A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

Mechanical forces are critical for regulating many biological processes such as cell differentiation, proliferation, and death. Probing the continuously changing molecular force through integrin receptors provides insights into the molecular mechanism of rigidity sensing in cells; however, the force information is still limited. Here, we built a coil-shaped DNA origami (DNA nanospring, NS) as a force sensor that reports the dynamic motion of single integrins as well as the magnitude and orientation of the force through integrins in living cells.

Autoregulation and dual stepping mode of MYA2, an Arabidopsis myosin XI responsible for cytoplasmic streaming.

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system.

The Synergic Role of Actomyosin Architecture and Biased Detachment in Muscle Energetics: Insights in Cross Bridge Mechanism Beyond the Lever-Arm Swing.

Muscle energetics reflects the ability of myosin motors to convert chemical energy into mechanical energy. How this process takes place remains one of the most elusive questions in the field. Here, we combined experimental measurements of in vitro sliding velocity based on DNA-origami built filaments carrying myosins with different lever arm length and Monte Carlo simulations based on a model which accounts for three basic components: (i) the geometrical hindrance, (ii) the mechano-sensing mechanism, and (iii) the biased kinetics for stretched or compressed motors.

Direct visualization of human myosin II force generation using DNA origami-based thick filaments.

The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highly-structured lattice. This complex imposes a geometric restriction on myosin in force generation. However, how single myosins generate force within the restriction remains elusive and conventional synthetic filaments do not recapitulate the symmetric bipolar filaments in sarcomeres.

Application of the fluctuation theorem for noninvasive force measurement in living neuronal axons.

Although its importance is recently widely accepted, force measurement has been difficult in living biological systems, mainly due to the lack of the versatile noninvasive force measurement methods. The fluctuation theorem, which represents the thermodynamic properties of small fluctuating nonequilibrium systems, has been applied to the analysis of the thermodynamic properties of motor proteins in vitro. Here we extend it to the axonal transport (displacement) of endosomes.

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA.

Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA.

Number Density Distribution of Small Particles around a Large Particle: Structural Analysis of a Colloidal Suspension.

Some colloidal suspensions contain two types of particles-small and large particles-to improve the lubricating ability, light absorptivity, and so forth. Structural and chemical analyses of such colloidal suspensions are often performed to understand their properties. In a structural analysis study, the observation of the number density distribution of small particles around a large particle (g) is difficult because these particles are randomly moving within the colloidal suspension by Brownian motion.

Myosin V is a biological Brownian machine.

Myosin V is a vesicle transporter that unidirectionally walks along cytoskeletal actin filaments by converting the chemical energy of ATP into mechanical work. Recently, it was found that myosin V force generation is a composition of two processes: a lever-arm swing, which involves a conformational change in the myosin molecule, and a Brownian search-and-catch, which involves a diffusive "search" by the motor domain that is followed by an asymmetric "catch" in the forward actin target such that Brownian motion is rectified. Here we developed a system that combines optical tweezers with DNA nano-material to show that the Brownian search-and-catch mechanism is the energetically dominant process at near stall force, providing 13 kBT of work compared to just 3 kBT by the lever-arm swing.

Switching of myosin-V motion between the lever-arm swing and brownian search-and-catch.

Motor proteins are force-generating nanomachines that are highly adaptable to their ever-changing biological environments and have a high energy conversion efficiency. Here we constructed an imaging system that uses optical tweezers and a DNA handle to visualize elementary mechanical processes of a nanomachine under load. We apply our system to myosin-V, a well-known motor protein that takes 72 nm 'hand-over-hand' steps composed of a 'lever-arm swing' and a 'brownian search-and-catch'.

Entropic potential field formed for a linear-motor protein near a filament: Statistical-mechanical analyses using simple models.

We report a new progress in elucidating the mechanism of the unidirectional movement of a linear-motor protein (e.g., myosin) along a filament (e.

Brownian search-and-catch mechanism for myosin-VI steps.

The cargo transporter myosin-VI processively walks along actin filaments using its two heads. Here we use single-molecule nanometry to show that the strong binding by myosin heads to actin is greatly accelerated (approximately 30-fold) when backward strain is applied to weakly bound heads during the actin search. We propose that the myosin head searches for the forward actin target by Brownian motion and catches the actin in a strain-dependent manner.

Thermal fluctuations biased for directional motion in molecular motors.

Recently developed single molecule measurements have demonstrated that the mechanisms for numerous protein functions involve thermal fluctuation, or Brownian motion. Protein interactions bias the random thermal noise in a manner such that the protein can perform its given functions. This phenomenon has been observed in molecular motor unidirectional movement where Brownian motion is used to preferentially bind the motor heads in one direction causing directional motility.

Biased Brownian motion mechanism for processivity and directionality of single-headed myosin-VI.

Conventional form to function as a vesicle transporter is not a 'single molecule' but a coordinated 'two molecules'. The coordinated two molecules make it complicated to reveal its mechanism. To overcome the difficulty, we adopted a single-headed myosin-VI as a model protein.

Single molecule measurements and molecular motors.

Single molecule imaging and manipulation are powerful tools in describing the operations of molecular machines like molecular motors. The single molecule measurements allow a dynamic behaviour of individual biomolecules to be measured. In this paper, we describe how we have developed single molecule measurements to understand the mechanism of molecular motors.

Cargo-binding makes a wild-type single-headed myosin-VI move processively.

Class VI myosin is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. Recently, however, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting a doubt on its processivity.

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps.