Two-dimensional time-resolved X-ray diffraction studies of live isometrically contracting frog sartorius muscle

Abstract

Results were obtained from contracting frog muscles by collecting high quality time-resolved, two-dimensional, X-ray diffraction patterns at the British Synchrotron Radiation Source (SERC, Daresbury, Laboratory). The structural transitions associated with isometric tension generation were recorded under conditions in which the three-dimensional order characteristic of the rest state is either present or absent. In both cases, new layer lines appear during tension generation, subsequent to changes from activation events in the thin filaments. Compared with the 'decorated' actin layer lines of the rigor state, the spacings of the new layer lines are similar whereas their intensities differ substantially. We conclude that in contracting muscle an actomyosin complex is formed whose structure is not like that in rigor, although it is possible that the interacting sites are the same. Transition from rest to plateau of tension is accompanied by approximately 1.6% increase in the axial spacing of the myosin layer lines. This is explained as arising from axial disposition of the interacting myosin heads in the actomyosin complex. Model calculations are presented which support this view. We argue that in a situation where an actomyosin complex is formed during contraction, one cannot describe the diffraction features as being either thick or thin filament based. Accordingly, the layer lines seen during tension generation are referred to as actomyosin layer lines. It is shown that these layer lines can be indexed as submultiples of a minimum axial repeat of approximately 218.7 nm. After lattice disorder effects are taken into account, the intensity increases on the 15th and 21st AM layer lines at spacings of approximately 14.58 and 10.4 nm respectively, show the same time course as tension rise. However, the time course of the intensity increase of the other actomyosin layer lines and of the spacing change (which is the same for both phenomena) shows a substantial lead over tension rise. These findings suggest that the actomyosin complex formed prior to tension rise is a non-tension-generating state and that this is followed by a transition of the complex to a tension-generating state. The intensity increase in the 15th actomyosin layer line, which parallels tension rise, can be accounted for assuming that in the tension-generating state the attached heads adopt (axially) a more perpendicular orientation with respect to the muscle axis than is seen at rest or in the non-tension-generating state. This suggests the existence of at least two structurally distinct interacting myosin head conformations.(ABSTRACT TRUNCATED AT 400 WORDS)

Labelling of Thin Filaments by Myosin Heads in Contracting and Rigor Vertebrate Skeletal Muscles

“…Experimentally, the ratio of these axial spacings varies between 0.992 and 1.017 (Huxley & Brown, 1967;Haselgrove, 1975;Squire & Harford, 1988;Craig et al, 1992;Bordas et al, 1993). Since an accurate measurement of the spacings of the 36 and 24nm layer-lines is difficult because of the diffuse nature of the layer-lines, the values cited in these literatures may be consistent with the explanation presented above.…”

“…1/24.3nm -l (l--9): during contraction (Bordas et al, 1993) and in rigor (Huxley & Brown, 1967;Haselgrove, 1975;Squire & Harford, 1988;Yagi, 1992). Also in an optical diffraction pattern from electron micrographs of rigor muscles (Craig, Alamo & Padron, 1992;Hirose et al, 1993).…”

“…1/10.4nm -1 (l--20): during contraction (Bordas et al, 1993) and in rigor (Wakabayashi et al, 1988). 1/9.94nm -1 (l--22): in rigor (Yagi, unpublished observation on the diffraction pattern taken from a frog striated muscle treated with N-ethylmaleimide).…”

“…Myosin heads cannot bind to actin maintaining their 14.5nm repeat because the thin filament does not have such a repeat. Q(z) = ~ q, cxp [-i(2rrsz/14.58) The symmetry of the thin filament in this muscle can be approximated by 80 subunits of actin in 37 turns of the genetic helix within an axial repeat of 218.7 nm (Squire & Harford, 1988;Bordas et al, 1993). This axial repeat is also 15 x 14.58 nm.…”

“…Although these repeats in the two filaments are incommensurate, it has been suggested that the myosin heads in contracting muscle interact with actin maintaining their axial repeat. This suggestion is mainly based on the high intensity of the 14.5 nm meridional reflection in the X-ray diffraction pattern (Yagi, O'Brien & Matsubara, 1981;Huxley, Faruqi, Kress, Bordas & Koch, 1982;Bordas et al, 1993) and in the optical diffraction pattern from electron micrographs (Tsukita & Yano, 1985;Hirose, Lenart, Murray, Franzini-Armstrong & Goldman, 1993).…”