. Cortical filaments are decorated with 5 nm gold-coupled secondary antibodies (arrows) reacting with primary murine antibodies against theM, 145 000 polypeptide. Bar, 0·2~-tm.
	Fig. 7. Identification ofthe nucleolar antigen recognized by the monoclonal antibody No- 114 (electrophoretical conditions were similar to those in Fig. 1; for details see Schmidt· Zachmann et al. 1984). Lane a, reference proteins include, in addition to those shown in Fig. 1, lane a, /3-galactosidase (M, 120000). Lane b, polypeptide pattern of isolated purified amplified nucleoli from X. laevis oocytes (Coomassie Blue-staining). Lane c, corresponding autoradiograph after immunoblotting reaction with antibody N o-114. This antibody reacts with a protein of M, 180 000 present in the nucleolar fraction (arrows in band c). M, are shown (X10-3). Fig. 8. Immunofluorescence microscopy of a frozen section throughX.laevis ovary after incubation with the monoclonal antibody No-114. The large nucleoli of oocytes and the smaller nucleoli of the surrounding follicle cells (/) show intense reaction. Bar, 50 11m. Fig. 9. Localization of theM, 180 000 protein by immunofluorescence microscopy of frozen sections through liver tissue ofX.laevis (A,B) and on permeabilized cultured kidney epithelial cells (line A6) grown on coverslips ( c-F). A,C,E,G. Phase-contrast optics; B,D,F, H, epifluorescence optics. Nucleoli of hepatocytes (arrows in B) and A6 cells (n) show strong fluorescence. In nuclei of erythrocytes, which are inactive in the synthesis of ribosomal RNA, only one to two small, dot-like areas are stained (arrowheads in B), representing the residual nucleolar structures characteristic of these cells ( cf. SchmidtZachmann et al. 1984). E,F. In actinomycin D-treated kidney epithelial cells (A6) the nucleolar components have segregated (denoted by arrow and pointer; E, 4h AmD), showing that the M, 180 000 protein recognized by monoclonal N o-114 is now located in the phase-contrast light hemisphere (F, arrow) which corresponds to the dense fibrillar component. G,H. A6 cells treated for 4h with actinomycin D and incubated then with monoclonal antibody RS1-105. In contrast to the No-114 antibody, the antibody RS1-105 (which recognizes ribosomal protein S,) binds to the phase-dark hemisphere, which corresponds to the granular component of the nucleolus (pointers in G and H). In addition, the ribosomes of the cytoplasm are stained. Bars: B, 50 11m; n,F,H, 10 11m.
	Fig. 10. Electron microscopic immunolocalization of theM, 180 000 polypeptide in X. laevis hepatocyte nuclei using monoclonal antibody o-114 and 5 nm gold-particlecoupled secondary antibodies. The dense fibrillar component (dfc) of the nucleolus is labelled. Fibrillar centres (not shown), the granular component (gc) and the surrounding chromatin (ch) are not significantly labelled . Bar, O·Z ,um.
