Comparison of the ability of freeze etch and freeze substitution to preserve actin filament structure

Abstract

In order to test the ability of freeze substitution to accurately preserve the ultrastructure of the actin component of the cytoskeleton, the structure of rotary shadowed actin filaments was compared following preparation by glutaraldehyde fixation and freeze etch or freeze substitution. Freeze substituted actin filaments were further processed by either etching away frozen organic solvent or critical-point-drying before rotary shadowing. Comparison of filament diameters showed no significant difference between actin filaments that were directly etched and those that were freeze substituted and then etched. However, freeze substituted and then critical-point-dried filaments were significantly larger in diameter than filaments that were directly etched in water. The long pitch (right-handed) two start helix was not affected by the different methods of preparation. However, the left-handed "genetic" helical repeat that was prominent in actin filaments prepared by freeze etch was more difficult to detect in freeze substituted specimen, especially following critical-point-drying. Although the organization and distribution of actin filaments in extracted cells was similar in both freeze substituted and freeze etched specimens, there were some detectable differences. In cells that were freeze substituted and then critical-point-dried, filaments appeared to intersect at greater angles and seemed more "taut." These results suggest that freeze substitution can preserve the overall morphology of actin filaments, but some chemical or physical modification of macromolecular surface structure may occur during the substitution process and these changes may be further exaggerated by subsequent processing steps.