Induction of chromatin condensation by nuclear expression of a novel arginine-rich cationic protein genetically engineered from the enhanced green fluorescent protein

In the interphase nuclei of cultured cells, chromatin is compacted and organized in higher-order structures through the condensation and decondensation processes. Chromosomes in the interphase nucleus are known to occupy distinct territories. The chromosome territory-interchromatin compartment model premises that the interchromatin compartment is separated from compact higher-order chromatin domains and expands in between these chromatin-organized territories. Chromatin in cultured cells is compacted under some conditions, such as the stress of heat shock and high osmolarity, and Src-mediated nuclear tyrosine phosphorylation. We report here that a novel arginine-rich cationic protein is generated by frameshift mutation of enhanced green fluorescent protein (EGFP). The arginine-rich cationic protein is highly hydrophilic and contains potential arginine-based nuclear localization signals. Expression of the arginine-rich cationic protein shows its predominant localization to the nucleus and induces striking chromatin condensation in the interphase, which might be involved in interchromatin spacing or euchromatinization. Thus, the arginine-rich cationic protein as a new tool would be useful for dissecting chromatin architecture dynamics.     

Image analysis of the chromatin organization in the nuclear domains of freeze fractured hepatocytes and lymphocytes

The complex organization of the interphase nucleus can be analyzed, by way of thin sectioning and also freeze-fracture. This approach has previously been utilized in association with image analysis to quantitatively describe the organization of isolated rat liver nuclei and nuclear matrices. The main nuclear domains which, in section, present marked differences due to their electron-density, can be identified in replicas with more complex procedures, based on the quantitative evaluation of the number of particles per unit area and mainly by using image analysis. A quantitative analysis of the nuclear substructures has been performed by way of image analysis on in situ nuclei of freeze-fractured cells presenting marked differences in the heterochromatin quantity, such as hepatocytes and lymphocytes. The replicated nuclear particles have been classified according to their diameter and the obtained histograms have been quantitatively evaluated. The nuclear domains, heterochromatin, interchromatin, nucleolus, present characteristic ratios among the three main classes of particles; that is, ribonucleoproteins, solenoid filaments and solenoid fibre aggregates. The typical patterns of the nuclear domains can be further stressed by selecting a single class of particles and by examining its topographic localization. While interchromatin and nucleolar domains present a similar quantitative pattern in hepatocytes and lymphocytes, the heterochromatin of lymphocytes contains a significative higher percentage of solenoid aggregates than that of hepatocytes.     

Intranuclear localization of phospholipids by ultrastructural cytochemistry.

The presence of phospholipids within the interphase nucleus and in isolated chromatin, previously demonstrated by analytical biochemical methods, has been only rarely documented by cytochemical procedures, especially at the ultrastructural level. By means of a gold-conjugated phospholipase technique, we investigated the fine localization of endogenous phospholipids in the different nuclear domains in rat pancreas and in cell cultures. To reduce possible removal or displacement of phospholipids, different specimen preparation procedures such as cryofixation, cryosectioning, and freeze-fracturing were utilized. Apart from slight differences in efficiency among these methods, phospholipids have been cytochemically identified in the same nuclear domains: the interchromatin granules and fibers and the dense fibrillar component of the nucleolus. These results suggest that the phospholipids are an actual nuclear component, not randomly distributed in the nucleoplasm but mainly localized in the nuclear domains involved in the synthesis, maturation, and transport of ribonucleoproteins.     

Localization of proteasomes and proteasomal proteolysis in the mammalian interphase cell nucleus by systematic application of immunocytochemistry

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.     

The Sm Core Domain Mediates Targeting of U1 snRNP to Subnuclear Compartments Involved in Transcription and Splicing

In the mammalian cell nucleus pre-mRNA splicing factors such as U snRNPs are concentrated in distinct subnuclear compartments named perichromatin fibrils (PFs), interchromatin granules (IGs), interchromatin granule-associated zones (IG-associated zones), and coiled bodies (CBs). The structural requirement for the localization of U snRNPs to these domains was investigated by microinjection of digoxygenin-labeled in vitro-reconstituted U1 snRNPs and mutants thereof and subsequent analysis by immunoelectron microscopy. Wild-type U1 snRNP was targeted, after injection into the cytoplasm, to the nucleus and localized in PFs, IGs, IG-associated zones, and CBs. Thus, microinjected U1 snRNP particles exhibited a subnuclear localization similar to that previously observed for endogenous U1 snRNPs. Specific U snRNP proteins were shown not to be essential for subnuclear targeting since U1 snRNP mutants that did not bind to 70K, A, or C peptides were distributed in the cell nucleus in a pattern indistinguishable from that of wild-type U1 snRNP. Moreover, the Sm core domain, common to all spliceosomal U snRNPs, was shown to be sufficient for appropriate subnuclear distribution. Thus, these observations indicate that the Sm core domain, previously shown to be essential for nuclear import of spliceosomal U1 snRNPs, is also important for mediating the targeting to distinct nuclear subcompartments.     

Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures

A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.     

Still Immunodetectable Nuclear RNPs Are Extruded from the Cytoplasm of Spontaneously Apoptotic Thymocytes

The fate of different nuclear ribonucleoprotein (RNP) components was investigated during spontaneous apoptosis of thymocytes using specific monoclonal antibodies against snRNPs, hnRNPs, and ribosomal proteins at light and electron microscopy levels and by flow cytometry. It was found that, during apoptosis, nuclear RNP-containing structures (perichromatin granules, interchromatin granules, and perichromatin fibrils) segregate in the interchromatin space and cluster into heterogeneous aggregates of granules in which some of the structures may still be recognized morphologically. Along with the progress of apoptosis, the clusters are extruded from the nucleus into the cytoplasm, from which they are finally released via cytoplasmic extrusions. At all these stages, RNPs inside the clusters are always recognized by specific antibodies, even when they bleb out of the cell surface, thus suggesting that degradation of RNPs might be only partial during apoptosis. This could be potentially harmful in genetically susceptible subjects, as the appropriate MHC class II molecules may capture and present normally cryptic self-peptides. It is tempting to speculate that this event might have implications in the etiology of autoimmune diseases.     

Half a century of "the nuclear matrix"

A cell fraction that would today be termed "the nuclear matrix" was first described and patented in 1948 by Russian investigators. In 1974 this fraction was rediscovered and promoted as a fundamental organizing principle of eukaryotic gene expression. Yet, convincing evidence for this functional role of the nuclear matrix has been elusive and has recently been further challenged. What do we really know about the nonchromatin elements (if any) of internal nuclear structure? Are there objective reasons (as opposed to thinly veiled disdain) to question experiments that use harsh nuclear extraction steps and precipitation-prone conditions? Are the known biophysical properties of the nucleoplasm in vivo consistent with the existence of an extensive network of anastomosing filaments coursing dendritically throughout the interchromatin space? To what extent may the genome itself contribute information for its own quarternary structure in the interphase nucleus? These questions and recent work that bears on the mystique of the nuclear matrix are addressed in this essay. The degree to which gene expression literally depends on nonchromatin nuclear structure as a facilitating organizational format remains an intriguing but unsolved issue in eukaryotic cell biology, and considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the in vivo situation.