Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actomyosin-ADP state on the bound leading head. When the isomerization precedes the 12-nm substep, the 36-nm step occurs without substeps.

The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization-depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments.

The homeostasis of body temperature and energy balance is one of the major principles in biology. Nanoscale thermometry of aqueous solutions is a challenging but crucial technique to understand the molecular basis of this essential process. Here, we developed a ratiometric nanothermometer (RNT) for intracellular temperature measurement in real time. Both the thermosensitive fluorophore, β-diketonate chelate europium(III) thenoyltrifluoroacetonate, and the thermoinsensitive fluorophore, rhodamine 101, which was used as a selfreference, are embedded in a polymeric particle that protects the fluorophores from intracellular conditions. The ratiometric measurement of single RNT spots is independent of the displacement of the RNT along the z-axis. The temperature is therefore determined at the location of each RNT under an optical microscope regardless of the dynamic movement of living cells. As a demonstration of the spot-by-spot intracellular thermometry, we successfully followed the temperature change in individual RNT spots in a single cell together with the Ca 2þ burst induced by the Ca 2þ ionophore ionomycin. The temperature increases differently among different spots, implying heterogeneous heat production in the cell. We then show that, in some spots, the temperature gradually decreases, while in others it remains high. The average temperature elevation within a cell is positively correlated to the increase in Ca 2þ , suggesting that the activity and/or number of heat sources are dependent on the Ca 2þ concentration.

The unbinding and rebinding of motor proteins and their substrate filaments are the main components of sliding movement. We have measured the unbinding force between an actin filament and a single motor molecule of muscle, myosin, in the absence of ATP, by pulling the filament with optical tweezers. The unbinding force could be measured repeatedly on the same molecule, and was independent of the number of measurements and the direction of the imposed loads within a range of +/- 90 degrees. The average unbinding force was 9.2 +/- 4.4 pN, only a few times larger than the sliding force but an order of magnitude smaller than other intermolecular forces. From its kinetics we suggest that unbinding occurs sequentially at the molecular interface, which is an inherent property of motor molecules.

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5-[␤,␥-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin-microtubule interaction during plus-and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility. Kinesin is a processive molecular motor that is essential for the transport of vesicles and organelles along a microtubule in various cells. Kinesin's processive movement has been explained by a mechanism that involves alternating between singleand double-headed bindings to a microtubule (1-5). Adjacent tubulin dimers of 8-nm length form consecutive binding sites (6), such that kinesin takes hundreds of 8-nm steps down a microtubule (7-10). Our recent single-molecule analysis of unbinding force (11) showed that conventional two-headed kinesin is involved in single-headed binding, both in the absence of nucleotides (nucleotide-free state) and in the coexistence of ADP and adenosine 5Ј-[␤,␥-imido]triphosphate (AMP-PNP) (ATP analogue), and double-headed binding in the presence of AMP-PNP (AMP-PNP state), which is consistent with the putative mechanism of kinesin motility.In the present study, we have measured the unbinding force of a single kinesin⅐microtubule complex under an optical microscope equipped with optical tweezers as was reported (11). To clarify the binding mode, we used one-headed kinesin heterodimers (12) in addition to conventional two-headed kinesin homodimers. Conventional two-headed homodimers or oneheaded heterodimers of kinesin molecules were attached to a polystyrene bead such that single kinesin binds to a single bead, and each bead was manipulated with optical tweezers on a microtubule that was adsorbed onto a coverslip (1, 9). An external load was imposed on the attached kinesin molecule by moving the bead toward the plus or the minus end of the microtubule. Here, we found that the two binding states exist in each head of kinesin depending on the nucleotide state. Also, we found that the dependence of the unbinding force on loading direction (where the unbinding force is smaller for the plus-end loading than for the minus-end loading) was independent of nucleotide states.We have analyzed the results for a weak and a strong bindi...

During cell division, many animal cells transform into a spherical shape and assemble a contractile ring composed of actin filaments and myosin motors at the equator to separate the cell body into two. Although actomyosin regulatory proteins are spatio-temporally controlled during cytokinesis, the direct contribution of cell shape and actomyosin activity to the contractile ring assembly remains unclear. Here, we demonstrated in vitro that actin polymerization inside cell-sized spherical droplets induced the spontaneous formation of single ring-shaped actin bundles in the presence of bundling factors. Despite a lack of spatial regulatory signals, the rings always assembled at the equator to minimize the elastic energy of the bundles. Myosin promoted ring formation by the dynamic remodelling of actin networks, and an increase in the effective concentration of myosin triggered ring contraction. These results will help us understand how animal cells coordinate cell shape and actomyosin activities to direct cytokinesis.

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of Ϸ72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through f luorescence polarization imaging of individual tetramethylrhodamine f luorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of Ϸ1 m, which is much greater than the 72 nm pitch. Thus, myosin does not ''walk'' on the helical array of actin protomers; rather it ''runs,'' skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each ''kick'' of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single f luorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.The actin filament is an array of actin protomers arranged in the form of two-start, right-handed helices with a pitch of Ϸ72 nm containing Ϸ13 protomers (1). If myosin tends to interact sequentially with one of the helical strands (the binding site on the other strand being on the opposite side), right-handed rotation of a sliding actin filament around its axis is expected. Indeed, in an in vitro motility assay in which the front end of a sliding filament was fixed on a surface, the middle part formed a left-handed superhelix, indicating right-handed rotation of the sliding rear part (2). However, in another assay where a marker (bead aggregate) was attached at the tail of a freely sliding filament, the filament slid over a long distance without rotating the tail beads (3). Quantitative resolution of this issue is important for the mechanism of motor function, because axial rotation is an indication of (i) sequential interaction of a myosin molecule with successive (or closely apposed) actin protomers, as stated above, and͞or (ii) the presence of a genuine torque component in the individual myosin-actin interaction not necessarily related to the helical structure. In the first assay above (2) where the superhelix formation was observed, the number of axial rotations could not be determined in the video images of limited resolution. A complication with the bead-tailed actin (3) was that the beads produced a large rotational, but not translational, friction that may have impeded the axial rotation. Here we show that the amount of rotation is small even in the absence of the external asymmetric load. Neither i nor ii appear to be essential components of the actomyosin motor. Quantitative measurement of the axial rotation without an impeding marker was achieved by continuo...

We report here the technique for detection and measurement of the temperature changes in single cells using a recently devised microthermometer (a glass micropipette filled with the thermosensitive fluorescent dye Europium (III) thenoyltrifluoroacetonate trihydrate). We found that the heat production in a single HeLa cell occurred with some time delay after the ionomycin-induced Ca(2+) influx from the extracellular space. The time delay inversely depended on extracellular [Ca(2+)], and the increase in temperature was suppressed when Ca(2+)-ATPases were blocked by thapsigargin. These observations strongly suggest that the enzymatic activity of Ca(2+)-ATPases in endoplasmic reticulum leads to the heat production. This study has therefore paved the way for studying the thermogenesis at the single-cell level.