Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres

Force generation and motion in skeletal muscle result from interaction between actin and myosin myofilaments through the cyclical formation and rupture of the actomyosin bonds, the crossbridges, in the overlap region of the sarcomeres. Actomyosin bond properties were investigated here in single intact muscle fibers by using dynamic force spectroscopy. The force needed to forcibly detach the cross-bridge ensemble in the half-sarcomere (hs) was measured in a range of stretching velocity between 3.4 ؋ 10 3 nm⅐hs ؊1 ⅐s ؊1 or 3.3 fiber length per second (l0s ؊1 ) and 6. cross-bridges ͉ rupture force ͉ detachment rate constant ͉ fast stretches A tomic force microscopy (1-4) and force spectroscopy (5, 6) have been used to reveal the properties of the weak noncovalent bond between receptor molecules and their ligands, and in particular dynamic force spectroscopy has been used to define in detail the properties of the single biotin-avidin bond (1-3, 7). As regarding molecular motors, the optical tweezers technique has allowed the investigation of the properties of an individual actomyosin bond and to measure the force and unitary step displacement generated (8-10). The properties of the single actomyosin bond under rigor conditions were also investigated by measuring the dependence of the bond lifetime on the applied load (11). It was found that bond lifetime was strongly reduced when a load was applied to the bond, in good quantitative agreement with the prediction of Bell theory (12). These results on single actomyosin interaction represent a great step forward in our understanding of molecular mechanism of force generation in muscle; however, they were generally obtained under experimental conditions that differ from those encountered by the same motors in native preparation. In living skeletal muscle, myosin heads (the molecular motors) are distributed over the myosin filament in a very regular fashion, and both actin and myosin filaments are arranged in a quasicrystalline lattice that maintains a precise geometrical relationship between myosin heads (or fraction S1 of myosin molecule) and the active sites regularly distributed along the actin filament (13). This remarkable order is lost when experiments are made on single molecules, and this may affect the bond properties and force developed. In addition, at physiological ionic strength, actin filaments tend to detach from myosin heads, and it is therefore necessary to perform the above experiments at low ionic strength. However, it is known that molecular motor performance strongly depends on ionic strength (14). The force generated is also strongly affected by myosin S1 orientation, which is not easily controlled in single-molecule experiments (14). This means that the force measured by the recording apparatus, usually along the actin filament axis, may not represent accurately the real capability of the motors when working in a regularly ordered array. The same is true in experiments in which an external load is applied to the single bond, because it is usually d...

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of actomyosin bond properties in intact skeletal muscle by force spectroscopy

Colombini

Bagni

Romano

et al. 2007

“…S3). Rather, splicing may introduce a "hook" or a "bend" in the LCBDs of myo1b a and myo1b b that may amplify torsional motions of the LCBD that are on the path to the transition state but are not apparent in the displacement measurements (19)(20)(21). Further structural and mechanical characterizations of the LCBD are required to resolve this issue.…”

Section: Discussionmentioning

confidence: 99%

Control of myosin-I force sensing by alternative splicing

Laakso

Lewis

Shuman

et al. 2009

Myosin-Is are molecular motors that link cellular membranes to the actin cytoskeleton, where they play roles in mechano-signal transduction and membrane trafficking. Some myosin-Is are proposed to act as force sensors, dynamically modulating their motile properties in response to changes in tension. In this study, we examined force sensing by the widely expressed myosin-I isoform, myo1b, which is alternatively spliced in its light chain binding domain (LCBD), yielding proteins with lever arms of different lengths. We found the actin-detachment kinetics of the splice isoforms to be extraordinarily tension-sensitive, with the magnitude of tension sensitivity to be related to LCBD splicing. Thus, in addition to regulating step-size, motility rates, and myosin activation, the LCBD is a key regulator of force sensing. We also found that myo1b is substantially more tension-sensitive than other myosins with similar length lever arms, indicating that different myosins have different tension-sensitive transitions.

“…Previous electron paramagnetic resonance (EPR) studies using spin labels on the myosin RLC have shown that the transition from relaxation to contraction is accompanied by a large rotation (Ϸ36°) of the light chain (LC) domain in a small fraction of myosin heads, which is consistent with the LC domain's action as a lever arm (2). Fluorescence polarization data are consistent with this model but do not provide sufficient resolution to detect clearly the change in orientation between relaxation and contraction (4,5). The EPR data (2,6) show that there are two equally populated orientations of RLC in relaxed muscle, suggesting that the two heads of myosin have LC domains with distinct orientations.…”

mentioning

confidence: 99%

Coordination of the two heads of myosin during muscle contraction

Lidke

Thomas

2002

We have used luminescence resonance energy transfer between regulatory light chains (RLC) to detect structural changes within the dimeric myosin molecule in contracting muscle fibers. Fully functional scallop muscle fibers were prepared such that each myosin molecule contained a terbium-labeled (luminescent donor) RLC on one head and a rhodamine-labeled (acceptor) RLC on the other. Time-resolved luminescence energy transfer between the two heads increased upon the transition from relaxation (ATP) to contraction (ATP plus Ca) and increased further in rigor (no ATP). Combined with experiments on mutant RLCs labeled specifically at other sites, these results support a model in which the forcegenerating weak-to-strong transition causes one myosin LC domain to tilt through a 30°angle toward the other, thus acting as a coordinated lever arm.