Ultrastructural and chemical study of the chromatin during spermiogenesis of the fish Scyliorhinus caniculus (L.) (author's transl)

Electron microscopic, cytochemical and biochemical techniques were applied to study structural aspects and changes in nuclear components during the spermiogenesis of Scyliorhinus caniculus. Five major stages of nuclear differentiation were recognized and characterized by variations in the organization and chemical properties of chromatin. Stage I is analogous to a somatic nucleus with heterogeneous chromatin. At the second stage, the nuclear content is dispersed but the chromatin fibers are of the same diameter as those of the stage I. The nuclear elongation begins at stage III, the DNP fibers running preferentially parallel to the long axis of the nucleus. During these early modifications of chromatin structure appear two new basic nuclear proteins (S 1 and S 2) which migrate faster than histones but typical histones remain assosciated with these nuclei. In later elongation stage (stage IV), the chromatin fibers organize in a helical form and fuse side by side giving lamellar systems which have a reticular structure. At the end of this stage, the nuclear material has become uniformly compact. These late variations in chromatin organization are parallel to the association of chromatin with new basic nuclear proteins (S 3, S 4, Z 1, Z 2 and Z 3). The cytochemical and electrophoretical properties of one of these proteins (S 4) which appears at the end of spermiogenesis are similar to those of a protamine. In stage V, the chromatin is homogeneous and the nucleus assumes a helical configuration beginning at the posterior end. The deoxyribonucleoproteins of the mature sperm show some novel chemical characters, including the appearance of a stable nuclear acidophilia with the ALFERT and GESCHWIND method and extraction with 0.25 N HCl of one of the basic protein fractions newly appeared in late spermiogenesis (Z 3), two other fractions (Z 1 and Z 2) being extracted with a more drastic procedure. The other fractions described before are no more detectable.     

Dehydrins: A commonalty in the response of plants to dehydration and low temperature

Among proteins that accumulate in plants in response to dehydrative forces or low temperature, dehydrins (late embryogenesis abundant [ Lea ] D11 family) have been the most commonly observed. Dehydrins are composed of several typical domains joined together in a few characteristic patterns, with numerous minor permutations. These domains include one or more putative amphipathic a -helix forming consensus regions, a phosphorylatable tract of Ser residues, and an N-terminal consensus sequence. Lesser conserved domains are also present at various positions, particularly between the putative a -helix forming domains, where they may occur as tandem repeats. This medley of permutations is mirrored by a wide size range of dehydrin polypeptides from less than 100 to nearly 600 amino acid residues. As of yet, the fundamental biochemical mode of action of dehydrins has not been demonstrated, but a number of immunolocalization and cell fractionation studies have established that dehydrins can be located in the nucleus or cytoplasm. Furthermore, it appears that these proteins associate with macromolecules ranging from nucleoprotein complexes in the nucleus to an endomembrane sheath in the cytoplasm. At present, all observations are consistent with a hypothesis that dehydrins are surfactants capable of inhibiting the coagulation of a range of macromolecules, thereby preserving structural integrity.     

Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb.

The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets.     

Development of the sperm head in the caddis fly Potamophylax rotundipennis (Insecta,Trichoptera)

The restructuring of the sperm head has been examined in a caddis fly, Potamophylax rotundipennis (Limnephilidae), using light and electron microscopy. The roughly spherical nuclei of young spermatids are transformed into needle-shaped elements in advanced spermatids. During this process, the nuclei transiently become sickle-shaped. Prominent structural changes occur within the nucleus during spermiogenesis. The chromatin of spherical and slightly elongated nuclei has an amorphous appearance, then coarse granules become apparent, chromatin threads are visible in fully elongated nuclei and finally lamellar elements appear. During the changes in chromatin texture, a dense layer, the chromatin rim, develops transiently. This feature of the chromatin surface is interpreted as the structural expression of exchanges between nucleus and cytoplasm. A microtubular manchette is formed at the cytoplasmic face of the nuclear envelope. Whereas the manchette covers the full perimeter of the nucleus in early stages of elongation, gaps in the palisade of microtubules appear before the nuclear diameter decreases and needle-shaped nuclei develop. It is possible that the intermittent deployment of manchette microtubules is involved in reducing the nuclear diameter towards the end of nuclear elongation. The delayed detachment of the chromatin from the posterior pole of the nucleus, observed at the onset of nuclear clongation, points to local modifications of the nuclear envelope responsible for the connection of the centriole adjunct and the flagellum with the posterior pole of the nucleus.     

BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication

We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.     

Mimicking the plant cell interior under water stress by macromolecular crowding: disordered dehydrin proteins are highly resistant to structural collapse

The dehydrins are a class of drought-induced proteins in plants that lack a fixed three-dimensional structure. Their specific molecular action, as well as the reason for their disordered character, is as yet poorly understood. It has been speculated, however, that the dehydrins are tuned to acquire a biologically active structure only under the conditions in which they normally function (i.e. upon dehydration). To test this hypothesis, we here investigate the effect of reduced water content and macromolecular crowding on three dehydrins from Arabidopsis (Arabidopsis thaliana). As a simplistic model for mimicking cellular dehydration, we used polyethylene glycol, glycerol, and sugars that plants naturally employ as compatible solutes (i.e. sucrose and glucose). Macromolecular crowding was induced by the large polysaccharides Ficoll and dextran. The results show that the dehydrins are remarkably stable in their disordered state and are only modestly affected by the solvent alterations. A notable exception is the dehydrin Cor47, which shows a small, intrinsic increase in helical structure at high concentrations of osmolytes. We also examined the effect of phosphorylation but found no evidence that such posttranslational modifications of the dehydrin sequences modulate their structural response to osmolytes and crowding agents. These results suggest that the dehydrins are highly specialized proteins that have evolved to maintain their disordered character under conditions in which unfolded states of several globular proteins would tend to collapse.     

