Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.     

Ultrastructural distribution of ribonucleoprotein complexes during mitosis. snRNP antigens are contained in mitotic granule clusters

The great majority of snRNP and hnRNP ribonucleoproteins have been shown to be confined to the nucleus except during periods of cell division. We have now determined the fine structure distribution of polypeptides associated with these RNP complexes during interphase and mitosis in mammalian tissue culture cells using immunoelectron microscopy. Many hnRNP antigens are found at the periphery of heterochromatin masses, known to be the sites of non-rRNP proteins initially surround areas of condensing chromatin and later become generally dispersed throughout the mitotic cell. The Sm protein antigens of snRNP complexes are found diffusely distributed in interphase nuclei as well as concentrated in fields of interchromatin granules (ICG). Proteins of snRNP complexes, unlike those of hnRNP, are associated with discernible cellular structures during mitosis. By prometaphase/metaphase, dense granular clusters are observed to contain a high concentration of snRNPs. These mitotic granule clusters (MGCs) are often in close proximity to chromosomal masses by late anaphase/telophase. The MGC structures are morphologically similar to interchromatin granule fields found in interphase nuclei. Furthermore, like interchromatin granules, they are sites of a high concentration of snRNP antigens and do not contain detectable hnRNP proteins or DNA.     

Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (C1, M(r) of 41,000; C2, M(r) of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone H1 kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.     

Phosphorylation of the nuclear lamins during interphase and mitosis

The nuclear lamina is a polymeric protein assembly that is proposed to function as an architectural framework for the nuclear envelope. Previous work suggested that phosphorylation of the major polypeptides of the lamina (the "lamins") may induce disassembly of this structure during mitosis. To further investigate the possible involvement of phosphorylation in regulation of lamina structure, we characterized lamin phosphorylation occurring in mammalian tissue culture cells during interphase and mitosis. Phosphorylation occurs continuously throughout all interphase periods (coordinately with nuclear envelope growth), and takes place mainly on the assembled lamina. When the lamina is disassembled during cell division, the lamins are modified with approximately 1-2 molecules of associated phosphate. This level of mitotic phosphorylation is 4-7-fold higher than the average interphase level. Lamin phosphate occurs predominantly as phosphoserine, and is distributed over numerous tryptic peptides, many of which are modified during both interphase and mitotic periods. Significantly, phosphorylation is the only detectable charge-altering postsynthetic modification of the lamins that occurs specifically during mitosis. The results of this study support the notion that phosphorylation is important for regulation of interphase and mitotic lamina structure.     

Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: immunological relationship with hnRNP P.

Serum from a patient showing symptoms related to autoimmunity was found to contain autoantibodies to the nuclear mitotic apparatus (NuMA) protein and to several novel nuclear antigens with estimated molecular weights of 40, 43, 72, 74 and 82 kDa. Using this serum for screening a human cDNA expression library a 2.5 kb cDNA clone was isolated which encoded the complete sequence of a protein of 633 amino acids. Sequence analysis revealed a modular structure of the protein: an acidic N-terminal region of approximately 150 amino acids was followed by three adjacent consensus sequence RNA binding domains located in the central part of the protein. In the C-terminal portion a nuclear localization signal and an octapeptide (PPPRMPPP) with similarity to a major B cell epitope of the snRNP core protein B were identified. This was followed by a glycine- and arginine-rich section of approximately 120 amino acids forming another type of RNA binding motif, a RGG box. Interestingly, three copies of a tyrosine-rich decapeptide were found interspersed in the RGG box region. The major in vitro translation product of the cDNA co-migrated in SDS-PAGE with the 82 kDa polypeptide that was recognized by autoantibodies. The structural motifs as well as the immunofluorescence pattern generated by anti-82 kDa antibodies suggested that the antigen was one of the proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Subsequently the 82 kDa antigen was identified as hnRNP R protein by its presence in immunoprecipitated hnRNP complexes and co-migration of the recombinant protein with this hitherto uncharacterized hnRNP constituent in two-dimensional gel electrophoresis. The concomitant autoimmune response to a hnRNP component of the pre-mRNA processing machinery and to NuMA, a protein engaged in mitotic events and reported to be associated with mRNA splicing complexes in interphase, may indicate physical and functional association of these antigens. Support for this notion comes from observations that concomitant or coupling of autoantibody responses to proteins which are associated with each other as components of subcellular particles are often found in autoimmune diseases.     

Nuclear protein kinase activities during the cell cycle of HeLa S3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.     

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS- polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000- 75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.     

Monoclonal antibodies to heterogeneous nuclear RNA-protein complexes. The core proteins comprise a conserved group of related polypeptides

Hybridomas secreting monoclonal antibodies that react with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins have been isolated by immunizing BALB/c mice with RNP particles isolated from chicken and screening the fusion products with mouse RNP complexes. The antibodies show varying affinities for the hnRNP core proteins that have been blotted onto nitrocellulose. The majority of the immunoglobulins react with all the core group proteins although several recognize subsets of the hnRNP polypeptides. The clones are specific for different antigenic determinants as shown by their inability to compete with one another for binding sites. A mild proteolytic digestion of hnRNP proteins generates fragments that have uniformly lost 12 kDa and contain the antigenic determinants recognized by several of the monoclonal antibodies. Thus, it appears the core proteins comprise a family of related polypeptides possessing underlying structural similarities. Polypeptides similar in number and molecular weights that have antigenic determinants cross-reactive with those of mouse RNP have been found in a number of organisms, thereby emphasizing their possible common structure and function in higher eukaryotes. No difference in the distribution within the cell of individual or groups of core proteins has so far been detected by indirect immunofluorescence.     

