西坂 崇之

Mycoplasma mobile is one of the fastest gliding bacteria, gliding with a speed of 4.5 μm s-1. This gliding motility is driven by a concerted movement of 450 supramolecular motor units composed of three proteins, Gli123, Gli349, and Gli521, in the gliding motility machinery. With general experimental setups, it is difficult to obtain the information on how each motor unit works. This chapter describes strategies to decrease the number of active motor units to extract stepwise cell movements driven by a minimum number of motor units. We also describe an unforeseen motility mode in which the leg motions convert the gliding motion into rotary motion, which enables us to characterize the motor torque and energy-conversion efficiency by adding some more assumptions.

F1-ATPase is a world's smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule which consists of one mutant β and two wild type βs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles which are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and give insights into the mechanism of driving unidirectional rotation.

Length control is a fundamental requirement for molecular architecture. Even small wall-less bacteria have specially developed macro-molecular structures to support their survival. <italic>Mycoplasma pneumoniae</italic>, a human pathogen, forms a polar extension called an attachment organelle, which mediates cell division, cytadherence, and cell movement at host cell surface. This characteristic ultrastructure has a constant size of 250–300 nm, but its design principle remains unclear. In this study, we constructed several mutants by genetic manipulation to increase or decrease coiled-coil regions of HMW2, a major component protein of 200 kDa aligned in parallel along the cell axis. HMW2-engineered mutants produced both long and short attachment organelles, which we quantified by transmission electron microscopy and fluorescent microscopy with nano-meter precision. This simple design of HMW2 acting as a molecular ruler for the attachment organelle should provide an insight into bacterial cellular organization and its function for their parasitic lifestyles.

A collective motion of self-driven particles has been a fascinating subject in physics and biology. Sophisticated macroscopic behavior emerges through a population in thousands or millions of bacterial cells, propelling itself by flagellar rotation and its chemotactic response. Here we found a series of collective motions accompanying successive phase-transitions in a non-flagellated rod-shaped soil bacterium, Flavobacterium johnsoniae, which was driven by a surface cell movement known as gliding motility. When we spot the cells on an agar plate with a low level of nutrients, the bacterial community exhibited vortex patterns that spontaneously appeared as lattice and integrated into a large-scale circular plate. All patterns exhibit with monolayer of bacteria, which enable to visualize an individual cell with a high resolution among a wide-range pattern two-dimensionally. The single cells moved at random orientation, but the cells connected with one another showed left-turn biased trajectories in starved environment. This feature is possibly due to the collision of cells inducing a nematic alignment of dense cells as self-propelled rods. Subsequently, each vortex oscillated independently, and then transformed to the rotating mode as an independent circular plate. Notably, the rotational direction of the circular plate was counterclockwise without exception. The plates developed accompanying rotation with constant angular velocity, suggesting that the mode is an efficient strategy for bacterial survival.ImportanceSelf-propelled bacteria propelled by flagella rotation often display highly organized dynamic patterns at high cell densities. Here we found a new mode of collective motion in non-flagellated bacteria: vortex patterns were spontaneously appeared as lattice and integrated into a large-scale circular plate comprising hundreds of thousands of cells, which exhibited unidirectional rotation in a counterclockwise manner and expanded in size on agar. A series of collective motions was driven by gliding motility of the rod-shaped soil bacterium Flavobacterium johnsoniae In a low nutrient environment, single cells moved at random orientation while cells at high density moved together as a unitary cluster. This might be an efficient strategy for cells of this species to find nutrients.

