Comparative histochemistry of a flatfish fin muscle and of other vertebrate muscles used for ultrastructural studies.

Because of the high degree of filament order in the myofibrils of fish skeletal muscles, and the resulting usefulness of such preparations (particularly flatfish fin muscles) in structural studies of muscular contraction, the fibre type composition of plaice fin muscle has been determined by conventional histochemical tests. As controls, and for comparison, fibre type distributions have also been studied in several other vertebrate skeletal muscles which are widely used for ultrastructural and mechanical studies. In view of the importance of single fibres in such studies and because much of the published information on fibre types is rather difficult to collate, we summarize here the fibre compositions of several muscles; comparable enzyme tests have been carried out on cryostat sections of rabbit psoas muscle, frog sartorius and semitendinosus muscles and plaice fin muscles. On this basis all four muscles are composed of more than one fibre type. We confirm that frog sartorius muscle is mainly a random mixture of two fast fibre types and show that there is also a third group of fibres which are small, metabolically rich and dark under acid m-ATPase tests. We confirm that the semitendinosus is composed of three fibre types, in three non-exclusive, concentric regions and that rabbit psoas muscle contains a mixture of at least three fibre types. The principal new findings of this work are that plaice fin muscle can be divided into four regions, some of which are composed of more than one fibre type, on the basis of its histochemical reactions. This division into regions changes seasonally. The system of classification devised by Dubowitz & Brooke (1973) for mammalian muscle, and which can be applied approximately to frog muscle, can also be applied to the fibres of plaice fin muscle provided that the test for lactate dehydrogenase is carried out in the presence of polyvinyl alcohol. These fibres do not easily fit the division into red, white and intermediate types normally used for fish myotomal muscles. Since none of these muscles is homogeneous, their complex nature must be borne in mind if they are to be used satisfactorily in structural and mechanical studies of muscular contraction involving the use of single fibres.

Three-dimensional structure of the insect (Lethocerus) flight muscle M-band.

The oval myosin filament profiles in transverse sections through the M-band of Lethocerus flight muscle are arranged in one of three orientations 60 degrees apart and point along the 11 directions of the hexagonal filament lattice. Relative orientations are not systematically related to give a superlattice structure, but neither are the orientations arranged completely randomly. In fact there is a nearly random structure with a slight bias towards adjacent filaments being identically oriented. This form of M-band structure is explained in terms of interactions between quasi-equivalent M-bridges. Its implications with regard to myosin crossbridge arrangement depend on the rotational symmetry of the crossbridge helix. For 6-stranded helices, 60 degrees rotations have no noticeable effect. However, in the case of the more likely 4-stranded structure, our results show that the crossbridge origins in the insect flight muscle A-band would be highly disordered. This disorder must be accounted for in interpreting both the flared-X crossbridge interactions seen in transverse sections of rigor insect flight muscle and the beautiful X-ray diffraction patterns from the same preparation. It is likely that in rigor insect muscle, some flared-Xs have the two heads of single myosin molecules interacting with two different actin filaments, whereas other flared-Xs have both of the myosin heads in one molecule interacting with the same actin filament.

Muscle structure, cryo-methods and image analysis.

Negatively stained cryo-sections from glutaraldehyde fixed, anti-freeze treated muscle, quench-frozen in Freon cooled by liquid nitrogen, show improved preservation of axial structure of the myofibrils compared with conventional plastic sections. Such sections are being used both to characterize the structural differences inthe M-bands of different vertebrate muscles and fibre types and also to define the axial distribution of myosin crossbridges and non-myosin 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.

Direct observation of a transverse periodicity in collagen fibrils.

Collagen fibrils have been extensively studied by electron microscopy, but only scant evidence has yet been obtained about their transverse structure. A transverse pediodicity of about 40 or 80 A might be expected if the microfibril model (until recently the common preference) is correct, but little direct evidence for such a periodicity has been obtained so far. We have studied electron micrographs of longitudinal cryosections of rat tail tendon. These appear very fibrous when studied by eye, but they produce at best only diffuse equatorial peaks in optical diffraction patterns. Nevertheless, we show here that like other fibrous protein structures, they do possess a strong transverse periodicity as revealed by their self-convolution functions (or auto-correlation functions). The observed periodicity is consistent with the presence of an 80 A structural unit in collagen fibrils in vivo.

High-voltage electron microscopy of crossbridge interactions in striated muscle.

In order to investigate the geometry of the interactions which myosin molecules make with actin filaments we have studied thick (0.2--0.5 micrometer) transverse sections of striated muscles in the 1 Me V electron microscope at Imperial College. Sections obtained from fixed relaxed frog sartorius muscle and both fixed relaxed and fixed rigor insect flight muscles, show regular electron opaque features between the thick and thin filament profiles. These are thought to be the overlapping images of the many levels of myosin heads that occur in such sections. From the appearances of these images, together with studies of thin transverse sections, it appears that of the possible interactions which one myosin molecule can make, namely that its two component heads interact with the same thin filament or with two different thin filaments, it is the former interaction (both heads on the same filament) which is predominant. Nevertheless appearances have been seen similar to those expected if an interaction of one molecule with two thin filaments occurs. It is concluded that both single filament and two filament interactions can occur depending on the steric convenience of the available actin subunits, but that the single filament interaction occurs in the majority of cases in the muscle states we have studied. Finally it is shown that the myosin filament profiles seen in thick transverse sections may be a very misleading guide to thick filament structure because of the influence which the myosin crossbridge have on the appearance of the profiles.

No polysaccharide demonstrated in filamentous structures in sieve elements by theiry's periodic acid-thiocarbohydrazide-silver proteinate method for electron microscopy.

By means of Thiery's Periodic acid-thiocarbohydrazide-silver proteinate method, for electron microscopy, it has been demonstrated that the filamentous structures of sieve elements contain no polysaccharide component.

Microfilaments and microtubules in elongating parenchyma cells of Nymphoides indica.

Observations on microfilaments and microtubules in elongating parenchyma cells of the central vascular bundle of Nymphoides indica (L.) O. Kuntze petiole are reported and discussed in relation to current concepts of the involvement of cellular organelles in cell wall synthesis.