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Molecular Structure, Dynamics, and Contractile Mechanism of Muscle

We use state-of-the-art electron microscopic techniques to understand how muscles contract. By studying the molecular structures of the actin and myosin filaments, whose interaction is responsible for contraction, we can elucidate the molecular mechanism of force generation and the processes responsible for regulating contraction. We are investigating systems as diverse as the rapidly contracting striated muscles of the skeleton and heart, and the smooth muscles of the internal organs (e.g. blood vessels), which are specialized to contract slowly and to maintain tension over long periods of time. These studies are adding to our basic understanding of muscle function, and also providing a structural basis for understanding muscle diseases caused by malfunction in the actin or myosin filaments.

Techniques: high resolution electron microscopy, 3D reconstruction, and atomic fitting

To decipher these filament structures in three dimensions at the molecular level, we use high resolution electron microscopy combined with computer image reconstruction. Specimens are observed by negative staining or cryo-electron microscopy, and 3D reconstructions of filaments are computed using helical or single particle methods. Atomic level detail is achieved by computationally 'fitting' atomic structures of filament subunits into the reconstruction. To study dynamic changes in filament structure that occur in active muscle, we have developed methods for capturing transient structural intermediates on the millisecond time scale for observation by EM.

Myosin filaments

Using these approaches we have recently achieved a major breakthrough in defining the 3D configuration of the key energy-transducing molecules, the myosin heads, on the surface of striated muscle myosin filaments (Woodhead et al., 2005). These results show for the first time, and in atomic detail, how myosin molecules are switched 'off', bringing about relaxation of muscle. The results suggest that the structure we observe is common to muscles of animals throughout most of the animal kingdom, and they provide a basis for understanding how these filaments are activated in contracting muscle. Our results also reveal for the first time how the tails of the myosin molecules are packed into the backbone of the thick filament, forming small 'subfilaments' that themselves assemble to form the thick filament core. This provides key background information for understanding how myosin filaments assemble in the cell.

Actin filaments

We have also made the first direct observations of how the protein tropomyosin, on the actin filament, regulates contraction by sterically blocking sites of myosin head attachment on actin filaments (Lehman et al., 1994; Xu et al., 1999; Pirani et al., 2005). We are currently determining the organization of the Ca2+-sensitive regulatory complex, troponin, on the thin filament, and how this changes on Ca2+ activation. These studies are revealing in atomic detail the molecular dynamics regulating muscle contraction.

Smooth muscle

In addition to our work on striated muscle, we have also shown that the myosin filaments of smooth muscle have a unique 'side-polar' structure, different from the helical organization in striated muscle. This structure helps to explain the characteristic ability of smooth muscles to undergo high degrees of shortening (Xu et al., 1996). Actin filaments from smooth muscle also differ from those in striated muscle, and we have gained new insights into their functioning in terms of the organization of their associated regulatory proteins (Hodgkinson et al., 1997; Lehman et al., 1997).

Current studies

We are currently determining the head organization in striated muscle myosin filaments from several key organisms, to test the generality of our model of the off state, and to determine whether subfilaments are a common feature of different species. We are imaging filaments at higher resolution to determine further details of their structure, and are carrying out tomographic studies of smooth muscle filaments to determine the three-dimensional details of their side-polar structure. In our studies of thin filaments, we are developing new methods of 3D reconstruction to reveal further details of the organization of troponin on actin, and we are combining the reconstructions with crystallographic structures of the thin filament components to produce a 3D thin filament model at the atomic level.

 

Myosin figure
 
Figure 1. 3D reconstruction and atomic fitting of (thick) myosin filament (from Woodhead et al., 2005). Left: 3D reconstruction showing arrangement of myosin heads on filament surface, and subfilaments running parallel to axis in filament backbone. Right: fitting of atomic structure of myosin heads (space-filling colored balls) into reconstruction of one pair of heads. The fitting reveals that the two heads interact with each other, preventing interaction with actin and thereby switching contraction off.
 

Actin figure

Figure 2. 3D reconstruction and atomic fitting of thin filament. Left: 3D reconstruction based on cryo images of thin filaments (from Xu et al., 1999). Actin in gold, tropomyosin in red (myosin blocking position), and green (non-blocking position). Right: fitting of actin atomic structure (yellow, α-carbon chain) into reconstruction of one actin subunit (blue wire). Highlighted in orange are amino acid clusters on actin that are blocked by tropomyosin in blocking position (white arrow). From Vibert et al., 1997.

