Pause of the target gliding microtuble on the virtual cathode.

We report the transient response of gliding microtubules on a virtual cathode. In vivo activities, microtubule-kinesin systems are known to act as motor proteins with respect to cell motility cytokinesis and cellular transport by hydrolyzing ATP molecules. With development of in vitro assays, motor proteins have been attracting much attention as a key component for highly efficient nano-transportation systems. The molecular functions based on structural states are affected by changing the ionic condition of the molecular functions and by changing the electrical field in solution because of electrical charges of the molecules. The virtual cathode, which was generated on the SiN display surface by a low energy electron beam, locally induced electrochemical reactions and electric field around the targeted molecules on the display surface, and then the gliding motions of the targeted microtubules were regulated. In this study, we demonstrated that the virtual cathode display temporally stops a selected gliding microtubule by only applying the virtual cathode to the microtubule. The pause mode of the microtubule was easily canceled by simply turning the virtual cathode off, and then the gliding motion was restarted.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

Design of Polymer Networks Involving a Photoinduced Electronic Transmission Circuit toward Artificial Photosynthesis.

Many strategies have been explored to achieve artificial photosynthesis utilizing mediums such as liposomes and supramolecules. Because the photochemical reaction is composed of multiple functional molecules, the surrounding microenvironment is expected to be rationally integrated as observed during photosynthesis in chloroplasts. In this study, photoinduced electronic transmission surrounding the microenvironment of Ru(bpy)3(2+) in a polymer network was investigated using poly(N-isopropylacrylamide-co-Ru(bpy)3), poly(acrylamide-co-Ru(bpy)3), and Ru(bpy)3-conjugated microtubules. Photoinduced energy conversion was evaluated by investigating the effects of (i) Ru(bpy)3(2+) immobilization, (ii) polymer type, (iii) thermal energy, and (iv) cross-linking. The microenvironment surrounding copolymerized Ru(bpy)3(2+) in poly(N-isopropylacrylamide) suppressed quenching and had a higher radiative process energy than others. This finding is related to the nonradiative process, i.e., photoinduced H2 generation with significantly higher overall quantum efficiency (13%) than for the bulk solution. We envision that useful molecules will be generated by photoinduced electronic transmission in polymer networks, resulting in the development of a wide range of biomimetic functions with applications for a sustainable society.

Controlling the bias of rotational motion of ring-shaped microtubule assembly.

Biomolecular motor system microtubule (MT)-kinesin is considered a building block for developing artificial microdevices. Recently, an active self-organization method has been established to integrate MT filaments into ring-shaped assembly that can produce rotational motion both in the clockwise and in the counterclockwise directions. In this work, we have investigated the effect of parameters such as MT and kinesin concentration, length, and rigidity of MT and type of kinesin (structure of tail region) on the preferential rotation of the ring-shaped MT assembly produced in an active self-organization. We elucidated that these factors can significantly affect the bias of rotation of the ring-shaped MT assembly, which seems to be related to the fluctuation of leading tip of moving MT filaments. This new finding might be important for designing handedness regulated artificial biomachine using the ring-shaped MT assembly in future.

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy.

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered-integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin-substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.

Spatiotemporal Control of Polymorphic Phase Transition of Glycine Crystals by Three-Dimensional Femtosecond Laser Ablation Processing.

Spatiotemporal control of the polymorphic phase transition of glycine crystals was demonstrated by three-dimensional (3D) processing with a focused femtosecond laser pulse as an external stimulus. We found that the transition from a metastable form (ß-form) to more stable ones (α- or γ-form) could be triggered from the irradiated area of not only the surface but also inside of glycine crystals. This 3D processing with a focused femtosecond laser pulse enabled us to precisely monitor the transition dynamics from a targeted position to the entire part of crystals. The systematic study with the space-selective phase transition method revealed that the phase transition inside of glycine crystals was significantly slower (e.g., ∼50 times) than that at the crystal surface, which indicates the crucial role of water molecules in air on the phase transition dynamics. We foresee that this laser method can be a practical tool for monitoring spatiotemporal dynamics of phase transition.

