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Cardiomyocyte (CM) nuclei are constantly exposed to mechanical stress, but how they maintain their nuclear shape remains unknown. In this study, we found that ventricular CM nuclei acquire characteristic prominent spatial organizations of heterochromatin (SOH), which are disrupted by high-level expression of H2B-mCherry in mice. SOH disruption was associated with nuclear softening, leading to extreme elongation and rupture under unidirectional mechanical stress. Loosened chromatin then leaks into the cytosol, causing severe inflammation and cardiac dysfunction. Although SOH disruption was accompanied by loosened higher-order genomic structures, the change in gene expression before nuclear deformation was mild, suggesting that SOH play major roles in nuclear structural integrity. Aged CM nuclei consistently exhibited scattered SOH and marked elongation. Furthermore, we provide mechanistic insight into the development and maintenance of SOH driven by chromatin compaction and condensate formation. These results highlight SOH as a safeguard of nuclear shape and genomic integrity against mechanical stress.

Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation1–6. Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis. Mitotic hyperphosphorylation of disordered repeat domains of Ki-67 generates alternating charge blocks in these domains and increases their propensity for liquid–liquid phase separation (LLPS). A phosphomimetic sequence and the sequences with enhanced charge blockiness underwent strong LLPS in vitro and induced chromosome periphery formation in vivo. Conversely, mitotic hyperphosphorylation of NPM1 diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution. Cell cycle-specific phase separation can be modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites.

The steady-state dynamics of karyopherin-dependent nuclear transport in a living cell is examined. The kinetic model established by a number of experimentally obtained parameters reveals how each step of the transport system contributes to maintaining steady-state cargo gradient and fluxes across the nuclear envelope.

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg2+or Ca2+at a scan rate of 1–2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.

This study demonstrates that the chromosome of the hyperthermophilic archaeon Thermococcus kodakarensisis organized into a heterogeneous structure created with histone and a novel protein TK0471/TrmBL2. TK0471/TrmBL2 plays dual roles as a chromosomal protein and as a global transcriptional repressor, and it is conserved in some archaeal and bacterial species.

Being distinct from bacteria and eukaryotes, Archaea constitute a third domain of living things. The DNA replication, transcription, and translation machineries of Archaea are more similar to those of eukaryotes, whereas the genes involved in metabolic processes show more similarity to their bacterial counterparts. We report here that TK0471/TrmB-like 2 (TrmBL2), in addition to histone, is a novel type of abundant chromosomal protein in the model euryarchaeon Thermococcus kodakarensis. The chromosome of T. kodakarensis can be separated into regions enriched either with histone, in which the genetic material takes on a "beads-on-a-string" appearance, or with TK0471/TrmBL2, in which it assumes a thick fibrous structure. TK0471/TrmBL2 binds to both coding and intergenic regions and represses transcription when bound to the promoter region. These results show that the archaeal chromosome is organized into heterogeneous structures and that TK0471/TrmBL2 acts as a general chromosomal protein as well as a global transcriptional repressor.

The cell-surface expression of the NaK-ATPase is tightly regulated at the ERGolgi transit level. The assembly of the ?- and ?-subunits is a prerequisite for the maturation of the enzyme and its exit from the ER. At present, the fate of the functional ATPase, once it reaches the plasma membrane, is obscure, although endocytosis, recycling, and degradation in the lysosome are known to be involved. In this study, we shall demonstrate the differential degradation of the NaK-ATPase subunits in the plasma membrane: that is, lysosomal degradation of ?-subunits and proteasomal degradation of ?-subunits, and propose the possibility of the subunit dissociation in the plasma membrane.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a ‘beads on a string’ structure with each nucleosome trapping 158±27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.

An altered ion‐transport stoichiometry from 3Na+,2K+to 2Na+,2K+is observed in a chimeric Na+,K+‐ATPase, which carries the Ca2+/calmodulin binding domain (CBD) of the plasma membrane Ca2+‐ATPase at its carboxy‐terminus [Zhao et al., FEBS Lett. 408 (1997) 271–275]. The ouabain‐resistant mutant of this chimera (ORα1‐CBD) was constructed to further investigate the effect of the CBD on ion‐transport properties. The ORα1‐CBD still shows the 2Na+,2K+stoichiometry. The loss of electrogenicity is accompanied by the disappearance of transient charge movements in the Na+/Na+exchange mode. We conclude that the binding of the third Na+ion, but not of the two others, in 3Na+,2K+transport mode apparently senses the electric field, and that the voltage‐dependent Na+binding is likely to be lost in the chimera with CBD.