Virus removal filters developed for the decontamination of small viruses from biotherapeutic products are widely used in basic research and critical step for drug production due to their long-established quality and robust performance. A variety of imaging techniques have been employed to elucidate the mechanism(s) by which viruses are effectively captured by filter membranes, but they are limited to 'static' imaging. Here, we propose a novel method for detailed monitoring of 'dynamic process' of virus capture; specifically, direct examination of biomolecules during filtration under an ultra-stable optical microscope. Samples were fluorescently labeled and infused into a single hollow fiber membrane comprising cuprammonium regenerated-cellulose (Planova 20N). While proteins were able to pass through the membrane, virus-like particles (VLP) accumulated stably in a defined region of the membrane. After injecting the small amount of sample into the fiber membrane, the real-time process of trapping VLP in the membrane was quantified beyond the diffraction limit. The method presented here serves as a preliminary basis for determining optimum filtration conditions, and provides new insights into the structure of novel fiber membranes.

Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of the rotational direction, which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150°. The flagellar helicity remained right-handed with a 1.3 μm pitch and 0.14 μm helix radius, which is consistent with the feature of a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not spread on a semi-solid surface. Based on these results, we concluded that a flagellar polymorphism is essential for spreading in structured environments.

Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts' gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni's flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni's opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.

The mechanism underlying Spiroplasma swimming is an enigma. This small bacterium possesses two helical shapes with opposite-handedness at a time, and the boundary between them, called a kink, travels down, possibly accompanying the dual rotations of these physically connected helical structures, without any rotary motors such as flagella. Although the outline of dynamics and structural basis has been proposed, the underlying cause to explain the kink translation is missing. We here demonstrated that the cell morphology of Spiroplasma eriocheiris was fixed at the right-handed helix after motility was stopped by the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the preferential state was transformed to the other-handedness by the trigger of light irradiation. This process coupled with the generation and propagation of the artificial kink, presumably without any energy input through biological motors. These findings indicate that the coexistence of two chiral helices is sufficient to propagate the kink and thus to propel the cell body.IMPORTANCE Many swimming bacteria generate a propulsion force by rotating helical filaments like a propeller. However, the nonflagellated bacteria Spiroplasma spp. swim without the use of the appendages. The tiny wall-less bacteria possess two chiral helices at a time, and the boundary called a kink travels down, possibly accompanying the dual rotations of the helices. To solve this enigma, we developed an assay to determine the handedness of the body helices at the single-wind level, and demonstrated that the coexistence of body helices triggers the translation of the kink and that the cell body moves by the resultant cell bend propagation. This finding provides us a totally new aspect of bacterial motility, where the body functions as a transformable screw to propel itself forward.

Mycoplasma pneumoniaeがもつユニークな運動装置である接着器官内部には複数のタンパク質の複合体である棒状の細胞骨格が局在しており、この構造は細胞の滑走運動中でも重要な役割を果たしていると考えられている。本研究ではこの細胞骨格の主要構成因子であるHMW2の長さを変化させることで細胞骨格の長さ制御に成功し、細胞骨格の全長を定義する「分子ものさし」の存在を明らかにした。(著者抄録)

Motor proteins are molecular machines that convert chemical energy into mechanical work. In addition to existing studies performed on the linear motors found in eukaryotic cells, researchers in biophysics have also focused on rotary motors such as F1-ATPase. Detailed studies on the rotary F1-ATPase motor have correlated all chemical states to specific mechanical events at the single-molecule level. Recent studies showed that there exists another ATP-driven protein motor in life: the rotary machinery that rotates archaeal flagella (archaella). Rotation speed, stepwise movement, and variable directionality of the motor of Halobacterium salinarum were described in previous studies. Here we review recent experimental work discerning the molecular mechanism underlying how the archaellar motor protein FlaI drives rotation by generation of motor torque. In combination, those studies found that rotation slows as the viscous drag of markers increases, but torque remains constant at 160 pN·nm independent of rotation speed. Unexpectedly, the estimated work done in a single rotation is twice the expected energy that would come from hydrolysis of six ATP molecules in the FlaI hexamer. To reconcile the apparent contradiction, a new and general model for the mechanism of ATP-driven rotary motors is discussed.

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.

Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 μm/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP the units catch and pull sialylated oligosaccharides fixed to solid surfaces. In this study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 picoNewtons after the addition of 0–0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. After the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 picoNewtons. Considering the 70-nm step size, this small-unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.