Mie-Resonant Nanophotonic-Enhancement of Asymmetry in Sodium Chlorate Chiral Crystallization.

Studies on chiral spectroscopy have recently demonstrated strong enhancement of chiral light-matter interaction in the chiral near-field of Mie resonance in high-refractive-index dielectric nanostructures by studies on chiral spectroscopy. This situation has motivated researchers to demonstrate effective chiral photosynthesis under a chiral near-field beyond circularly polarized light (CPL) as a chiral source. However, the effectivity of the chiral near-field of Mie resonance for chiral photosynthesis has not been clearly demonstrated. One major challenge is the experimental difficulty in evaluating enantiomeric excess of a trace amount of chiral products synthesized in the near-field. Here, by adopting sodium chlorate chiral crystallization as a phenomenon that includes both synthesis and the amplification of chiral products, we show that crystallization on a Mie-resonant silicon metasurface excited by CPL yields a statistically significant large crystal enantiomeric excess of ∼18%, which cannot be achieved merely by CPL. This result provides implications for efficient chiral photosynthesis in a chiral near-field.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Controlled cell adhesion using a biocompatible anchor for membrane-conjugated bovine serum albumin/bovine serum albumin mixed layer.

We report here a method for controlling cell adhesion, allowing simple yet accurate cell detachment from the substrate, which is required for the establishment of new cytometry-based cell processing and analyzing methods. A biocompatible anchor for membrane (BAM) was conjugated with bovine serum albumin (BSA) to produce a cell-anchoring agent (BAM-BSA). By coating polystyrene substrates with a mixture of BAM-BSA and BSA, controlled suppression of the substrate's adhesive properties was achieved. Hook-shaped nanoneedles were used to pick up cells from the substrate, while recording the cell-substrate adhesion force, using an atomic force microscope (AFM). Due to the lipid bilayer targeting property of BAM, the coated surface showed constant adhesion forces for various cell lines, and controlling the BAM-BSA/BSA ratio enabled tuning of the adhesion force, ranging from several tens of nano-Newtons down to several nano-Newtons. Optimized tuning of the adhesion force also enabled the detachment of cells from BAM-BSA/BSA-coated dishes, using a shear flow. Moreover, the method was shown to be noncell type specific and similar results were observed using four different cell types, including nonadherent cells. The attenuation of cell adhesion was also used to enable the collection of single cells by capillary aspiration. Thus, this versatile and relatively simple method can be used to control the adhesion of various cell types to substrates.

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Surface-limited reactions for spatial control of kinesin-microtubule motility assays using indirect irradiation of an electron beam.

Gliding of microtubules (MTs) on kinesins has been applied to lab-on-a-chip devices, which enable autonomous transportation and detection of biomolecules in the field of bioengineering. For rapid fabrication and evaluation of the kinesin-MT based devices, optical control techniques have been developed for control of kinesin activity and density; however, use of caged molecules lacks spatial controllability for long-term experiments, and direct irradiations of UV light onto kinesin-coated surfaces are inherently damaging to MTs due to their depth limit since the heights of the kinesin-MT systems are at the tens of a nanometer scale. Considering surface electric fields in electrolytic solutions are shielded at the nanometer scale due to Debye shielding, in this study, we show that fine spatial control of kinesin density and activity is enabled using surface-limited electrochemical reactions induced by indirect irradiations of an electron beam (EB). An EB is indirectly irradiated onto the kinesins through a 100-nm-thick silicon nitride membrane, and the electrons scattered in the membrane can cause localized electrochemical effects to the kinesins. We show that these localized electrochemical effects cause both ablation of kinesins and motility control of kinesin activity by changing the EB acceleration voltage. In particular, the latter is achieved without complete ablation of MTs, though the MTs are indirectly irradiated by the EB. As a demonstration of on-demand control of gliding MTs, we show the accumulation of the MTs on a target area by scanning the EB. The proposed accumulation technique will lead to rapid prototyping of microdevices based on MT-kinesin motility assay systems.

Mechanical guidance of self-condensation patterns of differentiating progeny.

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

Self-repairing filamentous actin hydrogel with hierarchical structure.

