Masahito Hayashi

Strain through the neck linker ensures processive runs: a DNA-kinesin hybrid nanomachine study

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA‐kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA‐kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C‐terminal or Mid position) and sub‐nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13‐amino‐acid chain positioned at the C‐terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter‐head coordination through the neck linker facilitates long‐distance walking.

Direct observation of the reversible unwinding of a single DNA molecule caused by the intercalation of ethidium bromide

Ethidium bromide (EtBr) is the conventional intercalator for visualizing DNA. Previous studies suggested that EtBr lengthens and unwinds double-stranded DNA (dsDNA). However, no one has observed the unwinding of a single dsDNA molecule during intercalation. We developed a simple method to observe the twisting motions of a single dsDNA molecule under an optical microscope. A short dsDNA was attached to a glass surface of a flow chamber at one end and to a doublet bead as a rotation marker at the other end. After the addition and removal of EtBr, the bead revolved in opposite directions that corresponded to the unwinding and rewinding of a dsDNA, respectively. The amount of intercalating EtBr was estimated from the revolutions of the bead. EtBr occupied 57% of base pairs on a single dsDNA at 1 mM of EtBr, indicating that EtBr molecules could bind at contiguous sites to each other. The isotherm of intercalation showed that negative cooperativity existed between adjoining EtBr molecules. The association constant of EtBr and dsDNA (1.9 (±0.1) × 105 M−1) was consistent with that of previous results. Our system is useful to investigate the twisting of a single dsDNA interacting with various chemicals and biomolecules.

Fully Automated On-Chip Imaging Flow Cytometry System with Disposable Contamination-Free Plastic Re-Cultivation Chip

We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 106 particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.

Multiple kinesin-14 family members drive microtubule minus end–directed transport in plant cells

Minus end–directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end–directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class VI kinesin-14, KCBP, is required for minus end–directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end–directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end–directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end–directed transporters in land plants.

Reversible Morphological Control of Tubulin-Encapsulating Giant Liposomes by Hydrostatic Pressure

Liposomes encapsulating cytoskeletons have drawn much recent attention to develop an artificial cell-like chemical-machinery; however, as far as we know, there has been no report showing isothermally reversible morphological changes of liposomes containing cytoskeletons because the sets of various regulatory factors, that is, their interacting proteins, are required to control the state of every reaction system of cytoskeletons. Here we focused on hydrostatic pressure to control the polymerization state of microtubules (MTs) within cell-sized giant liposomes (diameters ∼10 μm). MT is the cytoskeleton formed by the polymerization of tubulin, and cytoskeletal systems consisting of MTs are very dynamic and play many important roles in living cells, such as the morphogenesis of nerve cells and formation of the spindle apparatus during mitosis. Using real-time imaging with a high-pressure microscope, we examined the effects of hydrostatic pressure on the morphology of tubulin-encapsulating giant liposomes. At ambient pressure (0.1 MPa), many liposomes formed protrusions due to tubulin polymerization within them. When high pressure (60 MPa) was applied, the protrusions shrank within several tens of seconds. This process was repeatedly inducible (around three times), and after the pressure was released, the protrusions regenerated within several minutes. These deformation rates of the liposomes are close to the velocities of migrating or shape-changing living cells rather than the shortening and elongation rates of the single MTs, which have been previously measured. These results demonstrate that the elongation and shortening of protrusions of giant liposomes is repeatedly controllable by regulating the polymerization state of MTs within them by applying and releasing hydrostatic pressure.

Direct Observation of Strand Passage by DNA-Topoisomerase and Its Limited Processivity

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51±0.33 µm (11±6 turns) of a braid was unlinked in a burst of reactions taking 8±4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25–37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ∼100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ∼10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ∼10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.

A Non-Destructive Culturing and Cell Sorting Method for Cardiomyocytes and Neurons Using a Double Alginate Layer

A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

Continuous Concentration and Separation of Microparticles Using Dielectrophoretic Force in a V-Shaped Electrode Array

We have proposed and developed the novel principle of a V-shaped electrode array in a microfluidic pathway for continuous concentration and separation of particles by dielectrophoretic (DEP) force. The advantage of V-shape microelectrode arrays with a microfluidic flow for cell separation is that whole particles are concentrated into the center of a micropathway independent of the difference in their dielectric constants in the X–Y plane, while the particles are split between the top or bottom of the micropathway in the Z-axis direction depending on the differences in their dielectric constants and the applied AC frequency. After the application of a sinusoidal AC voltage of 1 MHz and 20 Vpp, both polystyrene spheres and Bacillus subtilis spores were concentrated at the tip of the V-shaped electrode at the center of microfluidic flow in the X–Y plane independent of their dielectric constant differences. They were also split into two directions in the Z-axis, i.e., polystyrene spheres rose to the top, and spores went down to the bottom depending on their dielectric constant differences and were successfully separated in two layered downstreams. The results indicate the potential of V-shaped electrode arrays for simple continuous purification of mixed particles depending on their dielectric constants.

Production of double-layered metal nanocups for artificial nanospace of biomolecular reaction

Nanocups (NCs), sub-micrometer semispherical bowls consisting of two different nanometer-thick metals on inner and outer layers, have been fabricated to mimic a localized nano-scale biochemical reaction environment for reactive biomolecules. Homogeneous polystyrene beads were used as a cast of the NCs, placed on a Si substrate, dried, and processed by oxygen plasma etching until the desired diameters and gaps among neighboring bead casts. For the fabrication of Au/Ni double-layered NCs, Au and Ni were sequentially deposited on upper halves of the bead surfaces by thermal evaporation with nanometer-order thickness control. The polystyrene casts were removed completely by UV–ozone oxidization reaction, and Au/Ni double-layered NCs were fabricated on a Si substrate. To orient the holes of the fabricated NCs to top for the substrate, poly(dimethylsiloxane) (PDMS) sol was dropped on the NCs placed on the Si substrate, hardened, and peeled off from the substrate, and then the NCs were placed on the PDMS surface with those holes turned-up. To examine the selective interaction of biomolecules on the inner layer of NCs as the artificial nanospace for biomolecular reactions, a thiolated target DNA was immobilized onto the inner layer of a Au/Ni NC as a model. The target DNA was labeled through hybridization reaction using small Au nanoparticles (NPs) on which a complementary probe DNA was immobilized. Both the surface-specific immobilization of the target DNA on the Au layer of the NC and the specific hybridization in NC nanospaces were confirmed by direct observations after those reactions using field emission scanning electron microscopy (FE-SEM), indicating that the inside of the fabricated NCs can be used as the artificial nanospace for studying localized biomolecular reactions.

Highly Concentrated Ethanol Solutions: Good Solvents for DNA as Revealed by Single‐Molecule Observation

Abstract We observed single DNA molecules at different ethanol concentrations by using fluorescence microscopy. Large single DNA molecules undergo reentrant conformational transitions from elongated coil into folded globule and then into elongated coil state, accompanied by the increase of the concentration of ethanol in a low‐salt aqueous environment. The second transition from globule into the coil state occurs at around 70u2009% (v/v) ethanol. From circular dichroism (CD) measurements, it is confirmed that the reentrant transition of the higher order structure proceeds together with the transitions of the secondary structure from B to C and, then, from C to A in a cooperative manner. The determined mechanism of the reentrant transition is discussed in relation to the unique characteristics of solutions with higher ethanol content, for which clathrate‐like nanostructures of alcohol molecules are generated in the surrounding water.