Title

Unfixed cryosections of striated muscle to study dynamic molecular events

UMMS Affiliation

Department of Cell Biology

Date

10-1-1994

Document Type

Article

Subjects

Actins; Animals; Freezing; Insects; Microscopy, Electron; Mollusca; Muscle Contraction; Muscle Relaxation; Muscles; Myosins; Nephropidae

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

The structures of the actin and myosin filaments of striated muscle have been studied extensively in the past by sectioning of fixed specimens. However, chemical fixation alters molecular details and prevents biochemically induced structural changes. To overcome these problems, we investigate here the potential of cryosectioning unfixed muscle. In cryosections of relaxed, unfixed specimens, individual myosin filaments displayed the characteristic helical organization of detached cross-bridges, but the filament lattice had disintegrated. To preserve both the filament lattice and the molecular structure of the filaments, we decided to section unfixed rigor muscle, stabilized by actomyosin cross-bridges. The best sections showed periodic, angled cross-bridges attached to actin and their Fourier transforms displayed layer lines similar to those in x-ray diffraction patterns of rigor muscle. To preserve relaxed filaments in their original lattice, unfixed sections of rigor muscle were picked up on a grid and relaxed before negative staining. The myosin and actin filaments showed the characteristic helical arrangements of detached cross-bridges and actin subunits, and Fourier transforms were similar to x-ray patterns of relaxed muscle. We conclude that the rigor structure of muscle and the ability of the filament lattice to undergo the rigor-relaxed transformation can be preserved in unfixed cryosections. In the future, it should be possible to carry out dynamic studies of active sacromeres by cryo-electron microscopy.

Rights and Permissions

Citation: Biophys J. 1994 Oct;67(4):1612-9.

Related Resources

Link to article in PubMed

PubMed ID

7819493