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Shige H Yoshimura

Publications and source records attributed to Shige H Yoshimura.

16 recordsLinked to original sources
PubMed2006

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL.

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.

Adenosine Diphosphate↗
PubMed2006

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy.

The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta.

Biomechanical Phenomena↗
PubMed2006

Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit.

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, approximately 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 approximately 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.

Animals↗
PubMed2006

Human small G proteins, ObgH1, and ObgH2, participate in the maintenance of mitochondria and nucleolar architectures.

The Obg subfamily protein is one of the P-loop small G proteins and is highly conserved in many organisms from bacteria to human. Two obg genes, obgH1 and obgH2, exist in the human genome. Both ObgH1 and ObgH2 showed similar GTPase activities (0.014 +/- 0.005 and 0.010 +/- 0.002/min for ObgH1 and ObgH2, respectively) to those of the bacterial Obg proteins and complemented the Obg function in Escherichia coli ribosome maturation, suggesting that the functions of Obg proteins are well conserved through evolution. Immunofluorescence microscopy of HeLa cells revealed that ObgH1 localizes in mitochondria, and ObgH2 in the dense fibrillar compartment region of the nucleolus. Knock-down of ObgH1 by RNAi induced mitochondria elongation, whereas knock-down of ObgH2 resulted in the disorganization of the nucleolar architecture. In conclusion, the two human Obg proteins have similar enzymatic activities that can complement bacterial Obg function, but show different cellular function(s) with different intracellular localizations.

Amino Acid Sequence↗
PubMed2005

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber.

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.

Chromatin↗
PubMed2004

Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy.

A small container of several to a few hundred microm3 (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of approximately 80 nm was found. In addition to this '80 nm fiber', a thinner '40 nm fiber' and a higher order 'loop' structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a 'coral reef structure' that also possessed the 80 nm fiber units, and, finally, into a 'tightly compacted nucleoid' that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid.

Bacterial Outer Membrane Proteins↗
PubMed2004

Molecular mechanisms of DNA end-loop formation by TRF2.

In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher-order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, approximately 1.8 (kb) with different end-structures and observed their higher-order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple-binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA-end structure (blunt, 3'-overhanging, telomeric, non-telomeric). However, when the DNA end carried the telomeric-3'-overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2-mediated DNA loop formation is independent of the end-structure and the 3'-overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end-loop formation by TRFs.

Binding Sites↗
PubMed2004

Atomic force microscopy demonstrates a critical role of DNA superhelicity in nucleosome dynamics.

Nucleosome is the most basic structural unit of eukaryotic chromosome, forming an 11 nm "beads-on-a-string" fiber. The molecular mechanism of chromatin folding toward higher-order structures (30 nm and thicker fibers) is speculative; however, it is thought to be critical for the regulation of transcription, replication, and chromosome propagation. We examined the relationship between the efficiency of the nucleosome formation and the physical properties of the template DNA. A series of plasmid DNA with different lengths (3, 5, 31, 56, or 106 kb) were prepared and, together with purified histones, used for the reconstitution of chromatin fibers by a salt-dialysis method. The reconstituted chromatin fibers were visualized and analyzed by atomic force microscopy (AFM). Based on the AFM images, the efficiency of the reconstitution was dependent on the length and the negative superhelical strain of the DNA used (i.e., the longer DNA had a higher efficiency in the reconstitution, because the longer plasmids retain much higher superhelical density than the shorter ones). These results suggest that the nucleosome dynamics are tightly coupled with the DNA superhelicity. This was further supported by the fact that the linearized or topoisomerase I-treated plasmids (relaxed circular) showed very low efficiency. Namely, the negative supercoiling promoted the efficient formation of the nucleosome but the positive supercoiling strongly inhibited it.

Centromere↗
PubMed2003

Channel induction by palytoxin in yeast cells expressing Na+,K+-ATPase or its chimera with sarco/endoplasmic reticulum Ca2+-ATPase.

