Tomotaka Komori

Simultaneous measurement of nucleotide occupancy and mechanical displacement in myosin-V, a processive molecular motor.

Adenosine triphosphate (ATP) turnover drives various processive molecular motors and adenosine diphosphate (ADP) release is a principal transition in this cycle. Biochemical and single molecule mechanical studies have led to a model in which a slow ADP release step contributes to the processivity of myosin-V. To test the relationship between force generation and ADP release, we utilized optical trapping nanometry and single molecule total internal reflection fluorescence imaging for simultaneous and direct observation of both processes in myosin-V. We found that ADP was released 69 +/- 5.3 ms after force generation and displacement of actin, providing direct evidence for slow ADP release. As proposed by several previous studies, this slow ADP release probably ensures processivity by prolonging the strong actomyosin state in the ATP turnover cycle.

A polysaccharide-based container transportation system powered by molecular motors

In living cells, the motor protein myosin, which is driven by ATP hydrolysis, intracellularly transports cargo such as vesicles and organelles 2] by moving along actin filaments. There have been many reports of how myosin can transport artificial cargoes such as polystyrene microspheres, 4] gold nanoparticles, and quantum dots. 7] Recently, cargo transportation systems powered by artificial nanomotors were actively studied. However, in these studies, the biomolecular or nanomotors are directly bound to the specific cargo. Therefore, these systems can be applied only to the delivery of certain types of cargo. Herein, we report the first container transportation system to be powered by biological motors. In this system, myosin is attached to a polysaccharide-based container that can hold a cargo. In fact, under physiological conditions, myosin binds to a container-like vesicle that holds a cargo. As the polysaccharide can form complexes with various cargoes such as carbon nanotubes and DNA, we envision that this novel container transportation system will expand the applicability of artificial intracellular transportation systems, including medically relevant procedures such as gene therapy. We have previously reported the very interesting “dynamic” properties of b-1,3-glucan polysaccharides, which are typified by schizophyllan (SPG). In nature, SPG adopts a triple-stranded helical structure (t-SPG), which dissociates into a single chain (s-SPG) upon dissolution in dimethyl sulfoxide (DMSO). The s-SPG chain can recover its original triple-stranded helix when DMSO is exchanged for water. These processes are referred to as denature (from t-SPG to sSPG) and renature (from s-SPG to t-SPG), respectively (Figure 1a). We found that b-1,3-glucans and their derivatives can act as 1D hosts that helically wrap nanomaterials such as carbon nanotubes, conjugated polymers, DNA, and gold nanoparticles, and allow these nanomaterials to be dissolved in water through the denature–renature process. Therefore, we selected SPG as the container for our system. A schematic representation of our novel container transportation system is shown in Figure 1b. The “cargo” is wrapped with the “container” and transported on the “rail” by “wheels”. We choose single-walled carbon nanotubes

Simultaneous Observation of the Lever Arm and Head Explains Myosin VI Dual Function

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

Background: The roles of conformational changes of actin in myosin motility are unclear. Results: A G146V mutation in actin, which perturbed its conformation, impaired force generation by myosin II, but not by myosin V. Conclusion: Conformational changes of actin involving Gly-146 have critical roles in motility of myosin II, but not of myosin V. Significance: The mechanism of motility may be different between myosin types. The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

Measurement system for simultaneous observation of myosin V chemical and mechanical events

Myosin V is an actin-based processive molecular motor driven by the chemical energy of ATP hydrolysis. Although the chemo-mechanical coupling in processive movement has been postulated by separate structural, mechanical and biochemical studies, no experiment has been able to directly test these conclusions. Therefore the relationship between ATP-turnover and force generation remains unclear. Currently, the most direct method to measure the chemo-mechanical coupling in processive motors is to simultaneously observe ATP-turnover cycles and displacement at the single molecule level. In this study, we developed a simultaneous measurement system suitable for mechanical and chemical assays of myosin V in order to directly elucidate its chemo-mechanical coupling.

Spontaneous Detachment of the Leading Head Contributes to Myosin VI Backward Steps

Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI.

Lever arm extension of myosin VI is unnecessary for the adjacent binding state.

Myosin VI is a processive myosin that has a unique stepping motion, which includes three kinds of steps: a large forward step, a small forward step and a backward step. Recently, we proposed the parallel lever arms model to explain the adjacent binding state, which is necessary for the unique motion. In this model, both lever arms are directed the same direction. However, experimental evidence has not refuted the possibility that the adjacent binding state emerges from myosin VI folding its lever arm extension (LAE). To clarify this issue, we constructed a myosin VI/V chimera that replaces the myosin VI LAE with the IQ3-6 domains of the myosin V lever arm, which cannot fold, and performed single molecule imaging. Our chimera showed the same stepping patterns as myosin VI, indicating the LAE is not responsible for the adjacent binding state.

Shrec Measurement of Myosin-VI Stepping Motion

Myosin-VI is a motor protein that plays an important role in a large variety of cellular events such as vesicle transport and anchoring actin bundles to the plasma membrane. Myosin-VI is thought to move processively as a dimer along an actin filament in a hand-over-hand fashion with large steps similar to myosin-V. However, unlike myosin-V, its step size is largely variable. Recently, we showed using FIONA method that myosin-VI does not have widely distributed step but rather has two step types, a regular large step (72nm) and short step (44nm).(Arimoto et al. Biophysical J. vol 96 p.139a) The large steps were consistent with the hand-over-hand model. The short steps, however, were not explained by canonical stepping model. We also showed that the fraction of short steps largely increases in the presence of ADP, suggesting the short and large steps are regurated in the ADP-dependent manner. In this study, in order to investigate the coordination of two heads during short and large steps, we performed an advanced multi-color FIONA technique called single-molecule high-resolution colocalization (SHREC) which involves labeling the two heads with differently-colored Q-dots. Now we are analyzing what is the condition that myosin-VI switches between two types of stepping manner. Furthermore, to clarify how myosin-VI switches between short and long steps, we are measuring myosin-VI movement under several ADP concentrations.