Akira Kakugo

Dynamic cell behavior on synthetic hydrogels with different charge densities

A series of poly(NaSS-co-DMAAm) hydrogels with ζ potentials (ζ) in the range −8.8–−20.5 mV and a constant Youngs modulus (E) of ca. 200 kPa were designed for studying the effect of the charge density of hydrogels on dynamic cell behavior. The ζ was adjusted by tuning the molar friction of negatively charged sodium p-styrene sulfonate (NaSS) contained in the monomer mixture [NaSS and N, N-dimethyl acrylamide (DMAAm)] for gelation. A critical ζ potential, which controls cell behavior, ζcrit = −14.0 mV, was observed. When ζ > ζcrit, the cells exhibited a small spreading area, fast migration velocity, and a large migration distance, with concurrent deficiency in actin fibers and less prominent focal adhesions. Conversely, when ζ ≤ ζcrit, the cells exhibited a large spreading area, slow migration velocity, and a short migration distance, with concurrent well-developed actin fibers and prominent focal adhesions. Furthermore, cells repeatedly oscillated in a stick–slip mode from a spreading shape to a round shape on the hydrogels with ζ > ζcrit, although were unable to proliferate. The cell behavior is well correlated with the adsorbed fibronectin on the hydrogel surfaces.

Prolongation of the Active Lifetime of a Biomolecular Motor for in Vitro Motility Assay by Using an Inert Atmosphere

Over the last few decades, the in vitro motility assay has been performed to probe the biophysical and chemo-mechanical properties as well as the self-organization process of biomolecular motor systems such as actin-myosin and microtubule-kinesin. However, aggression of the reactive oxygen species (ROS) and concomitant termination of the activity of biomolecular motors during investigation remains a drawback of this assay. Despite enzymatic protection that makes use of a combination of glucose, glucose oxidase, and catalase, the active lifetime of biomolecular motors is found to be only a few hours and this short lifetime restricts further study on those systems. We have solved this problem by using a newly developed system of the in vitro motility assay that is conducted in an inert nitrogen gas atmosphere free of ROS. Using microtubule-kinesin as a model system we have shown that our system has prolonged the active lifetime of the biomolecular motor until several days and even a week by protecting it from oxidative damage.

Antifouling activity of synthetic polymer gels against cyprids of the barnacle (Balanus amphitrite) in vitro

Barnacle (Balanus amphitrite) settlement on synthetic hydrogels with various chemical structures was tested in laboratory assays. The results demonstrated that cyprids settle less or not at all on hydrogels and PDMS elastomer compared with the polystyrene control. The low settlement on gels is most likely due to the ‘easy release’ of initially attached cyprids from the gel surfaces. This low adhesion of cyprids is independent of surface hydrophilicity or hydrophobicity, and of surface charge. The results also revealed that hydrogels can be categorized into two groups. One group showed an extremely strong antifouling (AF) performance that was independent of the elasticity (E) or swelling degree (q) of the gels. The second group showed relatively less strong AF performance that was E- or q-dependent. In the latter case, E, rather than the q, may be the more important factor for cyprid settlement.

Microtubule bundle formation driven by ATP : the effect of concentrations of kinesin, streptavidin and microtubules

Recently, a method was established for the formation of microtubule (MT) assemblies by an active self-organization (AcSO) process, in which MTs were crosslinked during sliding motion on a kinesin-coated surface, and this was coupled with adenosine triphosphate (ATP) hydrolysis. Streptavidin (ST) was the glue used to crosslink biotin-labeled MTs. Although most of the MT assemblies were in the bundle form, they varied in size, shape and motility, depending on the initial conditions used. In this paper, we systematically examined the effects of the concentrations of kinesin, ST and MT on the formation of MT bundles under the initial conditions of the process.

Selective formation of a linear-shaped bundle of microtubules.

