Biomedical subjects

C Toyoshima

Publications and source records attributed to C Toyoshima.

41 records · Page 3Linked to original sources

PubMed1984

Image analysis of the complex of actin-tropomyosin and myosin subfragment 1.

A three-dimensional image of the "rigor" complex of actin-tropomyosin-S1 was reconstituted from both low dose (10 electrons/A2) and high dose (greater than 500 electrons/A2) electron microscopic images of specimens embedded in unbroken and unbacked stain sheets of uranyl acetate over the holes of perforated carbon films. Myosin S1 shows multi-domain submolecular structure as has been earlier observed in actin-S1 ( Wakabayshi & Toyoshima, 1981) and actin-heavy meromyosin ( Katayama & Wakabayashi , 1981). The morphological unit of the actin-tropomyosin-S1 was found to be composed of at least three domains (domains A, B and D) and three regions (C, E and H). A myosin S1 molecule has a complex shape, which cannot be represented by a simple rod with one major axis. The shape of S1 should be approximated by at least two rods. The domain D is identified as the main part of S1. The angle between the major axis of this domain and the axis of actin helix was about 72 degrees, which is almost right angle. The angle between the axis of actin helix and major axis of the region E, which is less bulky than the domain D and makes no contact with actin, is much smaller than the value for the domain D. The resolution of reconstituted images from both high and low dose micrographs was improved so that the radial resolution became about 15 A and the axial one became about 25 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1981

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. II. The multi-domain structure of actin-myosin S1 complex.

A three-dimensional image of the "rigor" complex of actin and chymotryptic myosin subfragment-1 (S1) was reconstituted from electron micrographs of specimens embedded in unbroken and unbacked stain sheets of uranyl acetate over the holes of perforated carbon films to an effective resolution of 20 A radially and 26 A axially. The morphological unit of actin-S1 complex consists of at least three domains and myosin S1 shows a multi-domain submolecular structure. Possible ways to assign actin to one or more of these three domains are discussed. Two candidates for the shape of S1 molecule are also shown. Both candidates have a complex "embryo"-like shape. The solid model of the actin-S1 complex appears to be far less polar than that shown in the original electron micrographs or the projected density map of the reconstituted image shown by Toyoshima and Wakabayashi (1). The conspicuous polarity of the arrowhead pattern is related to the projected image of the spiral shape of the main part of the S1 molecule, which is almost at right angles to the helix axis. As one way to reconcile the non-tilted configuration of S1 in the rigor complex with sliding theory, the possibility of a pivoting mechanism (rotation of S1 head in the horizontal plane normal to the helix axis rather than in the vertical plane parallel to the helix axis) is discussed.

Actins↗

PubMed1979

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. I. Tilt angle of myosin subfragment-1 in the rigor complex.

A three-dimensional image of the "rigor" complex of actin and chymotryptic myosin subfragment-1 was reconstituted from electron micrographs of negatively stained specimens. Data went out to 20 A radially and 26 A axially. The reconstituted images allowed us to deduce the angle between the major axis of the main part of myosin subfragment-1 and the axis of the actin helix. The subfragment-1 molecules were attached to the actin filament in a configuration in which they were tilted by only about 15 degrees from the plane perpendicular to the axis of the actin helix. The implication of the smaller tilt angle than the commonly accepted value is discussed.

Actins↗

PubMedDate not supplied

Myosin subfragment-1 is sufficient to move actin filaments in vitro.

The rotating crossbridge model for muscle contraction proposes that force is produced by a change in angle of the crossbridge between the overlapping thick and thin filaments. Myosin, the major component of the thick filament, is comprised of two heavy chains and two pairs of light chains. Together they form two globular heads, which give rise to the crossbridge in muscle, and a coiled-coil rod, which forms the shaft of the thick filament. The isolated head fragment, subfragment-1 (S1), contains the ATPase and actin-binding activities of myosin (Fig. 1). Although S1 seems to have the requisite enzymatic activity, direct evidence that S1 is sufficient to drive actin movement has been lacking. It has long been recognized that in vitro movement assays are an important approach for identifying the elements in muscle responsible for force generation. Hynes et al. showed that beads coated with heavy meromyosin (HMM), a soluble proteolytic fragment of myosin consisting of a part of the rod and the two heads, can move on Nitella actin filaments. Using the myosin-coated surface assay of Kron and Spudich, Harada et al. showed that single-headed myosin filaments bound to glass support movement of actin at nearly the same speed as intact myosin filaments. These studies show that the terminal portion of the rod and the two-headed nature of myosin are not required for movement. To restrict the region responsible for movement further, we have modified the myosin-coated surface assay by replacing the glass surface with a nitrocellulose film. Here we report that myosin filaments, soluble myosin, HMM or S1, when bound to a nitrocellulose film, support actin sliding movement (Fig. 2). That S1 is sufficient to cause sliding movement of actin filaments in vitro gives strong support to models of contraction that place the site of active movement in muscle within the myosin head.

Actin Cytoskeleton↗

PubMedDate not supplied

Location of the ATPase site of myosin determined by three-dimensional electron microscopy.

Both ATP hydrolysis by myosin and the accompanying cyclic association-dissociation of actin and myosin are essential for muscle contraction. It is important for understanding the molecular mechanism of contraction to know the three-dimensional locations of the two major functional sites of myosin: the ATPase site and the actin-binding site. We have determined the position of the ATPase site of myosin using three-dimensional image reconstruction from electron micrographs and site-specific labelling with the avidin-biotin system. The ATPase site is about 5 nm from the tip of the myosin head and is about 4 nm away from the actin-binding site of myosin. This is the first report of the three-dimensional location of an enzyme active site by electron microscopy.

Actins↗