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T Yanagida

Publications and source records attributed to T Yanagida.

At least 127 records · Page 7Linked to original sources
PubMed1993

Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.

Actins↗
PubMed1993

Coupling between ATPase and force-generating attachment-detachment cycles of actomyosin in vitro.

We have developed a high resolution force measurement system in vitro by manipulating a single actin filament attached to a microneedle. The system could resolve forces less than a piconewton, and has time resolution in the submillisecond range. We have used this system to detect force fluctuations produced by individual molecular interactions. We observed large force fluctuations during isometric force generations. Noise analysis of the force fluctuations showed that the force was produced by stochastic and independent attachment-detachment cycles between actin and myosin heads, and one force-generating attachment-detachment cycle corresponded to each ATPase cycle. But, the force fluctuations almost completely disappeared during sliding at the velocities of 20 to 70% of the maximum one at zero load. The analysis indicated that myosin heads produced an almost constant force for most (probably > 70%) of the ATPase cycle time, i.e., the duty ratio > 0.7. Since the myosin step size was given as (velocity) x (the duty ratio) x (the ATPase cycle time, 30 ms), it was calculated to be 40 to 110 nm, corresponding to velocities of 20 to 70% of the maximum one (9 microns/s), respectively. These values are much greater than the displacement by a single attachment-detachment cycle of actomyosin (10-20 nm), indicating that multiple force-generating attachment-detachment cycles correspond to each ATPase cycle during sliding at velocities of > 20% of the maximum one. In conclusion, the coupling between the ATPase and the force-generating attachment-detachment cycles of actomyosin is not rigidly determined in a one-to-one fashion but is variable depending on the load.

Actins↗
PubMed1993

Nano-manipulation of actomyosin molecular motors in vitro: a new working principle.

Techniques have been recently developed that allow the direct observation of single actin filaments and their manipulation, using glass microneedles, in the nanometer range. Further development of these techniques has made possible the detection of subpiconewton-level forces of individual myosin heads. This in vitro motility model is sensitive in the submillisecond range and has allowed us to determine the force generation of an actomyosin motor directly at the molecular level. The results have led to a new conceptual framework for chemo-mechanical energy transduction in the molecular motor.

Actins↗
PubMed1993

Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament.

We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate and (b) copolymerization of unmodified actin monomers with monomers cross-linked internally with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Each of these modifications abolished the interaction of the modified monomers with myosin, so the remaining interactions were exclusively with unmodified monomers. The two modifications had similar effects on the interaction of actin with myosin in solution: decreased affinity of myosin heads for unmodified actin monomers, without a change in the Vmax of actin-activated myosin ATPase activity. However, modification (b) produced much greater inhibition of actin sliding on a myosin-coated surface, as measured by an in vitro motility assay. These results provide insight into the functional consequences of cooperative interactions within the actin filament.

Actins↗
PubMed1992

Orientational distribution of spin-labeled actin oriented by flow.

Previous studies on spin-labeled F-actin (MSL-actin), using saturation transfer electron paramagnetic resonance (ST-EPR), have demonstrated that actin has submillisecond rotational flexibility and that this flexibility is affected by the binding of myosin and its subfragments. This rotational flexibility does not change during the active interaction of myosin heads, actin, and adenosine triphosphate. However, these ST-EPR studies, performed on randomly oriented actin, would not be sensitive to orientational changes on the millisecond time scale or slower. In the present study, we have clarified these results by performing conventional EPR experiments on MSL-actin oriented by flow to detect changes in the orientational distribution. We have determined the orientational distribution of the spin labels relative to the magnetic field (flow direction) by comparing experimental EPR spectra to simulated EPR spectra corresponding to known orientational distributions. Spectra acquired during flow indicate two populations of probes: a highly ordered population and a disordered population. For the ordered population (28% of the total spin concentration), the angle between the actin filament axis and the nitroxide z axis (theta) fits a Gaussian distribution centered at 32.0 +/- 0.9 degrees, with a full width at half maximum of 20.7 +/- 3.9 degrees. The angle between the nitroxide x axis and the projection of the field in the xy plane (phi) is centered at 37.5 +/- 9.2 degrees with a full width of 24.9 +/- 10.7 degrees. This orientational distribution is not significantly changed upon the binding of phalloidin or myosin subfragment 1 (S1), indicating that these proteins do not affect the axial orientation of actin subunits. Spectra of spin-labeled S1 (MSL-S1) bound to actin oriented by flow have about the same orientational distribution as MSL-S1 bound to actin in oriented fibers. Thus, the oriented fraction of flow-oriented actin filaments has nearly the same high degree of alignment as the actin filaments in muscle fibers.

Actins↗
PubMed1992

The effectiveness of coronary angioscopy in detecting intraluminal pathologic changes.

