Mitsuhiro Sugawa

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.

Spontaneous Structural Changes in Actin Regulate G-F Transformation

Transformations between G- (monomeric) and F-actin (polymeric) are important in cellular behaviors such as migration, cytokinesis, and morphing. In order to understand these transitions, we combined single-molecule Förster resonance energy transfer with total internal reflection fluorescence microscopy to examine conformational changes of individual actin protomers. We found that the protomers can take different conformational states and that the transition interval is in the range of hundreds of seconds. The distribution of these states was dependent on the environment, suggesting that actin undergoes spontaneous structural changes that accommodate itself to polymerization.

Orientation of the γ Shaft and Catalytic β Subunit in F1-ATPase in the Intermediate State Revealed at the Single-Molecule Level

The enzyme FoF1-ATP synthase catalyzes the synthesis of ATP from ADP and inorganic phosphate (Pi) using proton-motive force (pmf) across a membrane. The F1 sector containing α3β3γδe subunits solely hydrolyzes ATP when isolated, is thus called F1-ATPase. Now it is well established that both Fo and F1-ATPase are rotary molecular motors sharing the common shaft: α3β3 cylinder in F1 rotates the γ shaft when ATP is cooperatively hydrolyzed in three catalytic sites located at α-β interfaces (Nishizaka et al., Nat. Struct. Mol. Biol., 2004), whereas reverse rotation of the shaft by pmf through Fo synthesizes ATP from ADP and Pi. The central question still remained unsolved is how these two different reaction, pmf and hydrolysis/synthesis, are coupled through the shaft from the structural point of view regarding the γ subunit. Because of asymmetric coiled-coil structure of the γ subunit, rotation of the γ subunit is expected to accompany the additional motion against the rotation axis during rotation. Here we scrutinize the rotation radius in an isolated α3β3γ subcomplex under optical microscope at the single-molecule level, and report the change of radius, which notably suggest the tilting motion of the shaft, estimated to be ∼4°, between two chemical states. In contrast, we have already reported that cooperative three-step motions in catalytic subunits of F1 correlate with 80° and 40° substep rotations, and thus revealed a previously undescribed set of β conformations, open, closed and partially closed, in the ATP-waiting dwells (Masaike et al., Nat. Struct. Mol. Biol., 2008). The conformation set presumably correlates the tilting of the shaft, and effectively transforms to the Fo rotation which couples to pmf.

Multiple Structural Forms of Actin in the Filamentous State

One of the most abundant proteins in eukaryotes is actin, a ubiquitous protein that plays a role in cell dynamics like cell migration. The dynamics of actin filament treadmilling is regulated by two actin structural states: globular actin (G-actin) and filamentous actin (F-actin). While G-actins crystal structure has been solved by several groups, F-actins has not. Although recently it was reported that the structure for the two actins differ (Oda et al), there is still much to resolve on the matter of their dynamic structures. Here we observed the dynamics of the actin structural states under various conditions by using single-molecule FRET in combination with total internal reflection fluorescence microscopy. To conduct these experiments, we first labeled actin residues 41 and 374, having substituted Gln 41 with Cys. The new Cys 41 site along with Cys 374 were used for site-directed labeling by SH-group reactive fluorescent dyes. We found that F-actin has at least two distinct states, and that the population distribution of these states was dependent on the ionic conditions. We are currently investigating these states by performing FRET measurements for observing the long -time transition between the two states.

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.

Fluctuation and Noises in Biosciences

From molecular and cellular to perception processes in biological systems, fluctuation is observed. Recently single molecule detection techniques have been developed and the dynamic changes of individual molecules can be measured and are studied in relation to the biological function. These results showed that thermal motion is biased in directional motion and reaction through the interaction with other protein molecules. When these molecules self‐assemble to higher levels of hierarchic structure of biological systems, unique operations emerge to make systems function more effective and adaptable. In this paper the roles of fluctuation are described in molecular motors, protein structures and cell signal transduction.