Yuichi Hiratsuka

A microrotary motor powered by bacteria

Biological molecular motors have a number of unique advantages over artificial motors, including efficient conversion of chemical energy into mechanical work and the potential for self-assembly into larger structures, as is seen in muscle sarcomeres and bacterial and eukaryotic flagella. The development of an appropriate interface between such biological materials and synthetic devices should enable us to realize useful hybrid micromachines. Here we describe a microrotary motor composed of a 20-μm-diameter silicon dioxide rotor driven on a silicon track by the gliding bacterium Mycoplasma mobile. This motor is fueled by glucose and inherits some of the properties normally attributed to living systems.

Utilization of myosin and actin bundles for the transport of molecular cargo.

The utilization of motor proteins for the movement and assembly of synthetic components is currently a goal of nanoengineering research. Application of the myosin actin motor system for nanotechnological uses has been hampered due to the low flexural rigidity of individual F-actin filaments. Here it is demonstrated how actin bundling can be used to affect the translational behavior of myosin-propelled filaments, transport molecules across a motor-patterned surface, and that the movement of bundled actin can be regulated photonically. These data suggest that actin bundling may significantly improve the applicability of the myosin motor for future nanotechnological applications.

Self-organized optical device driven by motor proteins

Significance Certain nanomaterials, such as protein molecules, produce various advanced functions when incorporated into an ordered system and, therefore, have large potential for use in engineering devices. To explore how to assemble a functional device from protein components, we have tried to create a molecular device inspired by a fish pigment cell, “melanophore.” We induced ordered assembly of protein molecules through self-organization of the proteins in a specific artificial microstructure and thereby succeeded in producing a melanophore-like optical device. We believe that self-organization of molecules in microstructures can be a powerful method for assembling functional molecular systems in future nanotechnology. Protein molecules produce diverse functions according to their combination and arrangement as is evident in a living cell. Therefore, they have a great potential for application in future devices. However, it is currently very difficult to construct systems in which a large number of different protein molecules work cooperatively. As an approach to this challenge, we arranged protein molecules in artificial microstructures and assembled an optical device inspired by a molecular system of a fish melanophore. We prepared arrays of cell-like microchambers, each of which contained a scaffold of microtubule seeds at the center. By polymerizing tubulin from the fixed microtubule seeds, we obtained radially arranged microtubules in the chambers. We subsequently prepared pigment granules associated with dynein motors and attached them to the radial microtubule arrays, which made a melanophore-like system. When ATP was added to the system, the color patterns of the chamber successfully changed, due to active transportation of pigments. Furthermore, as an application of the system, image formation on the array of the optical units was performed. This study demonstrates that a properly designed microstructure facilitates arrangement and self-organization of molecules and enables assembly of functional molecular systems.

Micrometer-sized molecular robot changes its shape in response to signal molecules

An amoeba-like molecular robot changes its shape in response to sequence-designed DNA signal molecules. Rapid progress in nanoscale bioengineering has allowed for the design of biomolecular devices that act as sensors, actuators, and even logic circuits. Realization of micrometer-sized robots assembled from these components is one of the ultimate goals of bioinspired robotics. We constructed an amoeba-like molecular robot that can express continuous shape change in response to specific signal molecules. The robot is composed of a body, an actuator, and an actuator-controlling device (clutch). The body is a vesicle made from a lipid bilayer, and the actuator consists of proteins, kinesin, and microtubules. We made the clutch using designed DNA molecules. It transmits the force generated by the motor to the membrane, in response to a signal molecule composed of another sequence-designed DNA with chemical modifications. When the clutch was engaged, the robot exhibited continuous shape change. After the robot was illuminated with light to trigger the release of the signal molecule, the clutch was disengaged, and consequently, the shape-changing behavior was successfully terminated. In addition, the reverse process—that is, initiation of shape change by input of a signal—was also demonstrated. These results show that the components of the robot were consistently integrated into a functional system. We expect that this study can provide a platform to build increasingly complex and functional molecular systems with controllable motility.

