Core histone charge and linker histone H1 effects on the chromatin structure of Schizosaccharomyces pombe.

Histones are highly conserved proteins among eukaryotes. However, yeast histones are more divergent in their sequences. In particular, the histone tail regions of the fission yeast, Schizosaccharomyces pombe, have fewer lysine residues, making their charges less positive than those of higher eukaryotes. In addition, the S. pombe chromatin lacks linker histones. How these factors affected yeast chromatin folding was analysed by biochemical reconstitution in combination with atomic force microscopy. Reconstitution of a nucleosome array showed that S. pombe chromatin has a more open structure similar to reconstituted human acetylated chromatin. The S. pombe nucleosomal array formed thinner fibers than those of the human nucleosomal array in the presence of mammalian linker histone H1. Such S. pombe fibers were more comparable to human acetylated fibers. These findings suggest that the core histone charges would determine the intrinsic characteristics of S. pombe chromatin and affect inter-nucleosomal interactions.

Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope.

Atomic force microscopy has been used to visualize nano-scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non-fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single-cell manipulation techniques.

Atomic force microscopy dissects the hierarchy of genome architectures in eukaryote, prokaryote, and chloroplast.

Because of its applicability to biological specimens (nonconductors), a single-molecule-imaging technique, atomic force microscopy (AFM), has been particularly powerful for visualizing and analyzing complex biological processes. Comparative analyses based on AFM observation revealed that the bacterial nucleoids and human chromatin were constituted by a detergent/salt-resistant 30-40-nm fiber that turned into thicker fibers with beads of 70-80 nm diameter. AFM observations of the 14-kbp plasmid and 110-kbp F plasmid purified from Escherichia coli demonstrated that the 70-80-nm fiber did not contain a eukaryotic nucleosome-like "beads-on-a-string" structure. Chloroplast nucleoid (that lacks bacterial-type nucleoid proteins and eukaryotic histones) also exhibited the 70-80-nm structural units. Interestingly, naked DNA appeared when the nucleoids from E. coli and chloroplast were treated with RNase, whereas only 30-nm chromatin fiber was released from the human nucleus with the same treatment. These observations suggest that the 30-40-nm nucleoid fiber is formed with a help of nucleoid proteins and RNA in E. coli and chroloplast, and that the eukaryotic 30-nm chromatin fiber is formed without RNA. On the other hand, the 70-80-nm beaded structures in both E. coli and human are dependent on RNA.

Fast-scanning atomic force microscopy reveals the molecular mechanism of DNA cleavage by ApaI endonuclease.

Newly developed fast-scanning atomic force microscopy (AFM) allows the dissection of molecular events such as DNA-enzyme reactions at the single-molecule level. With this novel technology, a model is proposed of the DNA cleavage reaction by a type IIP restriction endonuclease ApaI. Detailed analyses revealed that ApaI bound to DNA as a dimer and slid along DNA in a one-dimensional diffusion manner. When it encountered a specific DNA sequence, the enzyme halted for a moment to digest the DNA. Immediately after digestion, the ApaI dimer separated into two monomers, each of which remained on the DNA end and then dissociated from the DNA end. Thus, fast-scanning AFM is a powerful tool to aid the understanding of protein structures and dynamics in biological reactions at the single-molecule level in sub-seconds.

Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding.

The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

Histone core slips along DNA and prefers positioning at the chain end.

We studied the stability and dynamics of a model of a nucleosome, the fundamental unit for the packing of long DNA in eukaryotes, using a Brownian dynamics simulation. For the proper folding of a stiff polymer on a core particle, moderate attractive interaction is shown to be essentially important, which explains the empirical experimental protocol for the reconstitution of nucleosomes. The effect of the chain end on the positioning of the core particle is examined and compared with the experimental data by atomic force microscopy measurement. It is also suggested that the core particle exhibits sliding motion along the chain as a manifestation of Brownian motion.

P-type ATPase diversity and evolution: the origins of ouabain sensitivity and subunit assembly.

Molecular aspects of the diversity of P-type ATPases are explored in this review. From the substrate specificities among different ATPase molecules, the existence of isoforms within a single class of pump becomes evident and it is now recognized as a universal phenomenon. From the phylogenetic analyses using a vast collection of the deduced amino acid sequences for the P-type ATPase subunits, it also becomes evident that the divergence of substrate-specificity occurred early in the evolution and has been conserved ever since. Further extensive analyses identify a set of novel isoforms that retain an ancestral characteristic of the Na+/K+-(H+/K+-)ATPases in invertebrates.

Atomic force microscopy proposes a 'kiss and pull' mechanism for enhancer function. off.

The DNase I-hyper-sensitive sites (HS2-HS4) in the beta-globin gene enhancer region (locus control region; LCR) have been known as the target of Bach1/MafK heterodimers. We have demonstrated previously by utilizing atomic force microscopy (AFM) that Bach1/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 Bach1/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 Bach1/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.

Atomic force microscopy with carbon nanotube probe resolves the subunit organization of protein complexes.

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.

DNA phase transition promoted by replication initiator.

DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction. DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination. It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions. Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures. A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2). When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state. This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand. Furthermore, RepE54 is not wrapped by DNA repeatedly. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.

Atomic force microscopy sees nucleosome positioning and histone H1-induced compaction in reconstituted chromatin.

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 Xenopus 5S 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.

The Na+,K+-ATPase carrying the carboxy-terminal Ca2+/calmodulin binding domain of the Ca2+ pump has 2Na+,2K+ stoichiometry and lost charge movement in Na+/Na+ exchange.

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) 271-2751. The ouabain-resistant mutant of this chimera (ORalpha1-CBD) was constructed to further investigate the effect of the CBD on ion-transport properties. The ORalpha1-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 Ca2+/calmodulin binding domain of the Ca2+-ATPase linked to the Na+,K+-ATPase alters transport stoichiometry.

Using Xenopus oocytes as an expression system, we have investigated ion-transport and ouabain-binding properties of a chimeric ATPase (alpha1-CBD; Ishii and Takeyasu (1995) EMBO J. 14, 58-67) formed by the alpha1-subunit of chicken Na+,K(+)-ATPase (alpha1) and the calmodulin binding domain (CBD) of the rat plasma membrane Ca2(+)-ATPase. alpha1-CBD can be expressed and transported to the oocyte plasma membrane without the beta-subunit, and shows ouabain binding. In contrast to ouabain binding, this chimera requires the beta-subunit for its cation (Na+ and K+) transport activity. alpha1-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.