Time-resolved cryo-electron microscopy of vitrified muscular components

Abstract

Biological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo-electron microscopy shows great potential for the study of such time-dependent phenomena. This study examines the actin polymerization process using cryo-electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t less than 2 min), filaments exhibit a pronounced structural variation and frequently show a central low-density area. In the later stages of the polymerization, F-actin-ADP filaments have a more uniform appearance and rarely display a central low-density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F-actin-ATP and more probably F-actin-ADP-Pi) and filaments at steady state (F-actin-ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo-substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X-ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is possible using cryo-substitution.