1990 Fiscal Year Final Research Report Summary
Development of a New Optical Microscope System, which Resolves Motion of Motor Protein Molecules Less than Lnm on the Sub-Millisecond Time Scale.
| Project/Area Number |
63880034
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Osaka University |
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Principal Investigator |
YANAGIDA Toshio Osaka Univ. Faculty of Engineering Science, Professor, 基礎工学部, 教授 (30089883)
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| Co-Investigator(Kenkyū-buntansha) |
MASHIKO Masayuki Hamamatsu Photonics Corporation, Researcher, 研究員
HOTANI Kouichi Teikyo Univ. Faculty of Science, Professor, 理学部, 教授 (80025444)
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| Project Period (FY) |
1988 – 1990
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| Keywords | Actin / Myosin / Nanometer / Optical microscope |
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| Research Abstract |
We have developed a new system for measuring the force produced by a small number (1-100) of myosin molecules interacting with a single actin filament in vitro. The technique can resolve the motion of actin filaments less than 1 nm on the sub-millisecond time scale and thus detect fluctuations of force due to the individual molecular events. Development of this new system followed the design of techniques for force measurement in vitro motility assay by manipulating single actin filaments attached to glass microneedles under a fluorescence microscope.
Analysis of force fluctuations (noise) is a very powerful approach toward determining kinetic characteristics of individual myosin heads interacting with actin, especially when the number of interactions is small as in the present conditions. Under isometric conditions, we have observed large force fluctuations similar to membrane current fluctuations due to channel gating in electrophysiological systems with small numbers of channels.
The
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force fluctuations in the isometric condition are consistent with models incorporating stochastic and independent molecular events. Both a simplified, two state, ON-OFF model and the Huxley (1957) model simulate the amplitude and frequency spectrum of the observed cross-bridge noise. Using these models we determined the ON and OFF rates and the force generated by single myosin heads. The data are compatible with one fundamental mechanical interaction for each ATPase cycle.
On the other hand, when the actin filaments actively slide at velocities greater than -1 mum/s, fluctuations of force are much smaller. The data analysis indicates that myosin produces an almost constant force for greater than 90 % of the ATPase cycle time. This result indicates that during sliding, for each ATPase cycle there are many force-generating mechanical interactions between actin and myosin.
Thus, the present system allows us to directly examine how the ATPase cycle is coupled to the mechanical interactions of individual myosin heads with actin in vitro. These conclusions also apply directly to the molecular mechanism of muscle contraction. Less
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