Myosin filament structure in vertebrate smooth muscle.

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts.

Three-dimensional reconstruction of thick filaments from rapidly frozen, freeze-substituted tarantula muscle.

We have applied three-dimensional helical reconstruction techniques to images of myosin filaments of tarantula leg muscle obtained from rapidly frozen, freeze-substituted specimens. Computed Fourier transforms of filaments selected from longitudinal sections show up to six layer lines indexing on the 43.5-nm helical repeat of myosin crossbridges. The three-dimensional reconstruction, performed after separation of overlapped Bessel functions, shows four continuous strands of density on the surface of the filament, modulated by density at 14.5-nm intervals, corresponding to the myosin heads aligned approximately along the helical strands. In transverse viw, the reconstruction shows four projections and is similar in profile to myosin filaments seen in thin transverse sections of rapidly frozen muscle. The reconstruction is similar to that of negatively stained, isolated tarantula filaments except that in the latter there is an additional modulation of the helix density, which better resolves the two heads of each myosin crossbridge. Thus, the general arrangement of the myosin heads in the freeze-substituted specimens is preserved, although finer details of structure such as individual myosin heads are lost.

Steric-blocking by tropomyosin visualized in relaxed vertebrate muscle thin filaments.

Although widely accepted, the steric-blocking model of vertebrate skeletal muscle regulation has not been confirmed. Previous attempts to directly visualize tropomyosin in relaxed skeletal muscle and demonstrate that it interferes with the crossbridge-thin filament contractile cycle were unsuccessful. In the work reported here, tropomyosin was resolved in electron micrographs of native thin filaments isolated from relaxed vertebrate striated muscle. Three-dimensional helical reconstructions of these filaments showed continuous narrow strands of density, representing tropomyosin, which followed the outer domains of successive actin monomers. The results obtained from fitting the atomic model of filamentous actin to these reconstructions illustrate, and are consistent with, the mechanism of steric-blocking, since tropomyosin was found to be positioned on the actin surface of thin filaments over clusters of identifiable amino acids required for myosin crossbridge docking.