Assembly-disassembly of the nuclear lamina.

Recently, progress in the study of lamins has been made in three areas: signals required for targetting newly synthesized lamins to the correct subnuclear compartment have been identified; information on lamina assembly has been obtained from in vitro studies using bacterially expressed proteins; and a mechanistic explanation for how the nuclear lamina is diassembled at the onset of mitosis is emerging.     

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.     

Amoeba proteus: the nuclear periphery.

This study extends previous work on the nuclear envelope and associated structures. It illustrates that the cylindrical structures of the honeycomb lattice are not attached to the nuclear envelope, although generally perpendicular and closely apposed to it, and that there is a complex arrangement of fibrillar material between the cylinders of the lattice. The relationship of nuclear helices to these structures is described and the possible mode of their transfer from nucleus to cytoplasm is discussed.     

Cell cycle-dependent nuclear location of the matricellular protein SPARC: association with the nuclear matrix.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that inhibits cellular adhesion and proliferation. In this study, we report the detection of SPARC in the interphase nuclei of embryonic chicken cells in vivo. Differential partitioning of SPARC was also noted in the cytoplasm of these cells during discrete stages of M-phase: cells in metaphase and anaphase exhibited strong cytoplasmic immunoreactivity, whereas cells in telophase were devoid of labeling. Immunocytochemical analysis of embryonic chicken cells in vitro likewise showed the presence of SPARC in the nucleus. Furthermore, elution of soluble proteins and DNA from these cells indicated that SPARC might be a component of the nuclear matrix. We subsequently examined cultured bovine aortic endothelial cells, which initially appeared to express SPARC only in the cytoplasm. However, after elution of soluble proteins and chromatin, we also detected SPARC in the nuclear matrix of these cells. Embryonic chicken cells incubated with recombinant SPARC were seen to take up the protein and to translocate it to the nucleus progressively over a period of 17 h. These observations provide new information about SPARC, generally recognized as a secreted glycoprotein that mediates interactions between cells and components of the extracellular matrix. The evidence presented in this study indicates that SPARC might subserve analogous functions in the nuclear matrix.     

Androgen-induced nuclear accumulation of the estrogen receptor.

The effect of androgens on the nuclear uptake of both tritiated estradiol (3H-E2) and the estrogen receptor was studied in immature rat uteri. It was demonstrated that in vitro preincubation of immature rat uteri with various androgens (1 muM to 50 muM) followed by incubation with 3H-E2 (20 nM) resulted in a greatly decreased specific nuclear uptake of 3H-E2. Non-androgenic steroids had no effect. It was also confirmed that 5alpha-dihydrotestosterone (DHT) causes the accumulation of the estrogen receptor in the nuclei of uterine tissue. In vitro incubations of rat uteri with DHT (1muM and 50muM) were found to cause maximal nuclear estrogen receptor accumulation to the same degree as caused by estradiol, i.e. the nuclear uptake of approximately 100% of the estrogen receptor. Antiandrogens, which block the binding of androgens to the testosterone receptor in various tissues, did not inhibit the DHT - induced decrease in the 3H-E2 uptake by the uterine nuclei or the DHT - caused accumulation of the estrogen receptor in nuclei. These results seem to indicate that the uterine testosterone receptor has no role in the androgen - induced nuclear uptake of the estrogen receptor. However, the non-steroidal antiestrogens inhibited the DHT - induced nuclear accumulation of the estrogen receptor. This would seem to indicate that the estrogen - and androgen - induced nuclear accumulation of the estrogen receptor share a common mechanism.     

Pre-mRNA splicing and the nuclear matrix.

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.     

Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.

The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.     

Getting across the nuclear pore complex.

The nuclear pore complex (NPC) connects the cytoplasm and nucleus through the nuclear envelope and serves as the pipeline for moving material between the two compartments. Macromolecules that move through the NPC range in size from the very small (for example, ions and ATP) to the very large (for example, ribonucleoprotein particle complexes). Unlike translocation across other organelle membranes, proteins do not have to be unfolded to be transported through the NPC, and the NPC also routinely transports large, multicomponent substrates in both directions. This review focuses on current understanding of the different mechanisms by which macromolecules move across the NPC.     

Concentric nuclear inclusions.

Membrane bound concentric lamellar inclusions were found in the nuclei of pancreatic acinar cells of a patient with multiple primary neoplasms and in the parotid acinar cells of a patient with systemic lupus erythematosus. It is proposed that these inclusions form from rough cisternae sequestered in the nucleus.     

Ubiquitylation of the nuclear pore complex controls nuclear migration during mitosis in S. cerevisiae

Nuclear pore complexes (NPCs) correspond to large protein transport complexes responsible for selective nucleocytoplasmic exchange. Although research has revealed much about the molecular architecture and roles of the NPC subcomplexes, little is known about the regulation of NPC functions by posttranslational modifications. We used a systematic approach to show that more than half of NPC proteins were conjugated to ubiquitin. In particular, Nup159, a nucleoporin exclusively located on the cytoplasmic side of the NPC, was monoubiquitylated by the Cdc34/SCF (Skp1-Cdc53-F-box E3 ligase) enzymes. Preventing this modification had no consequences on nuclear transport or NPC organization but strongly affected the ability of Nup159 to target the dynein light chain to the NPC. This led to defects in nuclear segregation at the onset of mitosis. Thus, defining ubiquitylation of the yeast NPC highlights yet-unexplored functions of this essential organelle in cell division.     

Compositional bimodality of the nuclear genome of tobacco.

We have studied the compositional distribution of six genes (or small multigene families) and of one family of transposable elements, Tnt1, in DNA fractions from tobacco (Nicotiana tabacum) separated according to base composition. We have shown that gene distribution is bimodal and that such bimodality is due to the different base composition of the two parental genomes of tobacco (N.sylvestris and N.tomentosiformis) and to the different parental origin of the genes tested. These results indicate a physical separation and an absence of extensive recombination of the parental genomes, which have been together in the tobacco nucleus for a small span of their evolutionary life, and a conservation of their compositional patterns, including gene localization.     

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.