A chemically cross-linked filamentous actin (F-actin) gel consisting of globular actin (G-actin) as repeating units was prepared. The F-actin gel was cross-linked by covalent bonds, and the main chain is represented by the self-assembly of G-actin with a high-ordered hierarchical structure. The gel exhibited good mechanical performance with a storage modulus >1 kPa and undergoes reversible sol-gel transitions in response to changes in the salt concentration (chemical-induced sol-gel transition) as well as to shear strain (mechanical-induced sol-gel transition). Therefore, the gel exhibits self-repairing ability through dynamic polymerization and depolymerization across the structure hierarchies under repeated shear stress.

Thermoresponsive microtubule hydrogel with high hierarchical structure.

A thermoresponsive 3D microtubule hydrogel (MT gel) was prepared by simultaneous polymerization and chemical cross-linking of tubulins. The main chain of this gel is composed of cross-linked MTs, which consists of a cylindrical assembly of tubulin covalently connected by polyethylene glycol. This gel, which contains 10 mg/mL of tubulin, exhibits a storage modulus G' as high as 1 × 10(3), which is 10 times higher than the loss modulus G'' over a wide range of frequencies. The MT gel exhibits a reversible sol-gel transition by temperature changes at 4-37 °C via depolymerization and polymerization of the MT network. Notable effects of the presence of the cross-linkage on the process of polymerization and depolymerization of tubulin were experimentally observed, and the role of the cross-linkage was discussed.

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.

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.

Quantitative evaluation of the impact of artificial cell adhesion via DNA hybridization on E-cadherin-mediated cell adhesion.

Programmable cell adhesion with DNA hybridization is a promising approach for fabricating various tissue architectures without sophisticated instrumentation. However, little is known about how this artificial interaction influences the binding of cell adhesion proteins, E-cadherin. In this work, we designed a planar and fluid lipid membrane displaying E-cadherin and/or single-strand DNA with well-defined densities. Visualization of cells on membranes by fluorescence and interference microscopy revealed cell adhesion to be a two-step process: artificial adhesion by DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. Furthermore, we discovered that DNA hybridization can substantially facilitate E-cadherin-mediated cell adhesion. The promotive effect is probably due to the enforced binding between E-cadherin molecules in geometrical confinement between two membranes. Our in vitro model of cell adhesion can potentially be used to design functional synthetic molecules that can regulate cell adhesion via cell adhesion proteins for tissue engineering.

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).

Ring-shaped assembly of microtubules shows preferential counterclockwise motion.

In this paper, we reveal that microtubules (MTs), reconstructed from tubulin in vitro in the presence of guanosine-5'-triphosphate (GTP), have a ring or spiral shape on a motor protein-fixed surface, and these MTs show biased motion in the counterclockwise direction. By cross-linking these MTs during the sliding motion, we obtained large ring-shaped MT assemblies, 1 approximately 12.6 microm in diameter. The ratio of the rings rotating in the counterclockwise direction to those rotating in the clockwise direction was approximately 3/1. Under optimized conditions, the ratio was as high as 14/1. Thus, we successfully obtained aggregated MTs with a large hierarchic structure that shows a preferential motion, through a dynamic process in vitro.

Transduction of NeuroD2 protein induced neural cell differentiation.

NeuroD2, one of the neurospecific basic helix-loop-helix transcription factors, has the ability to induce neural differentiation in undifferentiated cells. In this paper, we show that transduction of NeuroD2 protein induced mouse neuroblastoma cell line N1E-115 into neural differentiation. NeuroD2 has two basic-rich domains, one is nuclear localization signal (NLS) and the other is basic region of basic helix-loop-helix (basic). We constructed some mutants of NeuroD2, ND2(Delta100-115) (lack of NLS), ND2(Delta123-134) (lack of basic) and ND2(Delta100-134) (lack of both NLS and basic) for transduction experiments. Using these proteins, we have shown that NLS region of NeuroD2 plays a role of protein transduction. Continuous addition of NeuroD2 protein resulted in N1E-115 cells adopting neural morphology after 4 days and Tau mRNA expression was increased. These results suggest that neural differentiation can be induced by direct addition of NeuroD2 protein.