	Fig. II . Characterization of karyoskeletal proteins of the nuclear envelope from X. laevis oocytes (SDS/PAGE, 10 % acrylamide). Lanes a, b, Coomassie Blue staining; lanes c-f, silver staining. Lane a, reference proteins : myosin heavy chain (M, 200 000), phosphorylase a (M, 94 000), BSA (M, 68 000), actin (M, 42 000) and chymotrypsinogen (M, 25 000). Lane b , residual polypeptides of 200 manually isolated nuclear envelopes after treatment with HS-T buffer. TheM, 68 000 polypeptide (arrowhead) is the major protein of this fraction . In addition, a minor polypeptide band of M, 100 000 and four faint polypeptide bands in theM, range of 180 000-250 000 are detectable. Lanes c-f, characterization of minor karyoskeletal polypeptides associated with the nuclear envelope as detected by silver staining. Lane c, reference proteins : {3-galactosidase (M, 120 000), BSA (M, 68 000) and actin (M, 42 000) . Lane d, polypeptide pattern of whole unextracted nuclear envelopes (20 manually isolated envelopes). Lanese, f, residual polypeptides of extracted nuclear envelopes (e, 30 envelopes; f, 150 envelopes; extraction as described in b) . In addition to the predominantM, 68 000 protein (arrowheads) four polypeptide bands in theM, range of 180 000-250 000 and one polypeptide of approximate J1!! , 100 000 are detectable in silver- stained gels (f; cf. Krohne eta! . 198 1) . Fig. 12. Immunological characterization of the nuclear lamina polypeptides of oocytes and erythrocytes of X. laevis (SDS/PAGE according to Thomas & Kornberg, 1975 ; 12% acrylamide gels). Lane a, karyoskeletal residue of mass- iso lated oocyte nuclei (arrowhead ; M , 68 000 polypeptide). Lane b, karyoskeletal residue of erythrocytes. Arrowheads denote lamina polypeptides L1 (M, 72 000) and Lu (M, 68 000) . Lanes a' , b' , corresponding autoradiograph after an immunoblot experiment using monoclonal antibody Lo46F7 , which recognizes only theM, 68 000 polypeptide of oocytes (a', arrowhead) , not the related lamina polypeptides of erythrocytes (b ' ). Lanes a", b", autoradiograph of an immunoblot experiment using monoclonal antibody PKB8, which reacts exclusively with the two nuclear lamina polypeptides of erythrocytes (b" , arrowheads), not with the M, 68 000 polypeptide of oocytes (a"; cf. Krohne et a!. 1984) .
	Fig. 13. Immunofluorescence microscopy of frozen sections through ovaries of X. laevis (A-F) and P. waltlii ( G-H) after incubation with antibodies against nuclear lamina proteins. A,C,E,G. Phase-contrast optics; B,D,F,H, epiftuorescence optics. Monoclonal antibody Lo46F7 (A,B) reacts exclusively with the nuclear envelope of oocytes whereas somatic cell nuclei are exclusively stained by the monoclonal antibody PKB8 (c,o). Antibodies to lamina proteins from rabbit antiserum (Stick & Hausen, 1980 ; Stick & Krohne, 1982) react with the nuclear envelope of both cell types (E,F). In somatic cells nuclear lamina antibodies react exclusively with the nuclear periphery (G,H; follicle cells stained with the rabbit antibodies as used in E and F). n, oocyte nucleus. Bars, 50 /),m.
	Fig . 14. Electron microscopic immunolocalization of the M, 68 000 polypeptide in isolated nuclear envelopes of Xenopus oocytes. Specifically bound antibodies were visualized by 5 nm gold-coupled secondary antibodies. Monoclonal antibody Lo46F7 shows exclusive reaction with the nuclear lamina (A) whereas polyclonal mouse antibodies (B; Stick & Krohne, 1982) react with both structures, the nuclear lamina and the pore complexes. Nuclear pore complexes are denoted by arrows (A,B). c, cytoplasmic side ; n, nucleoplasmic side. Bars, 0·2 /),m.
	Fig. IS . Biochemical characteri zation of nuclear lamina polypeptides from chicken erythrocytes . A . Nuclear lamina polypeptides (lamins A,B,C) after separation by twodimensional gel electrophoresis (for abbreviations see Fig. 3) and staining with Coomassie Blue. B-D. T wo-dimensional analysis of 125 1-labelled peptides obtained after tryptic digestion of laminA (B) , B (c) and C (D). E indicates electrophoresis in first dimension and c, chromatography in second dimension . Peptide maps of lam ins A and C show great similarity and are clearly different from that of !a min B.