One or more keywords matched the following items that are connected to Craig, Roger
Item TypeName
Academic Article The ultrastructural basis of actin filament regulation.
Academic Article The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity.
Academic Article Heterogeneity of Z-band structure within a single muscle sarcomere: implications for sarcomere assembly.
Academic Article Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants.
Academic Article Mini-thin filaments regulated by troponin-tropomyosin.
Academic Article Single particle analysis of relaxed and activated muscle thin filaments.
Academic Article Atomic model of a myosin filament in the relaxed state.
Academic Article A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle.
Academic Article Structural basis for the regulation of muscle contraction by troponin and tropomyosin.
Academic Article Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state.
Academic Article Ca2+ -induced tropomyosin movement in scallop striated muscle thin filaments.
Academic Article Head-head interaction characterizes the relaxed state of Limulus muscle myosin filaments.
Academic Article Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities.
Academic Article Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments.
Academic Article Ca2+ causes release of myosin heads from the thick filament surface on the milliseconds time scale.
Academic Article Molecular structure and organization of filaments in single, skinned smooth muscle cells.
Academic Article Protein switches in muscle contraction.
Academic Article Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation.
Academic Article An open or closed case for the conformation of calponin homology domains on F-actin?
Academic Article The structure of the vertebrate striated muscle thin filament: a tribute to the contributions of Jean Hanson.
Academic Article Structure and function of myosin filaments.
Academic Article Three-dimensional structure of vertebrate cardiac muscle myosin filaments.
Academic Article Head-head and head-tail interaction: a general mechanism for switching off myosin II activity in cells.
Academic Article Understanding the organisation and role of myosin binding protein C in normal striated muscle by comparison with MyBP-C knockout cardiac muscle.
Academic Article Tropomyosin and the steric mechanism of muscle regulation.
Academic Article Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms.
Academic Article Structural basis for the activation of muscle contraction by troponin and tropomyosin.
Academic Article Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle.
Academic Article A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments.
Academic Article Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function.
Concept Muscle Contraction
Concept Muscle Proteins
Concept Smooth Muscle Myosins
Concept Muscle Strength
Concept Muscles
Concept Muscle Relaxation
Concept Muscle, Smooth
Concept Muscle Fibers, Skeletal
Concept Muscle, Striated
Concept Muscle Fibers, Slow-Twitch
Concept Muscle Fibers, Fast-Twitch
Concept Muscle, Skeletal
Academic Article Structural changes induced in scallop heavy meromyosin molecules by Ca2+ and ATP.
Academic Article Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy.
Academic Article Direct determination of myosin filament symmetry in scallop striated adductor muscle by rapid freezing and freeze substitution.
Academic Article Caldesmon and the structure of vertebrate smooth muscle thin filaments. A minireview.
Academic Article Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments.
Academic Article Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar.
Academic Article Caldesmon and the structure of smooth muscle thin filaments: immunolocalization of caldesmon on thin filaments.
Academic Article Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.
Academic Article Steric-blocking by tropomyosin visualized in relaxed vertebrate muscle thin filaments.
Academic Article Unfixed cryosections of striated muscle to study dynamic molecular events.
Academic Article Myosin filament structure in vertebrate smooth muscle.
Academic Article Polymerization of myosin on activation of rat anococcygeus smooth muscle.
Academic Article Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.
Academic Article Three-dimensional reconstruction of thin filaments containing mutant tropomyosin.
Academic Article Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle.
Academic Article Purification of native myosin filaments from muscle.
Academic Article Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state.
Academic Article Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM.
Academic Article Through Thick and Thin--Interfilament Communication in Muscle.
Academic Article An invertebrate smooth muscle with striated muscle myosin filaments.
Academic Article An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits.
Academic Article Modulation of striated muscle contraction by binding of myosin binding protein C to actin.
Academic Article Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner.
Academic Article Molecular structure of muscle filaments determined by electron microscopy.
Academic Article The central role of the tail in switching off 10S myosin II activity.
Academic Article Lattice arrangement of myosin filaments correlates with fiber type in rat skeletal muscle.
Academic Article The myosin interacting-heads motif present in live tarantula muscle explains tetanic and posttetanic phosphorylation mechanisms.
Academic Article Cryo-EM structure of the inhibited (10S) form of myosin II.
Academic Article Relaxed tarantula skeletal muscle has two ATP energy-saving mechanisms.
Academic Article The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle.
Academic Article Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction.
Academic Article Structural basis of the super- and hyper-relaxed states of myosin II.
Academic Article Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle.
Academic Article Variants of the myosin interacting-heads motif.
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