Muscle structure, cryo‐methods and image analysis

Abstract

Negatively stained cryo-sections from glutaraldehyde fixed, anti-freeze treated muscle, quench-frozen in Freon cooled by liquid nitrogen, show improved preservation of the axial structure of the myofibrils compared with conventional plastic sections. Such sections are being used both to characterize the structural differences in the M-bands of different vertebrate muscles and fibre types and also to define the axial distribution of myosin crossbridges and nonmyosin proteins in the crossbridge region of the A-band. Combined with analysis of the transverse A-band structure from plastic sections, the cryo-sections are helping to reconstruct a three-dimensional picture of the molecular architecture of the A-band. This, in turn, is providing the necessary structural background with which to interpret the wealth of published X-ray diffraction data on muscle. Such data should reveal the nature of the contractile event itself.Since good X-ray diffraction patterns can be obtained from living muscles, these can be compared with optical diffraction patterns from muscle cryo-sections as a means of testing the degree of preservation in the sections. Muscle is therefore an excellent tissue with which to evaluate new cryo-techniques.

Time-resolved X-ray diffraction studies of myosin head movements in live frog sartorius muscle during isometric and isotonic contractions

“…the cluster of reflections at ca 21.5 nm, the reflection at ca 14.34-14.58 nm (spacing at rest and at P0, respectively) and the cluster at ca 10.7 nm; and the reflections at spacings of ca 30.75, 27.1, I7.45 and 15.3 7 nm. These are due to long-range periodicities within the sarcomere and originate from interference effects across the M and Z bands (Haselgrove, i975;Squire, 1981;Squire et al, 1982). Figure 4 shows meridional traces recorded during protocol 3 of muscles at: rest (A); 30% remnant tension (B); the end of the high velocity shortening, when the load is negligible (C); and, the second plateau of tension redeveloped after the muscles' length is clamped (D).…”

“…In these the resolution range is larger, so that in addition to the features described above for protocol 2, the higher orders of the myosin based reflections (up to and including the 10th order) are clearly resolved. These diffraction orders also show splitting of the main myosin reflections which is attributable to long-range interference effects across the M and Z bands (Haselgrove, 1975;Squire, 1981;Squire et al, 1982).…”

“…Because the high-angle half of the 21.5 nm cluster is at the position of sampling by the 43.02 nm repeat, it is natural that its intensity is strongly affected by such distortions. In addition, it may be worth noting that the second order of the 44.0 nm C-protein reflection, the reflections due to long-range periodicities, as well as some contribution from structures in the M-band may all contribute to the intensity in the 'hah' of the 21.5 nm cluster (Squire et al, 1982). Now, Fig.…”