Kiwamu Saito

A high-speed atomic force microscope for studying biological macromolecules

The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450–650 kHz) and small spring constants (150–280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 × 100 pixel2 image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm).

A High-Speed Atomic Force Microscope for Studying Biological Macromolecules in Action

The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in solution. However, there is no effective time axis with AFM; commercially available AFMs require minutes to capture an image, but many interesting biological processes occur at a much higher rate. Hence, what we can observe using the AFM is limited to stationary molecules, or those moving very slowly. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed various devices optimized for high-speed scanning. Combining these devices has produced an AFM that can capture a 100×100 pixel image within 80 ms, thus generating a movie consisting of many successive images of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica, in solution.

Orientation Dependence of Displacements by a Single One-Headed Myosin Relative to the Actin Filament

Displacements of single one-headed myosin molecules in a sparse myosin-rod cofilament were measured from bead displacements at various angles relative to an actin filament by dual optical trapping nanometry. The sparse myosin-rod cofilaments, 5-8 micron long, were synthesized by slowly mixing one-headed myosin prepared by papain digestion with myosin rods at molar ratios of 1:400 to 1:1500, so that one to four one-headed myosin molecules were on average scattered along the cofilament. The bead displacement was approximately 10 nm at low loads ( approximately 0.5 pN) and at angles of 5-10 degrees between the actin and myosin filaments (near physiologically correct orientation). The bead displacement decreased with an increase in the angle. The bead displacement at nearly 90 degrees was approximately 0 nm. When the angle was increased to approximately 150 degrees-170 degrees, the bead displacements increased to 5 nm. A native two-headed myosin showed similar size and orientation dependence of bead displacements as a one-headed myosin.

Movement of single myosin filaments and myosin step size on an actin filament suspended in solution by a laser trap

Movement of single myosin filaments, synthesized by copolymerization of intact myosin and fluorescently labeled light meromyosin, were observed along a single actin filament suspended in solution by a dual laser trap in a fluorescence microscope. The sliding velocity of the myosin filaments was 11.0 +/- 0.2 micron/s at 27 degrees C. This is similar to that of actin moving toward the center from the tip (the physiological direction) of myosin filaments bound to a glass surface but several times larger than that in the opposite direction (Ishijima and Yanagida, 1991; Yanagida, 1993). This indicates that the movement of myosin filaments is dominated by the myosin heads on one side of the myosin filament, which are correctly oriented relative to the actin filament. The incorrectly oriented myosin heads on the other side do not interfere with the fast movement. The step size (displacement produced during one ATPase cycle) of correctly oriented myosin was estimated from the minimum number of myosin heads necessary to produce the maximum velocity. This was determined by measuring the velocities of various lengths of myosin filaments. The minimum length of the myosin filaments moving near the maximum velocity was 0.30-0.40 microns, which contains 20 +/- 5 correctly oriented myosin heads. This number leads to a myosin step size of 71 +/- 22 nm. This value probably represents the lower limit, because all of the myosin heads on the filament would not always interact with the actin filament. Thus, the myosin step size is considerably larger than the length of a power stroke expected from the physical size of a myosin head, 10-20 nm (Huxley, 1957, 1969).

IMAGING AND NANO-MANIPULATION OF SINGLE BIOMOLECULES

We have developed a new technique for imaging single fluorescent dye molecules by refining epifluorescence and total internal reflection fluorescence microscopies. In contrast to previously reported single fluorescent molecule imaging methods, in which specimens were immobilized on an air-dried surface, our method enables video-rate imaging of single molecules in aqueous solution. This approach enabled us to directly image the processive movement of individual fluorescently labeled kinesin molecules along a microtubule. This method was also used to visualize individual ATP turnover reactions of single myosin molecules. The method can be combined with molecular manipulation using an optical trap. A single kinesin molecule attached to a polystyrene bead was brought into contact with a microtubule adsorbed onto the glass surface. The lifetime of bound Cy3-nucleotide in the absence or presence of the microtubule was 10 s or 0.08 s, respectively, showing that ATPase activity of the kinesin is strongly activated by microtubules. As the present system is equipped with a nanometer sensor, elemental steps of a single kinesin molecule can also be measured. By simultaneously measuring the individual ATP turnovers and elementary mechanical events of a single kinesin molecule, we will be able to obtain a clear answer to the fundamental problem of how the mechanical events are coupled to the ATPase reaction.

The crystal and molecular structure of cytosine-glycyl-glycine-copper(II) complex, a biologically important ternary coordination complex

Abstract The title compound was prepared and studied to gain some insight into the structural basis for the protein-nucleic acid-metal ion interaction. The crystal structure has been determined from three-dimensional diffractometer X-ray data using Cu Kα radiation. The crystals are monoclinic, space group P2 1 c , with cell dimension; a=10.642(1)A, b=8.081(1)A, c=17.792(1)A, β=124.29(1)o, z=4. Amino and amide nitrogen, carboxyl O(8) of glycylglycine, N(3) of cytosine and O(2) of adjacent cytosine molecule coordinate to the central copper ion to form a square pyramid. An additional weak interaction in complex molecule between copper and O(2) of cytosine is also observed. The complex molecules are held together by hydrogen- and coordination-bonds in crystalline state.

Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.

Single molecule imaging and nanomanipulation of biomolecules

Publisher Summary This chapter discusses the single molecule imaging and nanomanipulation of biomolecules. The methods described in the chapter are applied to examining single nucleotidase reactions of other enzymes. It is possible to suspend a single DNA in solution by manipulation with dual optical traps, as well as to see single fluorescently labeled RNA polymerases. Directly observing the reading process of the DNA genetic information by a single RNA polymerase molecule is not just a dream now, but realistic. The techniques for single molecule imaging and manipulation is very powerful for studies not only of motility of motor proteins, but also of molecular genetics, signal transduction and processing in cell, dynamic molecular process of proteins.

Dual‐colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti‐Stokes fluorescence

We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti‐Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine‐5‐iodoacetamide interacting with a BODIPY FL‐labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3‐labelled ATP by single myosin molecules and sliding of a BODIPY FL‐labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.

Average Energies Required per Scintillation Photon and Energy Resolutions in NaI(Tl) and CsI(Tl) Crystals for Gamma Rays

The average energies required to produce one scintillation photon Ws were determined for 662 keV gamma rays in NaI(Tl) and CsI(Tl) crystals to be 15.0±1.3 and 13.3±1.1 eV, respectively, from the absolute numbers of photoelectrons measured for several combinations of a crystal and a photomultiplier tube (PMT) used as a vacuum photodiode. The numbers of scintillation photons were obtained by calculating the collection efficiency of scintillation photons at the photocathode using Monte Carlo simulations and by determining experimentally the photon-to-photoelectron conversion efficiency at PMT photocathode. The values of Ws determined in the present study are in good agreement with the theoretical values presented recently. The factors affecting energy resolutions were also examined. The calculated resolution agrees well with that obtained experimentally.