Palytoxin (PTX) induces a cation channel through interaction with Na(+),K(+)-ATPase. It is unclear how this action relates to the enzyme catalytic activity. We examined whether the action of PTX depends on the catalytic domain specific for Na(+),K(+)-ATPase. Wild-type Na(+),K(+)-ATPase alpha-subunit (NNN) or its chimera (NCN), in which the catalytic domain was replaced with that of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, was co-expressed with beta-subunit in the yeast Saccharomyces cerevisiae. PTX (0.1-100 nM) increased K(+) efflux in NNN- or NCN-transfected cells to a similar degree but not in non-transfected cells. When ouabain-resistant NNN and NCN were expressed, PTX also increased K(+) efflux. Ouabain inhibited the effect of PTX in NNN or NCN cells but not in ouabain-resistant cells. These data suggest that the channel-forming action of PTX does not depend on the catalytic domain species.

Acrylamides↗
PubMed2003

On-substrate lysis treatment combined with scanning probe microscopy revealed chromosome structures in eukaryotes and prokaryotes.

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 approximately 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 approximately 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 approximately 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.

Cell Nucleus Structures↗
PubMed2003

Differential degradation of the Na+/K+-ATPase subunits in the plasma membrane.

The cell-surface expression of the Na(+)/K(+)-ATPase is tightly regulated at the ER/Golgi transit level. The assembly of the alpha- and beta-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 Na(+)/K(+)-ATPase subunits in the plasma membrane: that is, lysosomal degradation of alpha-subunits and proteasomal degradation of beta-subunits, and propose the possibility of the subunit dissociation in the plasma membrane.

Animals↗
PubMed2002

Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the head of SMC heterodimer.

Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented.

Adenosine Triphosphatases↗
PubMed2002

Vacuolar membrane dynamics revealed by GFP-AtVam3 fusion protein.

BACKGROUND: The plant vacuole is a multifunctional organelle that has various physiological functions. The vacuole dynamically changes its function and shape, dependent on developmental and physiological conditions. Our current understanding of the dynamic processes of vacuolar morphogenesis has suffered from the lack of a marker for observing these processes in living cells. RESULTS: We have developed transgenic Arabidopsis thaliana expressing a vacuolar syntaxin-related molecule (AtVam3/SYP22) fused with green fluorescent protein (GFP). Observations using confocal laser scanning microscopy demonstrated that the plant vacuole contained a dynamic membrane system that underwent a complex architectural remodelling. Three-dimensional reconstitution and time-lapse analysis of GFP-fluorescence images revealed that cylindrical and sheet-like structures were present in the vacuolar lumen and were moving dynamically. The movement, but not the structure itself, was abolished by cytochalasin D, an inhibitor of actin polymerization. This moving structure, which sometimes penetrated through the vacuolar lumen, possessed a dynamic membrane architecture similar to the previously recognized "transvacuolar strand." CONCLUSION: We propose two possible models for the formation of the vacuolar lumenal structure. Membrane structures including protruding tubules and reticular networks have recently been recognized in many other organelles, and may be actively involved in intra- and/or inter-organelle signalling.

Arabidopsis↗
PubMed2002

Chromatin reconstitution: development of a salt-dialysis method monitored by nano-technology.

The regulation of DNA replication and transcription is achieved by dynamic structural changes of chromatin in which a series of proteins will acquire accessibility to specific regions of the DNA strand. A combination of biochemistry and nano-technology is essential to address questions regarding the structural basis for such macromolecular mechanisms. In the present study, we established an efficient salt-dialysis method of chromatin reconstitution and employed atomic force microscopy (AFM) as a single-molecule-imaging technique, to monitor the efficiency of the reconstitution. At first, the reconstitution efficiency with short DNA molecules of several kilo-base pairs was low, although the salt dialysis yielded a "beads-on-a-string" structure of oligonucleosomes with each nucleosome trapping 158+/-27 bp DNA. However, the efficiency for nucleosome formation became higher when longer DNA molecules with a super-helical constraint were used. A statistical analysis of the obtained AFM images identified a first-order relationship between the efficiency of the reconstitution and the length of the super-coiled DNA used. A high efficiency of approximately 290 bp/nucleosome that is close to the in vivo situation was obtained with a approximately 100 kbp template DNA. This enabled the structure-function studies of long chromatin molecules under well-defined conditions.

Chromatin↗