The acute effects of olive oil, milk fat and safflower oil on postprandial lipemia and remnant lipoprotein metabolism were investigated.Eight Healthy male volunteers randomly underwent three types of oral fat-vitamin A loading tests. The test drink was a mixture of retinyl palmitate (RP)(50,000 IU of aqueous vitamin A/m2 body surface area) and one of the three types of oils (40 g of fat/m2 body surface area): olive oil (70.7% oleic acid of total fatty acids); milk fat (69.3% saturated fatty acid); safflower oil (74.2% linoleic acid).Olive oil significantly increased plasma triacylglycerol and RP concentrations 4 hours after fat loading, as compared to other fats. Increases of remnant like particle concentrations were higher after olive oil than after the other two fats.These results show that olive oil increases the magnitude of postprandial chylomicrons and chylomicron remnants compared to milk fat and safflower oil.

Using Xenopus oocytes as an expression system, we have investigated ion-transport and ouabain-binding properties of a chimeric ATPase (α 1 -CBD; Ishii and Takeyasu (1995) EMBO J. 14, 58-67) formed by the α 1 -subunit of chicken Na,K-ATPase (α 1 ) and the calmodulin binding domain (CBD) of the rat plasma membrane Ca-ATPase. α 1 -CBD can be expressed and transported to the oocyte plasma membrane without the β-subunit, and shows ouabain binding. In contrast to ouabain binding, this chimera requires the β-subunit for its cation (Na and K) transport activity. α 1 -CBD exhibits an altered stoichiometry of Na-K exchange. A detailed analysis of Na efflux, Rb uptake, pump current and ouabain binding suggests that the chimeric molecule can operate in an electrically silent 2Na-2K exchange mode and, with much lower probability, in its normal 3Na-2K exchange mode.

Using Xenopusoocytes as an expression system, we have investigated ion‐transport and ouabain‐binding properties of a chimeric ATPase (α1‐CBD; Ishii and Takeyasu (1995) EMBO J. 14, 58–67) formed by the α1‐subunit of chicken Na+,K+‐ATPase (α1) and the calmodulin binding domain (CBD) of the rat plasma membrane Ca2+‐ATPase. α1‐CBD can be expressed and transported to the oocyte plasma membrane without the β‐subunit, and shows ouabain binding. In contrast to ouabain binding, this chimera requires the β‐subunit for its cation (Na+and K+) transport activity. α1‐CBD exhibits an altered stoichiometry of Na+‐K+exchange. A detailed analysis of 22Na+efflux, 86Rb+uptake, pump current and ouabain binding suggests that the chimeric molecule can operate in an electrically silent 2Na+–2K+exchange mode and, with much lower probability, in its normal 3Na+–2K+exchange mode.

An altered ion-transport stoichiometry from 3Na +,2K +to 2Na +,2K +is observed in a chimeric Na +,K +-ATPase, which carries the Ca 2+/calmodulin binding domain (CBD) of the plasma membrane Ca 2+-ATPase at its carboxy-terminus [Zhao et al., FEBS Lett. 408 (1997) 271–275]. The ouabain-resistant mutant of this chimera (ORα1-CBD) was constructed to further investigate the effect of the CBD on ion-transport properties. The ORα1-CBD still shows the 2Na +,2K +stoichiometry. The loss of electrogenicity is accompanied by the disappearance of transient charge movements in the Na +/Na +exchange mode. We conclude that the binding of the third Na +ion, but not of the two others, in 3Na +,2K +transport mode apparently senses the electric field, and that the voltage-dependent Na +binding is likely to be lost in the chimera with CBD.

The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments. Direct measurements of the crowding states using a crowding-sensitive FRET (Förster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nuclear transport. Based on these findings, we propose a proximal control model of molecular crowding in vivothat is dynamically regulated at the molecular level.