Direct dissection of the angles of single fluorophores under an optical microscope has been a challenging approach to study the dynamics of proteins in an aqueous solution. For angle quantifications of single substrates, however, there was only one report (Nishizaka et al., 2014) because of difficulties of construction of experimental systems with active proteins working at the single-molecule level. We here show precise estimation of orientation of single fluorescent nucleotides bound to single tubulins that comprise microtubule. When single -headed kinesins immobilized on a glass surface drive the sliding of microtubules, microtubules show corkscrewing with regular pitches (Yajima et al., 2005 & 2008). We found, by using a three-dimensional tracking microscope, that S8A mutant kinesin also showed precise corkscrewing with a 330-m pitch, which is 13% longer than that of the wild type. The assay with the mutant was combined with a defocused imaging technique to visualize the rotational behavior of fluorescent nucleotide bound to corkscrewing microtubule. Notably, the defocused pattern of single TAMRA-GTP periodically changed, precisely correlating to its precession movement. The time course of the change in the fluorophore angle projected to the xy-plane enabled to estimate both the fluorophore orientation against microtubule axis and the precision of angle-determination of analyses system. The orientation showed main distribution with peaks at -40, 50 and 60. To identify their molecular conformations, the rigorous docking simulations were performed using an atomic-level structure modeled by fitting x-ray crystal structures to the cryo-electron microscopy map. Among isomers, 2'-O-EDA-GDP labeled with 5- or 6-TAMRA were mainly specified as possible candidates as a substrate, which suggested the hydrolysis of TAMRA-GTP by tubulins. (C) 2017 Elsevier Inc. All rights reserved.

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F-1-ATPase (F-1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-alpha(3)beta(3), while simultaneously monitoring rotations of the central shaft-gamma. In the ATP waiting dwell, two of three beta-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two beta-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F-1 structure and the rotation angle of the motor. Remarkably, a structure of F-1 in an epsilon-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the alpha beta-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of beta and the loosening/tightening motions at the alpha beta-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F-1-ATPase.

In this presentation, we explore the feasibility of plasmonic nanohole-based sub-diffraction-limited nanoscopy for biomolecular imaging. The technique utilizes near-field distribution localized by surface plasmon localization on metallic nanoholes which is used to sample molecular fluorescence. The optimum geometry of nanohole arrays was determined by numerical analysis. The localization sampling was applied to reconstructing sub-diffraction-limited images of gliding microtubules with a 76 nm effective resolution in the lateral direction. Extraordinary light transmission was also employed to address enhancement of axial resolution using nanohole arrays, based on which extraction of gliding motions of bacteria was demonstrated with an axial resolution down to 50 nm.

In this presentation, we explore the feasibility of plasmonic nanohole-based sub-diffraction-limited nanoscopy for biomolecular imaging. The technique utilizes near-field distribution localized by surface plasmon localization on metallic nanoholes which is used to sample molecular fluorescence. The optimum geometry of nanohole arrays was determined by numerical analysis. The localization sampling was applied to reconstructing sub-diffraction-limited images of gliding microtubules with a 76 nm effective resolution in the lateral direction. Extraordinary light transmission was also employed to address enhancement of axial resolution using nanohole arrays, based on which extraction of gliding motions of bacteria was demonstrated with an axial resolution down to 50 nm.

In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding Mycoplasma mobile (M. mobile). The results analyzed with multiple M. mobile bacteria show individual characters and reveal that M. mobile undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision.

Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (alpha beta gamma) as well as proteolytically generated two-headed (beta gamma) and one-headed (alpha) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in nnicrotubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed alpha beta gamma and one-headed alpha exhibited significantly longer pitch. In contrast, the pitch driven by two-headed beta gamma did not display this sensitivity. In the assays on lawns containing mixtures of alpha and beta gamma at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of a in the mixture increased. Even small proportions of alpha-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (alpha beta gamma) but is an intrinsic property of the subparticles of axonennal dyneins and also suggest that each subparticle may have distinct mechanical properties.