title: Heavy-meromyosin-decorated actin filaments: a simple method to preserve actin filaments for rotary shadowing.
abstract: It has become accepted that deep-freeze-drying at or below -90 degrees C is necessary to preserve the structure of supramolecular assemblies such as actin filaments (AFs) for metal shadowing. This has kept the metal shadowing technique from widespread use in the study of proteins complexed with AFs because of the limited availability of the apparatus for deep-freeze-drying. I report here that adsorption to freshly cleaved mica, treatment with buffered uranyl acetate in glycerol solution, rinsing, and removal of liquid eliminate the need of freeze-drying to preserve the structure of AFs. This technique, in combination with metal shadowing, was applied to the study of AFs decorated with heavy meromyosin (HMM). It was observed that (1) when HMM molecules are associated with single AFs in the majority of cases only one head of each HMM molecule makes contact at the point furthest from the neck region; (2) binding of HMM causes bundling of AFs, probably by the two heads of each molecule binding different filaments; and (3) the binding of HMM to the bundled AFs appears to be more stable than that to a single AF. This method of specimen preparation requires no freeze-drying and is therefore easily applicable to other large protein complexes.
authors.affiliation: Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
authors.forename: K
authors.initials: K
authors.lastname: Mabuchi
mesh_terms: D000199:Actins / Q000648:ultrastructure*; D000538:Aluminum Silicates; D000818:Animals; D005612:Freeze Drying; D007089:Image Enhancement; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000648:ultrastructure*; D009942:Organometallic Compounds; D011309:Preservation, Biological / Q000379:methods*; D011817:Rabbits
chemical_list: D000199:Actins; D000538:Aluminum Silicates; D015879:Myosin Subfragments; D009942:Organometallic Compounds; C005460:uranyl acetate; C011934:mica