The nuclear matrix: a heuristic model for investigating genomic organization and function in the cell nucleus.

Despite significant advances in deciphering the molecular events underlying genomic function, our understanding of these integrated processes inside the functioning cell nucleus has, until recently, met with only very limited success. A major conundrum has been the "layers of complexity" characteristic of all cell structure and function. To understand how the cell nucleus functions, we must also understand how the cell nucleus is put together and functions as a whole. The value of this neo-holistic approach is demonstrated by the enormous progress made in recent years in identifying a wide variety of nuclear functions associated with the nuclear matrix. In this article we summarize basic properties of in situ nuclear structure, isolated nuclear matrix systems, nuclear matrix-associated functions, and DNA replication in particular. Emphasis is placed on identifying current problems and directions of research in this field and illustrating the intrinsic heuristic value of this global approach to genomic organization and function.     

Nuclear deformations,from signaling to perturbation and damage

During cell growth and motility in crowded tissues or interstitial spaces, cells must integrate multiple physical and biochemical environmental inputs. After a number of recent studies, the view of the nucleus as a passive object that cells have to drag along has become obsolete, placing the nucleus as a central player in sensing some of these inputs. In the present review, we will focus on changes in nuclear shape caused by external and internal forces. Depending on their magnitude, nuclear deformations can generate signaling events that modulate cell behavior and fate, or be a source of perturbations or even damage, having detrimental effects on cellular functions. On very large deformations, nuclear envelope rupture events become frequent, leading to uncontrolled nucleocytoplasmic mixing and DNA damage. We will also discuss the consequences of repeated compromised nuclear integrity, which can trigger DNA surveillance mechanisms, with critical consequences to cell fate and tissue homeostasis.     

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast

Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.     

Comparative proteomic analyses of the nuclear envelope and pore complex suggests a wide range of heretofore unexpected functions

Since the discovery of several inherited diseases linked to the nuclear envelope the number of functions ascribed to this subcellular organelle has skyrocketed. However the molecular pathways underlying these functions are not clear in most cases, perhaps because of missing components. Several recent proteomic analyses of the nuclear envelope and nuclear pore complex proteomes have yielded not only enough missing components to potentially elucidate these pathways, but suggest an exponentially greater number of functions at the nuclear periphery than ever imagined. Many of these functions appear to derive from recapitulation of pathways utilized at the plasma membrane and from other membrane systems. Additionally, many proteins identified in the comparative nuclear envelope studies have sequence characteristics suggesting that they might also contribute to nuclear pore complex functions. In particular, the striking enrichment for proteins in the nuclear envelope fractions that carry phenylalanine-glycine (FG) repeats may be significant for the mechanism of nuclear transport. In retrospect, these findings are only surprising in context of the notion held for many years that the nuclear envelope was only a barrier protecting the genome. In fact, it is arguably the most complex membrane organelle in the cell.     

Cytological and functional aspects of telomere maintenance

The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity. Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions. However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions. Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis. Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer. Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr.

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.     

The discovery of labile methyl esters on proliferating cell nuclear antigen by MS/MS

The post-translational modification of proliferating cell nuclear antigen (PCNA) has been implicated in modulating its function for over 20 years. With multiple interacting partners, PCNA is involved in processes ranging from DNA replication and repair to cell cycle control and apoptosis. The ability of PCNA to distinguish between specific binding partners in different tasks is currently of intense interest, and several post-translational modifications have been reported to modulate its function. Unfortunately, these reports have produced contradictory information on the type(s) of modification present on the molecule. Here we report a detailed structural analysis of a single acidic PCNA isoform, cancer-specific polyferating nuclear anitgen (csPCNA), isolated from breast cancer cells by 2D-PAGE and LC-MS/MS. With this approach we fully characterized the csPCNA isoform and confidently identified a single post-translational modification, methyl esterification. Interestingly, the methyl esters consistently localized to 15 specific glutamic and aspartic acid residues of csPCNA. The methyl esterification of csPCNA represents a novel type of post-translational modification in mammalian cells that could ultimately hold the key towards unlocking its diverse functions.     

Practical aspects of donor cell selection for nuclear cloning

Choosing the right nuclear donor is the most critical decision in cloning by nuclear transfer (NT), or nuclear cloning, because the cloned animal will be a genetic copy of the donor cell genome used for NT. Both donor cell type and cell cycle stage are important methodological parameters and influence nuclear cloning efficiency. Cloning, however, is a multi-step procedure and the exact contribution of the nuclear donor to overall cloning success must be determined in comparative studies. This requires strict standardization of isolation, purification, and culture protocols, and application of stringent identification criteria in order to obtain a homogenous donor cell population. In all these respects, the standards in the cloning field are currently poor. The aim of this review is to provide a brief guideline for the major practical aspects of donor cell selection, cell cycle synchronization and preparation for NT.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.     

Isolation and fractionation of rat liver nuclear envelopes and nuclear pore complexes

The nuclear envelope is a double lipid bilayer that physically separates the functions of the nucleus and the cytoplasm of eukaryotic cells. Regulated transport of molecules between the nucleus and the cytoplasm is essential for normal cell metabolism and is mediated by large protein complexes, termed nuclear pore complexes (NPCs), which span the inner and outer membranes of the nuclear envelope. Significant progress has been made in the past 10 years in identifying the protein composition of NPCs and the basic molecular mechanisms by which these complexes facilitate the selective exchange of molecules between the nucleus and the cytoplasm. However, many fundamentally important questions about the functions of NPCs, the specific functions of individual NPC-associated proteins, and the assembly and disassembly of NPCs, remain unanswered. This review describes approaches for isolating and characterizing nuclear envelopes and NPC-associated proteins from mammalian cells. It is anticipated that these procedures can be used as a starting point for further molecular and biochemical analysis of the mammalian nuclear envelope, NPCs, and NPC-associated proteins.     

