Kunio Takeyasu

Carbon-nanotube tips for scanning probe microscopy: Preparation by a controlled process and observation of deoxyribonucleic acid

We report a controlled process to make carbon-nanotube tips for scanning probe microscopes. The process consists of three steps: (1) purification and alignment of carbon nanotubes using electrophoresis, (2) transfer of a single aligned nanotube onto a conventional Si tip under the view of a scanning electron microscope, and (3) attachment of the nanotube on the Si tip by carbon deposition. Nanotube tips fabricated using this procedure exhibit strong adhesion and are mechanically robust. Finally, the performance of these tips is demonstrated by imaging the fine structure of twinned deoxyribonucleic acid with tapping-mode atomic force microscopy in air.

Carbon nanotube tips for a scanning probe microscope: their fabrication and properties

We report a well controlled method to make carbon nanotube tips for a scanning probe microscope (SPM). A multiwalled carbon nanotube, which is purified by the electrophoresis, is transferred onto a conventional Si tip for a SPM using a scanning electron microscope (SEM) equipped with two independent specimen stages. The nanotube is fixed on the Si tip by electron beam deposition of carbon. A force curve measurement of nanotubes using the nanotube tips in the SEM reveals that Youngs modulus of a nanotube of 20 nm diameter is 1.1 TPa and the fixing of nanotubes by the carbon deposit is effective. The nanotube tips are used to image plasmid deoxyribonucleic acids on mica by tapping mode. The average resolution by using the nanotube tips is about two times higher than that by the best Si tips.

Autoimmune Disorders Associated with Gain of Function of the Intracellular Sensor MDA5

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

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.

The GTP binding protein Obg homolog ObgE is involved in ribosome maturation

Obg proteins belong to a subfamily of GTP binding proteins, which are highly conserved from bacteria to human. Mutations of obgE genes cause pleiotropic defects in various species but the function remained unclear. Here we examine the function of ObgE, the Obg homolog in Escherichia coli. The growth rate correlates with the amount of ObgE in cells. Co‐fractionation experiments further suggest that ObgE binds to 30S and 50S ribosomal subunits, but not to 70S ribosome. Pull‐down assays suggest that ObgE associates with several specific ribosomal proteins of 30S and 50S subunits, as well as RNA helicase CsdA. Purified ObgE cosediments with 16S and 23S ribosomal RNAs in vitro in the presence of GTP. Finally, mutation of ObgE affects pre‐16Sr‐RNA processing, ribosomal protein levels, and ribosomal protein modification, thereby significantly reducing 70S ribosome levels. This evidence implicates that ObgE functions in ribosomal biogenesis, presumably through the binding to rRNAs and/or rRNA‐ribosomal protein complexes, perhaps as an rRNA/ribosomal protein folding chaperone or scaffold protein.

Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy

The structure of Escherichia coli F0F1‐ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping‐mode operation. The majority of F0F1‐ATPases were visualized as spheres with a calculated diameter of , and a height of from the membrane surface. F0 sectors were visualized as two different ring‐like structures (one with a central mass and the other with a central hollow of depth) with a calculated outer diameter of . The two different images possibly represent the opposite orientations of the complex in the membranes. The ring‐like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1–3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 ± 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.

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 (∼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.

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.

Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly.

Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled‐coil with the terminal globular domains and the central hinge. Condensin and cohesin holo‐complexes contain three and two non‐SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo‐condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21‐bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled‐coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed double‐stranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non‐SMC heterotrimeric complex.