Kazuhiro Shikinaka

Integration of Motor Proteins – Towards an ATP Fueled Soft Actuator

We present a soft bio-machine constructed from biological motors (actin/myosin). We have found that chemically cross-linked polymer-actin complex gel filaments can move on myosin coated surfaces with a velocity as high as that of native F-actin, by coupling to ATP hydrolysis. Additionally, it is shown that the velocity of polymer-actin complex gel depends on the species of polycations binding to the F-actins. Since the design of functional actuators of well-defined size and morphology is important, the structural behavior of polymer-actin complexes has been investigated. Our results show that the morphology and growth size of polymer-actin complex can be controlled by changes in the electrostatic interactions between F-actins and polycations. Our results indicate that bio actuators with desired shapes can be created by using a polymer-actin complex.

Formation of motile assembly of microtubules driven by kinesins

Microtubule (MT) and kinesin are rail and motor proteins that are involved in various moving events of eukaryotic cells in natural systems. In vitro, the sliding motion of microtubules (rail) can be reproduced on a kinesin (motor protein)-coated surface coupled with adenosine triphosphate (ATP) hydrolysis, which is called a motility assay. Based on this technique, a method was recently established to form MT assemblies by an active self-assembly (AcSA) process, in which MTs are crosslinked during a sliding motion on a kinesin-coated surface. Streptavidin (ST) was employed as glue to crosslink biotin-labeled MTs. Various shapes, sizes, and motilities were formed with the AcSA MT assemblies, depending on the initial conditions. In this paper, we briefly review our recent work on the formation of MT assemblies on a kinesin-coated surface.

Hierarchical structures of the actin/polycation complexes, investigated by ultra-small-angle neutron scattering and fluorescence microscopy

By employing a combined method of ultra-small-angle & small-angle neutron scattering (USANS&SANS) and fluorescence microscopy (FM), we investigated the solution mixtures of filamentous actin (F-actin) and the synthesized cationic polymer poly-N-[3-(dimethylamino)propyl]acrylamide methylchloride quaternary (PDMAPAA-Q). The combined USANS&SANS method, covering a wide range of length scales from 10 micrometres to nanometres, plays a crucial role in elucidating the hierarchical structure of the complex structure, as it is in an aqueous solution. FM determined that the complex of F-actin and PDMAPAA-Q appears with a finite size (>10μm), referred to as a superbundle, and its morphology changes from a globular one to a stretched one by increasing the salt (KCl) concentration Cs from 0.01 to 0.3 M. USANS&SANS revealed that the superbundle is composed of a structural unit of a protobundle, in which PDMAPAA-Q binds F-actins in order to form a hexagonal lattice. The diameter of the protobundle (DII), determined by USANS&SANS, increases from 40 to 290 nm as Cs increases from 0.01 to 0.3 M. In order to explain the finite-size & hierarchical condensation observed in the actin/PDMAPAA-Q solution, we employ a scenario of imperfect charge neutralization between F-actins and PDMAPAA-Q. Due to the chain connectivity of PDMAPAA-Q, a spatial distribution of positive charges around the F-actins becomes inhomogeneous, so that the repulsive electrostatic interaction appearing in the protobundle limits bundle formation with an infinite-size. The morphology of the superbundles is controlled by the bending rigidity due to individual protobundles, which significantly increases as DII increases.

Photoinduced in situ formation of various F-actin assemblies with a photoresponsive polycation

We developed a novel in situ method for the control of F-actin assembly by using a synthetic photoresponsive polycation. The photoresponsive polycation mainly comprises a water-soluble cationic monomer and also contains a small amount of the monomer of a triphenylmethane leucohydroxide derivative (20 mol %), which is a well-known photochromic molecule that can be cationized in aqueous solution by ultra violet (UV) irradiation, thereby causing an increase in the total charge on the photoresponsive polycation. Thus, by exposure to UV radiation in aqueous solution, F-actin and the photoresponsive polycation start assembling into F-actin/photoresponsive polycation complexes of various morphologies such as bundles, coils, and networks, depending upon the concentrations of both the F-actin and salt. Further, localized UV irradiation can be applied in order to control the local formation of F-actin/photoresponsive polycation complexes. Thus, this technique provides a novel method for the spatiotemporal control of F-actin assembly and can be applied to investigate the unknown characteristics of F-actin.