Academic Background

Ph.D., London University, King's College, 1975

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.

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.