	Fig. 16. 8D8 /PAGE ( 10% acrylamide) according to Laemmli ( 1970) of nuclear content sub fractions from X. laevis oocytes. The nuclear contents were manually isolated in IM without MgClz (for details see scheme 1 in Materials and Methods). Lanes a-c, Coomassie Blue staining; lanes d-i, silver staining. Lane a, reference protein, actin (Mr 42000; a). Lane b, polypeptide pattern of the sediment derived from 50 nuclear contents (P1). Lane c, polypeptide pattern of the supernatant derived from 50 nuclear contents (8!). Lanes d, e, results of the extraction of pelletable material of nuclear contents (fraction P1) with H8-T buffer; d, polypeptides of 10 pelleted nuclear contents solubilized by H8-T buffer (84); e, protein that remains pelletable after extraction with H8-T buffer (Ps , residues from 70 nuclear contents). This fraction contains only one major protein, the Mr 145 000 polypeptide (arrowhead; for morphological comparison see Fig. 5). Lanes f, g, reference proteins denoted by dots: {3-galactosidase (Mr 120 000), phosphorylase a (Mr 94 000) and B8A (Mr 68 000). Lanes h, i, fractionation of proteins of nuclear contents (81 fraction) exposed to H8-T buffer; i, pelleted proteins of 81 fraction from 70 nuclear contents (P3; fractions P3 and Pz were practically identical in composition; no protein was found in the supernatant 83); h, protein of the 81 fraction that remains soluble after incubation with H8-T buffer (82 fraction; protein corresponding to one nuclear content). Fig. 17. Polypeptide composition of karyoskeletal residues (insoluble in H8-T buffer; fraction Ps) of nuclear contents ofX.laevis oocyte isolated in IM without (a, b) and with (d, e) 10mM-MgClz. 8D8/PAGE (10% acrylamide) seen after silver staining. Comparison between full-grown (a, d) and stage IV (b, e) oocytes. Lanes a, b, the residual fraction of nuclear contents isolated in IM without MgClz contains only one major protein, theM, 145 000 polypeptide (arrowheads). Lanes d, e, the karyoskeletal residue prepared from oocyte nuclear contents isolated in IM with 10 mM-MgClz contains several polypeptides in addition to theM, 145 000 protein (arrowheads; Mg-Ps fraction). This preparation contains large amounts of actin (a; for morphological comparison see Fig. 19B-D ). Lanes a, b, d, e, each lane contains protein corresponding to the residues from 70 extracted nuclear contents. Lane c, reference proteins (dots): {3-galactosidase (Mr 120 000) and B8A (M, 68 000). Lane f, reference proteins (dots): {3-galactosidase, phosphorylase a (M, 94000), B8A and actin (Mr 42000). Fig. 18. Effect of 10 mM-MgClz on protein composition of fraction 81 of nuclear contents from X. laevis oocytes (8D8/PAGE, Coomassie Blue staining). Lane a, protein corresponding to the supernatant from 50 nuclear contents (81). Lane b, little protein is pelleted in the 81 fraction (50 nuclear contents) when the incubation (15 min) was performed in IM without MgClz (control experiments). Lane c, when MgClz (final concentration 10 mM) was added to the 81 fraction (50 nuclear contents) and incubated for 15 min large amounts of actin appear in a pelletable form. Lane d, actin (a) from rabbit skeletal muscle (M, 42000).
	Fig. 19. A. Electron micrograph showing nuclear content material fromX.laevis oocytes isolated in IM containing 10 mM-MgClz. In addition to nucleoli (n) and MFBs a large number of microfilament bundles are visible (arrows). Insert on the right margin: higher magnification showing a microfilament bundle. B,C. Electron micrographs of ultrathin sections through the Mg-Ps fraction (see Fig. 17, lane d, for biochemical comparison). Oocyte nuclear contents were isolated in IM containing 10 mM-MgClz, washed in buffer containing 5 mM-EDT A, and then extracted with HS-T buffer. Compact residual nucleoli (n in B) are seen besides an extended fibrogranular meshwork. o. Negative staining of Mg-Ps fraction, demonstrating the fibrogranular nature of the.residual material, including long 5-6nm filaments. Bars: A-c, O·Sp;m; insert in A and o, 0·2p;m.