Hugh Esmor Huxley - US grants

The long-term objective of this research is to reach a detailed understanding of the molecular mechanism of muscle contraction, and at the same time to gain thereby important information about other cellular motile processes involving actin-myosin interactions. Understanding of muscle pathology must remain incomplete until we understand fully the basic mechanism which all the other ancillary processes in muscle cells have to support; a full understanding of cell movements during development, and of cell invasiveness, also requires basic knowledge of the motile mechanisms involved. The specific aim of the project is to obtain as much information as possible about the structural changes taking place in the myosin crossbridges in muscle as they interact with actin to produce the sliding force between the arrays of actin and myosin filaments. The techniques that will be employed in this work are those of low angle X-ray diffraction and electron-microscopy. The X-ray work will exploit the new sources of high intensity synchrotron radiation at such places as the Brookhaven National Laboratory to carry out detailed time-resolved studies of the changes in the muscle diffraction pattern which reflect the structural events in contraction. The electron-microscope work will make use of rapid freezing techniques to obtain direct images of muscle filaments and crossbridges in chosen states of contraction. Both approaches call for substantial technical developments but, if successful, these new techniques will be of considerable value in other biological fields. The results of these structural studies should provide a detailed framework of understanding for the very active research on contraction and motility being pursued by many other workers using biochemical, biophysical, genetical and physiological techniques.

This research is intended to elucidate the mechanism by which force is developed by the interaction of myosin and actin molecules in muscle (and in a number of other motile systems) and will focus on the dynamics of the changes taking place in the crossbridges in striated muscle during contraction. It has long been established that muscle contraction is brought about by a sliding filament mechanism, in which partially overlapping arrays of actin and myosin filaments are acted on by a relative sliding force which leads to shortening of the muscle. It is generally believed that this force is generated by a cyclic interaction of myosin crossbridges with sites on the actin filaments. However, the structural details of the process have proved to be particularly difficult to establish, partly because of the transient and unsynchronized nature of many of the changes taking place, and partly because of the technical difficulty of obtaining submicroscopic structural information about any rapidly changing biological system. The development of rapid freezing techniques by Heuser and others has now made it possible in principle to obtain electron microscope images of these transient structural states and suitable variations of these techniques will be developed to enable correlations to be made with evidence derived from time-resolved x-ray diffraction studies. These approaches should provide important new information about the structural behavior of the crossbridges as they interact with actin and develop force. When combined with new information from biochemical and physiological studies, the results of the present work should materially advance our understanding of this very fundamental biological mechanism.

This application is for partial funding of a symposium 'Skeletal muscle - crossbridges' that forms part of the XXXII International Congress of Physiological Sciences, Glasgow, August 1-5, 1993. This congress is organized as a series of Symposia with strong themes that run throughout the meeting. Chairpersons and organizers for each symposium have been selected because of their contributions to the symposium theme. The list of speakers includes major contributors in the several exciting developments that are presently occurring in skeletal muscle research. Two of the contributions will set these developments into the context of existing knowledge (H.E. Huxley, Simmons). These new developments arise principally out of: 1) atomic resolution structures of actin and myosin, 2) time resolved structural changes of crossbridges especially from low angle X-ray diffraction and freeze-substitution electron microscopy. These advances in turn depend in part on the novel mechanical techniques developed by Lombardi and co-workers. 3) in vitro motility and force measurements of individual myosin and actin filaments. These are techniques that permit individual cross-bridge events to be analyzed. They also permit insights afforded by molecular genetics to be fully exploited in muscle research. This meeting is timely and sets an exciting stage for the other muscle related symposia of the meeting. In this the meeting is unique. It allows and encourages the advances made in skeletal muscle research (where the highly organized sarcomer structure makes this muscle amenable to advanced biophysical techniques) to be transmitted in a direct way to other muscle researchers. In this way it complements the more traditionally interactive meetings on skeletal muscle (such as The Contractility Gordon Conference, also in 1993) where nearly all the participants are expert in one particular area of muscle research. Anticipated attendance is expected to be between 5000 and 6000, ensuring exposure to a wide range of both basic and clinical scientists involved in muscle physiology. The importance of this symposium is that it launches the exciting developments and results of the past three years in skeletal muscle into the general awareness of a much wider body of scientists. This knowledge will percolate in a broad way into research on health related muscle disease and dysfunction.

DESCRIPTION: X-ray diffraction studies of muscle contraction mechanism The long-term objectives of this research are to reach a full and detailed understanding of the molecular mechanism of muscle contraction and thereby to better understand the many other motile mechanisms involving actin-myosin type interactions which are utilized in a wide variety of important cellular functions. Understanding of muscle pathology, whether in dystrophy or in heart diseases, must incomplete until we fully understand the basic processes involved in the essential contractile function of the tissue. A full understanding of the operation of motor proteins involved in other cell movements, whether during development or healing or metastasis, also requires basic knowledge of the motile mechanisms involved. The specific aim of the project is to obtain as much information as possible about the structural changes in the myosin crossbridges as they interact with actin filaments to produce force and sliding movement during contraction. Although much is known about the structure of the components of this system. the actual manner in which relative forte and displacement is produced still remains unknown. The X-ray work will take advantage of the extremely high flux now available from certain synchrotron radiation X-ray sources, and of other developments in X-ray technology, to analyze new and informative aspects of the diffraction diagrams which can now be explored, especially during transient states, to characterize the nature of the crossbridge movements involved. The results of these studies on intact muscles and on skinned muscle fibers should greatly facilitate arriving at a full understanding of the motor process, using all the remarkably detailed information now becoming available from structural, biochemical and physiological investigations.