Three approaches to assembling nano-bio-machines using molecular motors

Efforts to use protein molecular motors as nanoactuators are making rapid progress. For instance, it is now possible to carry out directional transport of small cargo along microtracks or microchannels using kinesin-microtubule systems, which could be the basis of micro-conveyor belts or molecular shuttles. However, the applicability of protein-based devices is limited by their poor stability in artificial environments. In addition, assembly of complex, intelligent microdevices or systems will likely require bottom-up self-assembly, and we still do not have sufficient knowledge to rationally design self-assembling protein-based microdevices or systems. One approach to solving the problems associated with protein-based systems is to use DNA-based nanodevices, which are amenable to rational design. Indeed, ingenious design has enabled realization of DNA-based nanoactuators and self-assembled micropatterns of various shapes. One also could use cells, organelles, or tissues as preassembled motile units, and several motile devices have already been realized using this approach. In addition to being less prone to the assemaly problems, cell-based microdevices have the advantage that the motile units reproduce themselves, and genetically encoded functional modifications can be replicated effortlessly. These protein-based, DNA-based, and cell-based systems each have distinct advantages and disadvantages, so that hybrid devices combining the best characteristics of all three would seem the most likely to succeed.

Sequential parylene lift-off process for selective patterning of biological materials

This paper describes a method of selective patterning of various types of biological materials with parylene lift-off process. Multiple parylene thin sheets with a microhole array were formed on a glass substrate, and were then sequentially peeled off during the pattering process. Using this method we have achieved high density patterning of different kinds of beads, lipids and proteins on the same substrate, respectively. We have also patterned proteins on a parylene sheet, and rolled up the sheet to a cylindrical structure as a demonstration of 3D protein chips.

Shape control of filamentous motor proteins for bio-nano driving units

This paper describes a technique for controlling the shape of filamentous motor proteins for the bio-nano driving units in MEMS devices. In this experiment, we have used Actin, a protein to construct cytoskeleton actin monomers (G-actin) polymerize in high salt condition and form filaments (F-actin); the filaments move when they bind with the motor protein (Myosin) in ATP (adenosine tri-phosphate) solution. Fascin, a putative bundling protein, tightly bundles several F-actins together to form tight bundles of actin [1]. When G-actin and the fascin solution was confined and polymerized in the polydimethylsiloxane (PDMS) or parylene micro chambers, we found that the polymerized actin bundles followed the geometry of chambers, and then formed several shapes, such as circles, rods, triangles or squares. Since the bundled actins still have motility, we believe this technique is useful for forming a desired pattern of bio-molecular motors toward the actuation of MEMS/NEMS devices.

Amino acids 519–524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change

Residues 519–524 of Dictyostelium myosin II form a small surface loop on the actin binding face, and have been suggested to bind directly to actin through high affinity hydrophobic interactions. To test this hypothesis, we have characterized mutant myosins that lack this loop in vivo and in vitro. A mutant myosin in which this loop was replaced by an Ala residue (Δ519–524/+A) was non-functional in vivo. Replacement with a single Gly residue instead of Ala yielded partial function, suggesting that structural flexibility, rather than hydrophobicity, is the key feature of the loop. The in vivo phenotype of the mutant enabled us to identify a number of additional amino acid changes that restore function to the Δ519–524/+A mutation. Intriguingly, many of these, including L596S, were located at some distances away from the 519–524 loop. We have also isolated suppressors for the L596S mutant myosin, which was not functional in vivo. The suppressors for Δ519–524/+A and those for L596S showed complementary charge patterns. In ATPase assays, Δ519–524/+A S1 showed very low activity and little enhancement by actin, whereas L596S S1 was hyper active and displayed enhanced affinity for actin. In motility assays, Δ519–524/+A myosin released actin filaments upon addition of ATP and was unable to support movements. L596S myosin was also inactive, but in this case actin filaments stayed immobile even after the addition of ATP. Transient kinetic measurements demonstrated that Δ519–524/+A S1 is not only slower than wild type to bind actin filaments, but also slower to dissociate from actin filaments. Based on these results, we concluded that the 519–524 loop is not a major actin binding site but aids actin binding by facilitating a critical conformational change.

Feasibility study on an optical device operated through self-organizations of microtubules and kinesin motors

Inspired by melanophores, we envisioned an optical device which was switched between bright and dark states, reversibly. We proposed the working principle of the device which utilized self-organizations of microtubules and kinesin motors. By using a computer simulation, we investigated the feasibility of the envision device. The simulation showed that the working principle was feasible. The simulation also indicated physical properties governing the operation times.

Towards a microrotary motor driven by motor proteins

Biological molecular motors, motor proteins, have a number of unique advantages over artificial motors, including efficient conversion of chemical energy into mechanical work and the potential for self-assembly into larger structures. This paper describes a bio- hybrid micro rotary motor using kinesin/ microtubule motor. We developed a simple fabrication process for a micro-structure by one-time deposition of parylene and a selective immobilization of protein molecules onto the specific region of its 3D microstructure. Using the parylene micro-rotor, we are challenging to make the micro motor, which smoothly and stably rotates by biological motor at least for few hours.