The yeast Apq12 protein affects nucleocytoplasmic mRNA transport

An important step in mRNA biogenesis is the export of mRNA from the nucleus to the cytoplasm. In this work, we provide evidence that the previously uncharacterized gene APQ12 functions in nucleocytoplasmic mRNA transport in Saccharomyces cerevisiae. First, apq12delta strains manifest 3' hyperadenylated mRNA similar to other previously characterized RNA export mutants. Second, bulk poly(A)+ RNA is retained in the nucleus in apq12delta cells. Third, an Apq12p-GFP chimeric protein is localized to the nuclear periphery. Fourth, mRNA in apq12delta cells is stabilized, consistent with a defect in the rate of nuclear export. Interestingly, apq12delta mutants are severely compromised for growth and display atypical cell morphology. Because this aberrant cell morphology is not seen with other viable export mutants, Apq12p must have either an additional cellular function, or preferentially impinge on the export of mRNAs regulating cell growth. Together, these findings support a role for APQ12 in nucleocytoplasmic transport of mRNA.     

Coupling of cell structure to cell metabolism and function

The fact that cells make directed decisions regarding how to use energy, i.e., where to direct intracellular particles or where to move, suggests that energy can be, and is, harnessed in specific ways. It is now well established that the chemical reactions of the cell do not occur in nonorganized soup, but rather in the context of ordered structure. The physical components that make up this ordered structure of the cell are part of the tissue matrix, which consists of the dynamic linkages between the skeletal networks of the nucleus (the nuclear matrix), the cytoplasm (the cytoskeleton), and the extracellular environment (the extracellular matrix). To understand gene function and how the energy of the cell is directed towards accomplishing the tasks directed by DNA (gene expression), a further understanding of how cell structure is tied to cellular energy and function is required. We propose that the structural components of the cell harness cellular energy to direct cell functions by providing a dynamic bridge between thermodynamics and gene expression. © 1994 Wiley-Liss, Inc.     

The skeletal function of non‐genic nuclear DNA: new evidence from ancient cell chimaeras

DNA can be divided functionally into three categories: (1) genes — which code for proteins or specify non-messenger RNAs; (2) semons — short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g. loops or domains) or selfish genetic elements; (3) secondary DNA — which does not function by means of specific sequences. Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton. The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity. This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes. Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function. Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei. Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether junk or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes. New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated independently by secondary symbiogenesis (i.e., the merger of two radically different eukaryote cells) several hundred million years ago. In both groups the nucleus and plasma membrane of the former algal symbiont persist as the nucleomorphs and periplastid membrane, respectively. The fact that nucleomorphs have undergone a 200- to 1000-fold reduction in genome size and have virtually no secondary DNA shows that selection against non-functional nuclear DNA is strong enough to eliminate it very efficiently; therefore, the large amounts of secondary DNA in the former host nuclei of these chimaeras, and in nuclei generally, must be being maintained by positive selection. The divergent selection for secondary DNA in the nucleus and against it in nucleomorphs is readily explicable by the skeletal DNA hypothesis, given the different spectrum of gene functions that it encodes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Structural organization of the endoplasmic reticulum

The endoplasmic reticulum (ER) is a continuous membrane system but consists of various domains that perform different functions. Structurally distinct domains of this organelle include the nuclear envelope (NE), the rough and smooth ER, and the regions that contact other organelles. The establishment of these domains and the targeting of proteins to them are understood to varying degrees. Despite its complexity, the ER is a dynamic structure. In mitosis it must be divided between daughter cells and domains must be re-established, and even in interphase it is constantly rearranged as tubules extend along the cytoskeleton. Throughout these rearrangements the ER maintains its basic structure. How this is accomplished remains mysterious, but some insight has been gained from in vitro systems.     

BAG-1, an anti-apoptotic tumour marker

BAG-1 is a multifunctional and anti-apoptotic or anti-cell death protein that interacts with a variety of cellular proteins and affects their functions. On the cell surface, it binds to the cytosolic domain of the growth factor receptors and enhances the protection from cell death triggered by growth factor receptors. In the cytosol, it binds to Bcl-2 and heat shock protein, and modulates their functions. In the nucleus, it binds to a variety of nuclear hormone receptors and inhibits hormone-induced apoptosis. BAG-1 is widely overexpressed in a variety of tumour cell lines and cancer tissues. In addition, differential expression of BAG-1 isoforms has been observed. Preclinical studies indicate that overexpression of BAG-1, especially its nuclear and cytoplasmic isoforms, may be useful as a prognostic and/or predictive biomarker. Pilot clinical studies have demonstrated that overexpression of nuclear BAG-1 may be associated with a shorter survival in breast and laryngeal carcinomas. Conversely, overexpression of cytoplasmic BAG-1 may be associated with a better clinical outcome in early stage breast cancer and in non-small cell lung cancer. Further large-scale clinical studies are warranted to establish the role of BAG-1 as a novel prognostic and/or predictive biomarker in the clinical management of these common malignancies.