Force-generating cross-bridges during ramp-shaped releases: evidence for a new structural state.

Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5 degrees C, force transients were slowed sufficiently to record diffraction patterns in several 2-4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATPgammaS] and at 1 degrees C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.

Structural changes in the myosin filament and cross-bridges during active force development in single intact frog muscle fibres: stiffness and X-ray diffraction measurements.

Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4 degrees C and 2.15 microm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T(0)) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (S(M3) and S(M6)) increase by 1.5% from their resting values, with time courses that lead that of force by approximately 10 and approximately 20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by approximately 10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere)(-1) shortening at the velocity (V(0)) that maintains zero force. Shortening at V(0) transiently reduces S(M3) following the latent period and delays the subsequent increase in S(M3), but only delays the S(M6) increase without a transient decrease. Shortening at V(0) imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (I(M3)), whereas the intensity of the M6 reflection (I(M6)) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.

Dynamics of the self-assembly of unilamellar vesicles.

We have studied the transient stages in the formation of unilamellar vesicles with millisecond time resolution. The self-assembly was initiated by rapid mixing of equimolar amounts of anionic and zwitterionic micelles and the transient micellar entities were probed by time-resolved small-angle x-ray scattering. Within the mixing time, original micelles transformed to disklike micelles which evolved further to a critical size and then closed to form monodisperse unilamellar vesicles within a second. Subsequent growth led to an unexpected broadening of the vesicle size distribution.

Real-time simultaneous wide- and small-angle fibre diffraction.

A combination of two independent imaging area-detector systems controlled by a single data-acquisition system, provides a powerful system for X-ray diffraction studies of time-resolved phenomena over a wide q range, in samples with intrinsic or induced structural orientation. With this system we have observed a transient, tensile-stress-induced, orthorhombic-to-monoclinic transition in high-density polyethylene.