Two cases of cerebral arterial occlusive disease with telangiectasia are described. The patients are white women (ages 39 and 25), who developed symptoms following prolonged use of oral contraceptives. Their courses were slow but progressive. This condition with its characteristic angiographic picture was originally reported in Japan as a variety of cerebral arterial occlusive disease with predilection for young women. Such cases have been reported following nonspecific arteritis, meningitis, radiation therapy, trauma, and in association with neurofibromatosis. A slowly progressive occlusion in the supraclinoid portion of the internal carotid artery of young patients may be the primary lesion. In the two cases described here, prolonged use of oral contraceptives may have been related to the occlusive disease and subsequent telangiectasia.

Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the ‘on-substrate’ lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, ∼40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80–100 nm. Thus, these results suggest that the ∼40 nm fiber would be a stable structural unit and fold the 80–100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes.

The proper function of the genome largely depends on the higher‐order architecture of the chromosome. To understand the detailed chromosome structure in a native state, we developed an on‐substrate procedure of subcellular fractionation suitable for the observation by atomic force microscopy (AFM). HeLa cells on a coverslip were successively treated with a detergent and a high‐salt solution to remove the cytoplasmic and nucleoplasmic materials. A closer observation of the nucleus by AFM revealed that the interphase chromosome is composed of a granular unit of ∼80 nm in diameter. Subsequent mild treatment with deoxyribonuclease I (10 U ml−1) exposed these units more clearly, which enabled us to uncover the 80‐nm granules forming a fibre of ∼80 nm width. In the cytoplasmic regions, cytoskeletal fibres with varying widths (10–70 nm) were observed. These observations suggest that the 80 nm granular fibre is a fundamental structural unit of the interphase chromosome. This on‐substrate procedure was also applied to Escherichia coli. Cells attached on a coverslip were successively treated with lysozyme and detergent to partially release the nucleoid onto the substrate. The AFM observation revealed that the ∼80 nm fundamental structural unit forms a granular fibre similar to that of HeLa cells. These results suggest that the fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

The DNase I-hyper-sensitive sites (HS2–HS4) in the β-globin gene enhancer region (locus control region; LCR) have been known as the target of Bachl/MafK heterodimers. We have demonstrated previously by utilizing atomic force microscopy (AFM) that Bachl/MafK mediates the formation of a looped-DNA structure in the LCR fragment. Here we perform further detailed analyses of the loop structure formed between each HSs by AFM, and propose a novel model for the enhancer/protein interaction: the Bachl/MafK heterodimer preferentially binds to HS2 with highest affinity and to HS3 with lower affinity. However, they assemble to each other to form a stable complex of four heterodimers and mediate a DNA-loop formation. Once the DNA loop is formed between HS2 and HS3, the Bachl/MafK complex at the HS3 side leaves the HS3 and starts to slide along the DNA strand towards HS2 with the other side of the complex fixed at the HS2 region. This ‘kiss and pull’ model will contribute to understand the function of regulatory proteins at enhancer regions in terms of higher-order structure of DNA, e.g. nucleosomes and chromatin.

Among many scanning probe microscopies, atomic force microscopy (AFM) is a useful technique to analyse the structure of biological materials because of its applicability to non-conductors in physiological conditions with high resolution. However, the resolution has been limited to an inherent property of the technique; tip effect associated with a large radius of the scanning probe. To overcome this problem, we developed a carbon nanotube probe by attaching a carbon nanotube to a conventional scanning probe under a well-controlled process. Because of the constant and small radius of the tip (2.5–10 nm) and the high aspect ratio (1 : 100) of the carbon nanotube, the lateral resolution has been much improved judging from the apparent widths of DNA and nucleosomes. The carbon nanotube probes also possessed a higher durability than the conventional probes. We further evaluated the quality of carbon nanotube probes by three parameters to find out the best condition for AFM imaging: the angle to the tip axis; the length; and the tight fixation to the conventional tip. These carbon nanotube probes, with high vertical resolution, enabled us to clearly visualize the subunit organization of multi-subunit proteins and to propose structural models for proliferating cell nuclear antigen and replication factor C. This success in the application of carbon nanotube probes provides the current AFM technology with an additional power for the analyses of the detailed structure of biological materials and the relationship between the structure and function of proteins.