Nuclear matrix proteins are carried within peripheral material of mitotic chromosomes

Immunofluorescent analysis has shown that autoimmune sera M-222 and M-260 are bound to interphase nuclei and mitotic chromosomes of the pig embryo kidney cell culture. The fluorescent stain is diffuse in nuclei and forms a thin fluorescent area around each nucleolus, whereas the nucleolar cores are unstained. The periphery of each mitotic chromosome is stained distinctly. After removal of histones and DNA by the cell treatment with 2 M NaCl and DNase I, the Hoechst 33258 staining of nuclei and chromosomes disappears completely, whereas the pattern of staining with antibodies is not changed as compared with normal cells. Electron microscopy revealed in interphase nuclei after such treatment only lamina, residual nucleoli, and the intranuclear matrix network, and antibodies are bound just to these elements. Molecular mass of proteins bound to these antibodies was determined by immunoblotting. Serum M-260 contained antibodies to a single 65 kDa polypeptide, whereas antibodies to two polypeptides of 47 and 65 kDa were found in M-222. After chromatin removal and revealing nuclear protein matrix, M-222 binds only to 65 kDa polypeptides. Thus, peripheral chromosomal material is involved in transfer of the nuclear matrix polypeptide to daughter nuclei during mitosis.     

Partial purification and characterization of mitotic factors from HeLa cells

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.     

HnRNP core proteins: Synthesis,turnover and intracellular distribution

Our present data indicate that the M_r 34–40,000 polypeptides which are involved in the binding of a large fraction of hnRNA sequences, including mRNA, are for the most part metabolically stable species in mouse ascites tumor cells. An exception to this generalization is the smallest of 30S RNP core polypeptides, the M_r 34,000 protein, which has a relatively high turnover rate. The relationship of the various synthesis and degradation rates to the physiological state of mammalian cells remains to be determined, as does the pathway of assembly and disassembly of RNP substructures during re-utilization of the proteins and during their turnover. Immunofluorescent studies, which have confirmed the expected nucleoplasmic or euchromatic localization of the RNP core proteins, have also indicated that these species are stable during mitosis, at which time they are dispersed through the cell away from the condensed chromosomes. The proteins appear to relocate in the nucleus as soon as the nuclear envelope is reformed.     

Intermediates in adenovirus assembly.

Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII.     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.     

Nup2 requires a highly divergent partner,NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region

Chromatin and nuclear pore complexes (NPCs) undergo dramatic changes during mitosis, which in vertebrates and Aspergillus nidulans involves movement of Nup2 from NPCs to the chromatin region to fulfill unknown functions. This transition is shown to require the Cdk1 mitotic kinase and be promoted prematurely by ectopic expression of the NIMA kinase. Nup2 localizes with a copurifying partner termed NupA, a highly divergent yet essential NPC protein. NupA and Nup2 locate throughout the chromatin region during prophase but during anaphase move to surround segregating DNA. NupA function is shown to involve targeting Nup2 to its interphase and mitotic locations. Deletion of either Nup2 or NupA causes identical mitotic defects that initiate a spindle assembly checkpoint (SAC)–dependent mitotic delay and also cause defects in karyokinesis. These mitotic problems are not caused by overall defects in mitotic NPC disassembly–reassembly or general nuclear import. However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis. Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.     

Segregation of unreplicated chromosomes in Saccharomyces cerevisiae reveals a novel G1/M-phase checkpoint.

Saccharomyces cerevisiae dbf4 and cdc7 cell cycle mutants block initiation of DNA synthesis (i.e., are iDS mutants) at 37 degrees C and arrest the cell cycle with a 1C DNA content. Surprisingly, certain dbf4 and cdc7 strains divide their chromatin at 37 degrees C. We found that the activation of the Cdc28 mitotic protein kinase and the Dbf2 kinase occurred with the correct relative timing with respect to each other and the observed division of the unreplicated chromatin. Furthermore, the division of unreplicated chromatin depended on a functional spindle. Therefore, the observed nuclear division resembled a normal mitosis, suggesting that S. cerevisiae commits to M phase in late G1 independently of S phase. Genetic analysis of dbf4 and cdc7 strains showed that the ability to restrain mitosis during a late G1 block depended on the genetic background of the strain concerned, since the dbf4 and cdc7 alleles examined showed the expected mitotic restraint in other backgrounds. This restraint was genetically dominant to lack of restraint, indicating that an active arrest mechanism, or checkpoint, was involved. However, none of the previously described mitotic checkpoint pathways were defective in the iDS strains that carry out mitosis without replicated DNA, therefore indicating that the checkpoint pathway that arrests mitosis in iDS mutants is novel. Thus, spontaneous strain differences have revealed that S. cerevisiae commits itself to mitosis in late G1 independently of entry into S phase and that a novel checkpoint mechanism can restrain mitosis if cells are blocked in late G1. We refer to this as the G1/M-phase checkpoint since it acts in G1 to restrain mitosis.     

Structural Relationship among the Rice Glutelin Polypeptides

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the α and β subunits are proteolytic products of the 51 kD precursor protein. The poly(A)⁺ RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.     

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with ³²P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.