So Nishikawa

Simple Dark-Field Microscopy with Nanometer Spatial Precision and Microsecond Temporal Resolution

Molecular motors such as kinesin, myosin, and F(1)-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems. Here, we describe a simple dark-field imaging system that employs objective-type evanescent illumination to selectively illuminate a thin layer on the coverslip and thus yield images with high signal/noise ratios. Only by substituting the dichroic mirror in conventional objective-type total internal reflection fluorescence microscope with a perforated mirror, were nanometer spatial precision and microsecond temporal resolution simultaneously achieved. This system was applied to the study of the rotary mechanism of F(1)-ATPase. The fluctuation of a gold nanoparticle attached to the gamma-subunit during catalytic dwell and the stepping motion during torque generation were successfully visualized with 9.1-mus temporal resolution. Because of the simple optics, this system will be applicable to various biophysical studies requiring high spatial and temporal resolution in vitro and also in vivo.

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.

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.

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.

GLU 46 of ribonuclease T1 is an essential residue for the recognition of guanine base

The Glu 46 of ribonuclease T1, which is assumed to interact with Nl of the guanine residue in RNA by a hydrogen bond from the result of X-ray analysis, was changed to alanine by site-directed mutagenesis and its function examined. The nucleolytic activity of the Ala 46 mutant enzyme against pGpC decreased to 0.4% of that of the wild-type enzyme, on the other hand its activity against pApC increased. This result suggests that the Glu 46 is essential for the recognition of the guanine base but that it also interferes with the recognition of the adenine base.

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.

Motor function of unconventional myosin.

Myosins are motor proteins that interact with actin filaments and convert energy from ATP hydrolysis into mechanical force. In addition to the well-characterized conventional, filament forming, two-headed myosin II of muscle and non-muscle cells, a number of myosin-like proteins have recently been discovered. Based upon their amino acid sequences, these newly found “myosins” do not seem to form myosin filaments, thus they are often called “unconventional” myosins. The discovery of these “myosin-like motor proteins” has fundamentally expanded the potential physiological importance of myosins in diverse biological processes such as chemotactic motility, endocytosis, exocytosis, phagocytosis, vesicular trafficking, secretion, etc. The myosins are classified based upon phylogenetie sequence comparisons of the motor domain (Cheney et al, 1993; Goodson and Spudich, 1993; Mooseker and Cheney, 1995; Cope et al, 1996; Titus, 1997; Hodge and Cope, 2000) and divided into at least 18 classes. In vertebrates, it has been shown that eleven classes of myosin, (including conventional filament forming myosin) are expressed. The N-terminal domains of these classes of unconventional myosins are relatively conserved and contain the primary force production machinery, whereas the C-terminal tail domains are highly divergent and are thought to function as targeting sites that bind to the cellular partner molecules. Between the motor and the diverse tail domains of myosin, there are neck regions that are composed of various numbers of light chain binding motifs (Mermall et al, 1998).

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.

Imaging of cooperative motion on a simulated energy landscape

The transport through a modulated energy landscape is a classical problem that, in different forms, arises in different biological systems, such as protein folding and the motion of molecular motors. The calculations become increasingly difficult if a realistic potential landscape is taken into account and in many experimental settings the control of parameters is not possible. We propose and experimentally demonstrate an experimental apparatus that allows direct simulation and imaging of stochastic molecular motion on a time-dependent energy landscape.