SPring-8, the large synchrotron radiation facility

Release Date

06 Aug, 2002

August 6, 2002
Japan Synchrotron Radiation Research Institute (JASRI)

 A skeletal muscle (striated muscle) consists of many muscle cells (muscle fibers, diameter, 50 - 100 µm)(Fig. 1a). A muscle cell in turn consists of many myofibrils (diameter, 1 - 2 µm)(Fig. 1b). Each myofibril is made up of many sarcomeres (the smallest functional unit of muscle; length, ~2 µm) connected in series (Fig. 1c). In each sarcomere, the myofilaments (polymer of contractile protein molecules, either actin or myosin) are arranged in a regular hexagonal lattice (Fig. 1d). Because of this arrangement, a number of reflections indexed to the hexagonal lattice can be recorded when irradiated with X-ray beams. The obtained diffraction pattern contains rich information about the structure of the contractile proteins.
Previously, the smallest muscle specimen from which X-ray diffraction patterns could be recorded had been single muscle fibers. The diffraction pattern recorded from a single muscle fiber (equatorial reflections) are essentially a rotary-averaged pattern from which information about the orientations of the lattice planes has been lost (Fig. 2b, c). This is because a single muscle fiber still contains thousands of myofibrils, and their lattice planes are randomly oriented. To separate the reflections arising from different lattice planes, one must record a diffraction pattern from a single myofibril.
The flight muscle of insects, such as bumblebee (Fig. 2a), is known to have unusually regular arrangement of contractile proteins in the sarcomere. By using this muscle, the research group was able to record diffraction patterns from single myofibrils and to separate reflections arising from different lattice planes of a single lattice (Fig. 3b, c). The patterns were obtained from a specimen whose volume was only 1/1,000 of that of the previously used smallest muscle specimen (single muscle fibers).
 The technical background for this achievement is as follows:
(1) The use of X-ray microbeams generated by pinholes (diameter, 2 µm). X-ray microbeams have been used to record diffraction patterns from human hair and other dried biological macromolecules. However, hydrated intracellular protein assemblies are much more susceptible to radiation damage and this is the first example that clear diffraction patterns were recorded by X-ray microbeams from such assemblies. The exposure time was only 5 s. Such a short exposure time was made possible by the high-flux, low-emittance beams from the third-generation synchrotron facility and sensitive detectors. The BL40XU beamline (which became operational recently) can produce even brighter X-ray beams, and its use may make possible the real-time observation of the structural changes of contractile proteins in a lattice.
(2) End-on diffraction. To record diffraction patterns from fibers, the specimen is usually placed perpendicular to the incident X-ray beams. In case of recording from a single myofibril, diffraction theories predict that reflections from only one lattice plane will be recorded at the same time. To circumvent the problem, the myofibril was irradiated end-on along its axis (Fig. 2c, d). This method of diffraction recording has a merit that one does not have to isolate a single myofibril from a muscle fiber, and all that is needed is to shoot at a myofibril in a muscle fiber (although it is also technically difficult). By using this method, one can record reflections from all lattice planes at the same time.
 In the diffraction patterns recorded in this way (Fig. 3b, c), the reflections from each lattice plane appear as isolated spots. This is surprising when one considers that the length of the specimen in the beampath is ~3 mm. This is because, in order for the reflections to appear as spots, the lattice planes of the ~1,000 sarcomeres in the 3 mm long myofibril must be strictly in register. Even a 0.1° twist between two neighboring sarcomeres would reduce the diffraction pattern to a rotary-averaged picture as shown in Fig. 2c. The length of 3 mm is almost equal to that of the whole length of the flight muscle of bumblebee. Therefore, one may say that a myofibril in the flight muscle of bumblebee is a single crystal grown in the body of the insect.
 Besides the actin-myosin contractile apparatus, there are many other functional protein assemblies in the cell, such as mitotic spindles, cilia, flagella and membrane skeletons. However, they are much smaller than the contractile apparatus of muscle (mostly in micrometer ranges) and their structural analysis by X-ray diffraction has been impractical. However, the present achievement makes it a real possibility that the structure of these protein assemblies can be analyzed by X-ray diffraction. In the post-genome era, it will be increasingly important to analyze the structure and function of proteins in large-scale assemblies. The techniques used here will serve as powerful tools to achieve these goals.

 The present study was performed, in collaboration with Dr. Tetsuro Fujisawa at RIKEN Harima Institute, as a part of the research project under SPring-8 Joint Research Promotion Scheme of Japan Science and Technology Corporation.

Explanation of technical terms

Actin and myosin:
 The two major contractile proteins in muscle. Each protein polymerizes to form myofilaments. Muscle contraction occurs as the actin and myosin filaments slide past each other. Actin is a globular protein. Myosin consists of a globular head and a filamentous tail region. The head contains binding sites for actin and ATP, which servers as energy source. The tail can polymerize to form the myosin filament backbone.

Hydration:
 A state of material in which, as in living cells, its constituent molecules interact strongly with water. In case of enzymes, they are capable of enzymatic reactions while hydrated.

Membrane skeleton:
 Cytoskeleton lying immediately beneath the plasma membrane. It serves to keep the shape of the cell. Major constituent proteins include actin and spectrin.

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