The Structure of Certain Muscle Fibrils as Revealed by the Use of Electron Stains

Abstract

Fibrils from certain molluscan muscles, in particular the adductor muscles of the clam Venus mercenaria, were examined with the electron microscope and found to possess periodic variations in structure. In order to make these structural variations visible, it was necessary to treat the fibrils with reagents of high electron scattering power (electron stains). Phosphotungstic acid was found to be particularly suitable. This stain combines with specific regions in the fibrils, forming a remarkably regular geometrical pattern of which the most prominent feature is a regular cross striation, representing a fiber-axis spacing of about 145A. Within each stained band, the stain is more highly concentrated in spots spaced about 193A from center to center across the band. A line drawn through any such spot parallel to the fiber axis passes through other similar spots, spaced five cross bands apart, making the length of the fiber-axis period precisely five times the fiber-axis spacing. X-ray diffraction data obtained by Bear from the intact muscles are compared with the electron microscope observations. The small angle diffractions are in close agreement with those which would be expected from the observed structure except for the magnitude of the lateral spacing. Electron microscope values for this spacing are significantly smaller than the 325A indicated by the x-ray data, probably as a result of lateral shrinkage in the vacuum-dried electron microscope specimens. No significant difference in axis spacing has been observed in fibrils isolated from muscles fixed with alcohol in contracted and extended states.

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…The structural characteristics of paramyosin containing thick filaments are the filament (or sometimes just its core) possessing one or more of 1) a prominent 14.5 nm periodicity (amphioxus: Flood et al, 1969;Yongshui and Zuxun, 1979;mollusc: Elliott et al, 1957;Philpott et al, 1960;Ishii and Takahashi, 1983), 2) a ribbon-like structure (Nematomorpha: Lanzavecchia et al, 1977), 3) long, typically 72 nm, periodicity, sometimes with a multiple of 14.5 nm substructure (Echinodermata: Baccetti and Rosati, 1968;annelid: Lanzavecchia, 1972;Camatini et al, 1976;Nematomorpha: Swanson, 1971b;Lanzavecchia et al, 1977;Deitiker and Epstein, 1993;Epstein et al, 1995;mollusc: Bear, 1944;Jakus et al, 1944;Hall et al, 1945;Schmitt et al, 1947;Bear and Selby, 1956;Hanson et al, 1957;Hodge, 1959;Kahn and Johnson, 1960;Elliott, 1964bElliott, , 1968bSobieszek, 1973;Heumann, 1973;Eshleman et al, 1982), or 4) a checkerboard pattern (a 'Bear-Selby net') with a repeat distance typically of 5 × 14.5 = 72 nm (Fig. 3A) (mollusc: Lanzavecchia, 1966Lanzavecchia, , 1972Szent-Györgyi et al, 1971;Heumann, 1973;Nonomura, 1974;Elliott, 1979;Castellani et al, 1983;Ishii and Takahashi, 1983;Bennett and Elliott, 1984;Panté, 1994;…”

“…Most early work favored a paracrystalline or stacked flat layer (ribbon) structure (Hall et al, 1945;Bear and Selby, 1956;Elliott et al, 1957;Elliott and Lowy, 1961;Elliott, 1964a;Lanzavecchia, 1966). This was followed by a period in which helical properties were believed to be present in large diameter thick filaments (Elliott and Lowy, 1969;Elliott, 1971), giving rise to models in which the paramyosin dimers were arranged to form a sheet that was then either rolled up like a rug to form the paramyosin filament (Elliott and Lowy, 1970;Lanzavecchia, 1977), or the filament was composed of concentrically nested cylinders of paramyosin sheets (Heumann, 1980).…”

Electron Stains

“…Clearly, if the stain contains heavy atoms, fewer molecules are required to cause a given increase in density. Early work by Hall (17) and Hall et al (18) suggested the use of heavy metal ions as electron stains.…”

The Reactivity and Staining of Tissue Proteins With Phosphotungstic Acid

“…Phosphotungstic acid (PTA) has long been used as a protein precipitant (19), and Hall et al (9,10) introduced it for positive staining of whole muscle and collagen fibrils. From these and other studies (5-7, 11-13, 18), it was concluded that PTA acted as an anionic stain for the positively charged groups of protein.…”