By using rigid microtubules (MTs) prepared by polymerization with guanylyl-(alpha, beta)-methylene-diphosphonate GMPCPP, giant straight-shaped MT bundles were selectively obtained through a dynamic self-assembly process. We demonstrate the effect of the rigidity on the shape and motility of MT bundle composed of GMPCPP-polymerized MTs (GMPCPP-MTs) compared with control MTs that were polymerized with GTP and stabilized with paclitaxel.

Antifouling properties of tough gels against barnacles in a long-term marine environment experiment

In the marine environment, the antifouling (AF) properties of various kinds of hydrogels against sessile marine organisms (algae, sea squirts, barnacles) were tested in a long-term experiment. The results demonstrate that most hydrogels can endure at least 2 months in the marine environment. In particular, mechanically tough PAMPS/PAAm DN and PVA gels exhibited AF activity against marine sessile organisms, especially barnacles, for as long as 330 days. The AF ability of hydrogels toward barnacles is explained in terms of an ‘easy-release’ mechanism in which the high water content and the elastic modulus of the gel are two important parameters.

Formation of well-oriented microtubules with preferential polarity in a confined space under a temperature gradient.

Tubulin polymerization in a confined space under a temperature gradient produced well-oriented microtubule assemblies with preferential polarity. We analyzed the structure and polarity of these assemblies at various levels of resolution by performing polarized light microscopy (millimeter order), fluorescence microscopy (micrometer order), and transmission electron microscopy (nanometer order).

Formation of ring-shaped assembly of microtubules with a narrow size distribution at an air–buffer interface

Biopolymers such as actin, microtubules and DNA are well known for their fascinating in vivo self-organization phenomena. Considerable efforts have been devoted to mimicking their organization process in vitro that produced ring-shaped or toroid structures in an irreversible manner. However, understanding the factors that lead to formation of such assembled structures deserves more investigation to achieve a unified insight into the assembly process, particularly of the microtubules. Here, we report an active assembly process of microtubules (MTs) at an air–buffer interface that resulted in ring-shaped microtubule structures with a narrow size distribution and a high yield. Using an “air–buffer interface control system” combined with the newly developed “inert chamber system (ICS)” we have also successfully observed the reversible conformational transition between ring- and linear-shaped microtubules at the air–buffer interface. This is the first ever direct in situ observation of a reversible assembly process of MTs and probably provides us with valuable discernment to understand the in vivo organizational behavior of biopolymers.

Growth of ring-shaped microtubule assemblies through stepwise active self-organisation

The microtubule (MT)–kinesin system is a promising candidate for constructing artificial biomachines. The active self-organisation (AcSO) method has been developed to integrate MT filaments into highly organised assembled structures. The creation of ring-shaped MT assemblies is one of the outcomes of the organisation process and holds prospects for use in future nano-technological applications. However, making use of ring-shaped MT assemblies in practical applications requires further control of the size of these assemblies, which has not yet been addressed. In this work, we demonstrated AcSO of MTs in a stepwise manner inside an inert atmosphere. We show that in an inert atmosphere, AcSO could be performed several times (at least nine times), and as a result, this method successfully increased the thickness of ring-shaped MT assemblies.

Controlled Clockwise–Counterclockwise Motion of the Ring-Shaped Microtubules Assembly

The microtubule (MT)-kinesin system has been proposed as the building block of biomolecular motor based artificial biomachines. Considerable efforts have been devoted to integrate this system that produced a variety of ordered structures including the ring-shaped MT assembly which is being considered as a promising candidate for the further development of the biomachines. However, lack of proper knowledge that might help tune the direction of motion of ring-shaped microtubule assembly from counterclockwise to clockwise direction, and vice versa, significantly restricted their potential applications. We report our success in controlling the direction of rotational motion of ring-shaped MT assembly by altering the preparation conditions of microtubules. The change in the direction of rotation of MT rings could be interpreted in terms of the accompanied structural rearrangement of the MT lattice. For achieving handedness-regulated efficient biomachines having tunable asymmetric property, our study will be significantly directive.