Single carbon nanotube-based reversible regulation of biological motor activity.

Because of their small size and high thermal conductivity, carbon nanotubes (CNTs) are excellent candidates for exploring heat transfer at the level of individual molecules in biological research. With a view toward examining the thermal regulation of single biomolecules, we here developed single CNTs as a new platform for observing the motile activity of myosin motors. On multiwall CNTs (diameter ∼170 nm; length ∼10 µm) coated with skeletal-muscle myosin, the ATP-driven sliding of single actin filaments was clearly observable. The normal sliding speed was ∼6 µm/s. Locally irradiating one end of the CNT with a red laser (642 nm), without directly irradiating the active myosin motors, accelerated the sliding speed to ∼12 µm/s, indicating the reversible activation of protein function on a single CNT in real time. The temperature along the CNT, which was estimated from the temperature-dependence of the sliding speed, decreased with the distance from the irradiated spot. Using these results with the finite element method, we calculated a first estimation of the thermal conductivity of multiwall CNTs in solution, as 1540 ± 260 (Wm(-1) K(-1)), which is consistent with the value estimated from the width dependency of multiwall CNTs and the length dependency of single-wall CNTs in a vacuum or air. The temporal regulation of local temperature through individual CNTs should be broadly applicable to the selective activation of various biomolecules in vitro and in vivo.

Temperature changes in brown adipocytes detected with a bimaterial microcantilever.

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell's thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell's heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4-7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell's state by measuring thermal characteristics.

Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis.

The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB.

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.