Chromatin as a regulative architecture of the early developmental functions of mammalian embryos after fertilization or nuclear transfer

Nuclear transfer of a somatic nucleus into an enucleated oocyte has demonstrated in several mammalian species that the chromatin of a differentiated nucleus can be reprogrammed so as to be able to direct the full development of the reconstructed embryo. This review focus on the timing of the early events that allow the return of somatic chromatin to a totipotent state. Our understanding of the modifications associated with chromatin remodeling is limited by the low amount of biological material available in mammals at early developmental stages and the fact that very few genetic studies have been conducted with nuclear transfer embryos. However, the importance of several factors such as the covalent modifications of DNA through the methylation of CpG dinucleotides, the exchange of histones through a reorganized nuclear membrane, and the interaction between cytoplasmic oocyte components and nuclear complexes in the context of nuclear transfer is becoming clear. A better characterization of the changes in somatic chromatin after nuclear transfer and the identification of oocyte factors or structures that govern the formation of a functional nucleus will help us to understand the relationship between chromatin structure and cellular totipotency.     

Nuclear apoptotic changes: an overview

Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post-translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components.     

Dynamics of chromatin decondensation reveals the structural integrity of a mechanically prestressed nucleus

Genome organization within the cell nucleus is a result of chromatin condensation achieved by histone tail-tail interactions and other nuclear proteins that counter the outward entropic pressure of the polymeric DNA. We probed the entropic swelling of chromatin driven by enzymatic disruption of these interactions in isolated mammalian cell nuclei. The large-scale decondensation of chromatin and the eventual rupture of the nuclear membrane and lamin network due to this entropic pressure were observed by fluorescence imaging. This swelling was accompanied by nuclear softening, an effect that we quantified by measuring the fluctuations of an optically trapped bead adhered onto the nucleus. We also measured the pressure at which the nuclear scaffold ruptured using an atomic force microscope cantilever. A simple theory based on a balance of forces in a swelling porous gel quantitatively explains the diffusive dynamics of swelling. Our experiments on decondensation of chromatin in nuclei suggest that its compaction is a critical parameter in controlling nuclear stability.     

Repair of O6-ethylguanine DNA lesions in isolated cell nuclei. Presence of the activity in the chromatin proteins

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6-ethylguanine and 7-ethylguanine during a subsequent incubation at 37 degrees C. The rate of O6-ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6-ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7-ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7-ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase. The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6-ethylguanine from an added ethylated DNA. No free O6-ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6-ethylguanine were released either, indicating that the disappearance of O6-ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease. Activities capable of removing O6-ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2-3-times the repair activity of the chromatin proteins.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.     

Study of the structural and structure-functional subdomains in the interphase nucleus by photostabilization

The role of the nuclear matrix components in the organization of structural and functional domains of interphase nuclei was studied using irradiation with blue light in the presence of a photosensibilized agent (Ethidium bromide). Nuclear domain resistance to extractive solution (2 M NaCl) treatment served as a criterion of irradiation-induced stabilization of different nuclear domains. The following results have been obtained: 1) the structural organization of the complexes of chromatin and clusters of replication does not depend on the state of the nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear matrix by Cu(2+)-ions is not sufficient for the organization of chromatin domains; 3) irradiation in the presence of Ethidium bromide stabilizes domains of the nuclei, but does not lead to stabilization of the nuclear matrix internal network. Hence, the irradiation prevented extraction from the nuclear domains of nonhistone proteins which were not standard matrix proteins. Based on the results obtained, a hypothesis was proposed about a coexistence of two groups of nonhistone proteins in the cell nucleus. The first group includes proteins of the nuclear matrix involved in immobilization of scafford attachment regions and active genes. The second group includes some hypothetical structural proteins participating only in compaction of DNA of condensed chromatin.     

Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes

Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal–nuclear–chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations.     

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.     

Dehydrin stress proteins in <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. needles under conditions of extreme climate of Yakutia

This is the first study to investigate stress proteins dehydrins with the use of specific antibodies in the Scots pine (Pinus sylvestris L.) needles and their changes in the annual cycle under extreme climate of Yakutia. No pronounced polymorphism of major dehydrins (14–15 and 66 kDa) has been found during the winter dormancy period of P. sylvestris. A clear correlation between the seasonal variations in dehydrins and changes in the water content in needles was revealed. Consistently high levels of dehydrins was retained throughout the period of low negative temperatures. It is assumed that dehydrins can participate in the formation of P. sylvestris L. resistance to the permafrost conditions.     

Chromatin decondensation and nuclear reprogramming by nucleoplasmin

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.