Takashi Ohki

Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms

The crescent‐shaped BAR (Bin/Amphiphysin/Rvs‐homology) domain dimer is a versatile protein module that senses and generates positive membrane curvature. The BAR domain dimer of human endophilin‐A1, solved at 3.1 Å, has a unique structure consisting of a pair of helix–loop appendages sprouting out from the crescent. The appendages short helices form a hydrophobic ridge, which runs across the concave surface at its center. Examining liposome binding and tubulation in vitro using purified BAR domain and its mutants indicated that the ridge penetrates into the membrane bilayer and enhances liposome tubulation. BAR domain‐expressing cells exhibited marked plasma membrane tubulation in vivo. Furthermore, a swinging‐arm mutant lost liposome tubulation activity yet retaining liposome binding. These data suggested that the rigid crescent dimer shape is crucial for the tubulation. We here propose that the BAR domain drives membrane curvature by coordinate action of the crescents scaffold mechanism and the ridges membrane insertion in addition to membrane binding via amino‐terminal amphipathic helix.

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions.

Early stages of energy transduction by myosin: Roles of Arg in Switch I, of Glu in Switch II, and of the salt-bridge between them

On the basis of the crystallographic snapshots of Rayment and his collaborators [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972], we have understood some basic principles about the early stages of myosin catalysis, namely, ATP is drawn into the active site, over which the cleft closes. Catalyzed hydrolysis occurs, and the first product (orthophosphate) is released from the backdoor of the cleft. In the cleft-closing process, the active site incidentally signals its movement to a particular remote tryptophan residue, Trp-512. In this work, we expand on some of these ideas to rationalize the behavior of a mutated system in action. From the behavior of recombinant myosin systems in which Arg-247 and Glu-470 were substituted in several ways, we draw the conclusions that (i) the force between Arg-247 and γ-phosphate of ATP may assist in closing the cleft, and incidentally in signaling to the remote Trp, and (ii) in catalysis, Glu-470 is involved in holding the lytic H2O (w1). We also propose that w1 and also a second water, w2, enter into a structure that bridges Glu-470 and the γ-phosphate of bound ATP, and at the same time positions w1 for its in-line hydrolytic attack.

Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin–AcGFP in Z discs

Nanoscale imaging of cultured cardiomyocytes allows the quantitative assessment of changes in the length of single sarcomeres during contractile events.

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca(2+) and incubated overnight at 4 degrees C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca(2+)-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.

Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function.

¡Vive la différence! In cardiac contraction, the reduction in sarcomere length—rather than length itself—determines contractile force.

Directional Bleb Formation in Spherical Cells under Temperature Gradient

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C μm(-1); 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients.

Modulation of the mechano-chemical properties of myosin V by drebrin-E

The regulation of actin filament networks by various proteins has essential roles in the growth cone dynamics. In this study we focused on the actin-myosin interaction which has been suggested to be an important player in the neurite extension. We examined in vitro how the decoration of actin filaments with a side-binding protein, drebrin-E, affects the motile properties of an intracellular transporter myosin V. Single myosin V molecules landed on the drebrin-E-decorated actin filaments with a lower frequency and ran over shorter distances; however, their velocities were normal. Furthermore, the analysis of the movement of myosin V molecules in the optical trap revealed that the decoration of actin filaments with drebrin-E markedly increased the load-sensitivity of the myosin V stepping. These results are attributable to the delay in the attachment of the motors leading head (ADP·P(i) state) to actin, induced by the competitive binding of drebrin-E to actin, whereas the rate of ADP release from the trailing head (the rate-limiting step in the ATPase cycle of myosin V) is unaffected. Our study indicates that, in addition to the regulation of binding affinity of myosin V, drebrin-E also modulates the chemo-mechanical coupling in the motile myosin V molecules, presumably affecting the movement of the growth cone.

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain

We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

The Force Production by the Depolymerization Activity of MCAK

During cell division the replicating chromosomes must be precisely arranged and separated polewards. Though many cellular processes involving motility require force generation by motor proteins, the chromosome movement is suggested to use the energy stored at plus ends of the microtubules to which kinetochores are attached. This energy is converted into the chromosome movement via passive couplers, whereas the role of the microtubule-based motors is supposed to be limited to the regulation of microtubule dynamics. The microtubule-depolymerizing kinesins, such as mitotic centromere-associated kinesin (MCAK), which is a founding member of kinesin-13 family, facilitate microtubule dynamics in a spindle and are required for the chromosome congression and segregation; however, the key question - whether the depolymerizing activity generates tension to pull the chromosomes - has remained unsolved. To probe the link between the generation of tension and the microtubule-disassembling activity of MCAK, we developed a single-molecule assay to examine the interaction between a single microtubule and multiple MCAK molecules under the fluorescence microscope equipped with the dual-trap optical tweezers. Here we show that the microtubule-disassembling activity of MCAK generates significant tension. The depolymerization force increases with the number of interacting MCAK molecules. These results provide a simple and attractive model for the generation of the driving force and the regulation of chromosome segregation in a spindle by the activity of MCAK.