西坂 崇之

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase II alpha, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51+/-0.33 mu m (11+/-6 turns) of a braid was unlinked in a burst of reactions taking 8+/-4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being similar to 100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of similar to 10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break similar to 10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.

Sub-diffraction-limited imaging of fluorescent monomers on sliding microtubules in vitro by nanoscale localization sampling (NLS) is reported. NLS is based on periodic nanohole antenna arrays that create locally amplified electromagnetic hot spots through surface plasmon localization. The localized near-field hot spot temporally samples microtubular movement for enhanced spatial resolution. A fourfold improvement in spatial resolution compared to conventional wide-field microscopy is demonstrated. The resolution enhancement is achieved by imaging rhodamine-labeled microtubules that are sampled by the hot spots to provide sub-diffraction-limited images at 76 nm resolution in the direction of movement and 135 nm orthogonally. The intensity distribution produced by the NLS is measured to be broader than that of conventional imaging, which is consistent with the improvement of imaging resolution. Correlation studies between neighboring nanoantennas are also performed. This confirms the possibility of measuring microtubular transport dynamics. NLS can be useful for moving objects that have a high labeling density or for performing fluctuation spectroscopy in small volumes, and may allow super-resolution on demand by customizing nanoantenna structures for specific resolution needs.

F1-ATPase, a water-soluble portion of ATP synthase, is a fully reversible rotary molecular machine in which a central γ subunit rotates inside a cylinder made of three α and three β subunits alternately arranged. This motor rotates counterclockwise by hydrolyzing ATP in three catalytic sites but synthesizes ATP when forced to rotate clockwise by an external force. Single-molecule studies have revealed how the chemical reactions that occur in the three catalytic sites are coupled to mechanical rotation. © 2012 Elsevier B.V. All rights reserved.

F-1-ATPase is a water-soluble portion of FoF1-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit gamma has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit gamma between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a similar to 4 degrees outward tilting of the gamma-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the gamma-subunit and provides a new constraint condition of the ATP-waiting conformation of F-1-ATPase, which has not been determined as an atomic structure from x-ray crystallography.

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically Conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells. (C) 2011 Elsevier Inc. All rights reserved.

F 1-ATPase is the smallest rotary molecular motor ever found. Unidirectional rotation of the γ-shaft is driven by precisely coordinated sequential ATP hydrolysis reactions in three catalytic sites arranged 120° apart in the cylinder. Single-molecule observation allows us to directly watch the rotation of the shaft using micron-sized plastic beads. Additionally, an advanced version of "total internal reflection fluorescence microscope (TIRFM)" enables us to detect binding and release of energy currency through fluorescently labeled ATP. In this chapter, we describe how to set up the system for simultaneous observation of these two critical events. This specialized optical setup is applicable to a variety of research, not only molecular motors but also other single-molecule topics. © 2011 Springer Science+Business Media, LLC.

The final goal of our group is to establish the missing link between chemical reaction and mechanical event in molecular motors. To achieve this, we have developed advanced versions of conventional optical microscopes and applied them into single-molecule techniques. In this chapter we present two studies: one is about the kinesin-microtubule system and the other F(1)-ATPase. These techniques are applicable to other molecular machines, hopefully in more sophisticated ways, and we hope to investigate this in future studies.

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non-cargo proteins during COPII vesicle formation using single-molecule microscopy combined with an artificial planar lipid bilayer. Single-molecule analysis showed that the Sar1p-Sec23/24p-cargo complex, but not the Sar1p-Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non-cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non-cargo proteins from the COPII vesicles. The EMBO Journal (2009) 28, 3279-3289. doi: 10.1038/emboj.2009.269; Published online 17 September 2009

Rotation of the central shaft gamma subunit in a molecular motor F(1)-ATPase is assumed to correlate with and probably be driven by domain motions of the three catalytic beta subunits. Here we observe directly these b motions through an attached fluorophore, concomitantly with 80 degrees and 40 degrees substep rotations of c in the same single molecules. We show the sequence of conformations that each b subunit undergoes in three-step bending, a similar to 40 degrees counterclockwise turn followed by two similar to 20 degrees clockwise turns, occurring in synchronization with two substep rotations of gamma. The results indicate that most previous crystal structures mimic the conformational set of three b subunits in the catalytic dwells. Moreover, a previously undescribed set of beta conformations, open, closed and partially closed, is revealed in the ATP-waiting dwells. The present study thus bridges the gap between the chemical and mechanical steps in F(1)-ATPase.

Rotation of the central shaft γ subunit in a molecular motor F 1-ATPase is assumed to correlate with and probably be driven by domain motions of the three catalytic β subunits. Here we observe directly these β motions through an attached fluorophore, concomitantly with 80° and 40° substep rotations of γ in the same single molecules. We show the sequence of conformations that each β subunit undergoes in three-step bending, a ∼40° counterclockwise turn followed by two ∼20° clockwise turns, occurring in synchronization with two substep rotations of γ. The results indicate that most previous crystal structures mimic the conformational set of three β subunits in the catalytic dwells. Moreover, a previously undescribed set of β conformations, open, closed and partially closed, is revealed in the ATP-waiting dwells. The present study thus bridges the gap between the chemical and mechanical steps in F1-ATPase. © 2008 Nature Publishing Group.

Mitotic kinesin Eg5 is a homotetrameric molecular motor that cross-links and slides microtubules. The extent to which Eg5 moves processively is not clear. Here we use three-dimensional tracking of a quantum dot attached to the microtubule in a motility assay to directly visualize the corkscrew motion of a sliding microtubule. We show that the rotational pitch of microtubule sliding conveniently reports on the processivity of the driving motors, confirming that two-headed Eg5 is much less processive than two-headed kinesin-1.

Mitotic kinesin Eg5 is a homotetrameric molecular motor that cross-links and slides microtubules. The extent to which Eg5 moves processively is not clear. Here we use three-dimensional tracking of a quantum dot attached to the microtubule in a motility assay to directly visualize the corkscrew motion of a sliding microtubule. We show that the rotational pitch of microtubule sliding conveniently reports on the processivity of the driving motors, confirming that two-headed Eg5 is much less processive than two-headed kinesin-1. © 2008 Nature Publishing Group.

F1-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F1 that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F1 is forced to rotate slowly, that release of Cy3-ADP occurs at similar to 240 degrees after it is bound as Cy3-ATP at 0 degrees. This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P-i in its individual actions at a single molecule level. The F, portion of ATP synthase, also called F-1-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of Delta G(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.