We have evaluated the feasibility of percutaneous transluminal coronary angioscopy for detecting intraluminal pathological changes as a diagnostic tool, and investigated the pathogenesis of two acute coronary disorders, acute myocardial infarction and unstable angina. Twelve patients with an acute coronary disorder and 20 patients who underwent percutaneous transluminal coronary angioplasty were selected for this comparison between the diagnostic accuracy of angioscopy and arteriography. One hundred and thirty patients were investigated by angioscopy as follows in order to investigate the pathogenesis of their acute coronary disorders: 22 within 8 h of onset acute myocardial infarction; 28 from one day to 2 months since onset recent myocardial infarction; 37 with an old myocardial infarction; 26 with unstable angina; and 23 with stable angina. Our results have indicated thrombi were detected more frequently by angioscopy than by arteriography (p less than 0.01). Also, thrombi, intimal irregularities, and xanthomatous atheromas were observed more frequently in patients with acute myocardial infarction, recent myocardial infarction, and unstable angina. It is concluded that coronary angioscopy is a much more sensitive method for detecting intraluminal changes, and that a thrombus overlying a rupture in the lining of plaque plays a major role in an acute coronary disorders, and that the fragile, lipid-rich gruel atheroma may precede its rupture.

Coronary Angiography↗
PubMed1992

[Apparent depth by the stereograms without binocular retinal disparity: is Lau effect artifact?].

We examined the idea that, when the Zöllner figure in one eye and the principal lines in the figure in the other eye were presented, the perceived depth is due to "phenomenal" disparity. The phenomenal disparity is defined as apparent displacement between two illusory "oblique" principal lines in one eye and two "parallel" principal lines in the other. Observes were asked whether depth was seen or not at upper or lower part of the two perceived lines and, if seen, which line was closer. Seventeen observers showed that only 9% of their responses was consistent with the idea, although they reported depths for disparity stimuli. This suggests that the dichoptic presentation of such figures should not be considered as that requires the processing of binocular disparity.

Adult↗
PubMed1991

Sub-piconewton force fluctuations of actomyosin in vitro.

A new system has been developed for measuring the forces produced by a small number (less than 5-150) of myosin molecules interacting with a single actin filament in vitro. The technique can resolve forces of less than a piconewton and has a time resolution in the submillisecond range. It can thus detect fluctuations of force caused by individual molecular interactions. From analysis of these force fluctuations, the coupling between the enzymatic ATPase activity of actomyosin and the resulting mechanical impulses can be elucidated.

Actins↗
PubMed1991

Simulation of the electroretinographic slow PIII component by injection of K+ -free Ringer's solution in the frog retina.

The light-induced hyperpolarization of the photoreceptor results in a decrease in extracellular potassium ion concentration, [K+]. A decrease in [K+] in the photoreceptor layer was caused by injecting a small amount of K+-free Ringer's solution in the isolated frog retina, and the distribution of the field potential as induced by K+-free Ringer's solution was mapped. The depth profile of the response resembled that of the slow PIII component of the electroretinogram. These results support the notion that the slow PIII is generated by the Müller cells as they hyperpolarize in response to a light-induced decrease in [K+] in the photoreceptor layer.

Animals↗
PubMed1991

Mechanism of the active movement of actin: is actin sliding directly or indirectly related to binding with myosin and activation of myosin ATPase?

In the present work we examined the effect of crosslinking of polymerized and monomeric actin with glutaraldehyde, EDC and DSS on: 1) binding of actin to HMM in solution; 2) activation of HMM ATPase; 3) sliding movement of actin on glass-attached myosin; 4) properties of actin itself, like polymerizability and exchangeability of tightly bound nucleotide. The obtained data show that inhibition of sliding cannot be explained only by changes in the extent of activation of HMM ATPase and binding of actin to HMM; this result emphasizes the role of structural properties of actin in the mechanism of movement generation.

Actins↗
PubMed1991

Chemomechanical coupling in actomyosin system: an approach by in vitro movement assay and kinetic analysis of ATP hydrolysis by shortening myofibrils.

On the basis of our recent studies of the sliding distance of actin filaments during one ATP cycle on the surface of myosin-coated glass surface and ATP hydrolysis by rapidly shortening myofibrils, the molecular mechanism of chemomechanical coupling is considered. We conclude that the myosin head can repeat many active cyclic interactions with actins to drive the actin filaments over a long distance during one ATP cycle, and that the distance is variable depending on the load.

Actomyosin↗
PubMed1990

Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility.

The effects of crosslinking of monomeric and polymeric actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), disuccinimidyl suberate (DSS) and glutaraldehyde on the interaction with heavy meromyosin (HMM) in solution and on the sliding movement on glass-attached HMM were examined. The Vmax values of actin-activated HMM ATPase decreased in the following order: intact actin = EDC F-actin greater than DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than EDC G-actin. The affinity of actin for HMM in the presence of ATP decreased in the following order: DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than intact actin greater than EDC F-actin greater than EDC G-actin. However, sliding movement was inhibited only in the case of glutaraldehyde-crosslinked F and G-actin and EDC-crosslinked G-actin. Interestingly, after copolymerization of "non-motile" glutaraldehyde or EDC-crosslinked monomers with "motile" monomers of intact actin sliding of the copolymers was observed and its rate was independent of the type of crosslinked monomer, i.e. of the manner of their interaction with HMM. These data strongly indicate that inhibition of the sliding of actin by crosslinking cannot be explained entirely by changes in the Vmax value or affinity for myosin heads. We conclude that movement is generated by interaction of myosin with segments of F-actin containing a number of intact monomers, and the mechanism of inhibition involves an effect of the crosslinkers on the structure of F-actin itself.

Actins↗