204 



F. S. SJOSTRAND AND EBBA ANDERSSON 



each of the primary filaments. The arrangement is, 

 however, not completely identical with that in verte- 

 brate muscle; in insect muscle, the secondary fila- 

 ments are located midway between two primary 

 filaments, and not symmetrically between three pri- 

 mary filaments as in vertebrate striated muscle. The 

 cross-sections also show in many places the bridges 

 between the primary and secondary filaments. 



It is apparent from the cross-sectional views that 

 longitudinal sections which are very thin and which 

 pass successively through different layers of lattice 

 should in some areas contain only secondary fila- 

 ments. This is in fact often observed. Detailed exa- 

 mination of such sections also confirms that the 

 secondary filaments are not merely part of the pri- 

 mary filaments in adjacent layers. 



Examination of the details of the structure in the 

 neighbourhood of the Z-line is now proceeding. 



Discussion. — The appearance of the double array 

 of filaments in insect flight muscle confirms that its 

 structure bears many resemblances to that of verte- 

 brate striated muscle, and suggests that the contrac- 

 tion mechanism may be similar to the one suggested 

 for the latter type of muscle. Hodge interpreted his 

 results rather diff"erently. However, reference to his 

 published electron micrographs shows that the cross- 

 sections could well be interpreted in many areas 

 as showing the secondary filaments heavily cross- 



bridged to the primary filaments, and located midway 

 between each pair of them. Whether the "bridge" or 

 the "filament" appearance of the secondary material 

 predominates will depend very critically on the degree 

 of staining, the section thickness, the orientation, 

 and the exact plane of sectioning. The micrographs of 

 longitudinal sections published by Hodge show the 

 intact bridges much less clearly than do the cross- 

 sections and the general contrast and orderliness is 

 considerably less than on the cross-sections; the 

 non-appearance of secondary filaments in these 

 sections is not, we feel, completely conclusive. The 

 system of cross-bridges clearly provides a means by 

 which the secondary filaments could be pulled along 

 relative to the primary ones, as indeed has already 

 been suggested for vertebrate striated muscle. We 

 have observed the filaments and the bridges together 

 with such consistency that we feel that the secondary 

 filaments are indeed a completely genuine structure. 



References 



1. Hanson, J., /. Biophys. Biochem. Cytol. (1957, in press). 



2. Hanson, J. and Huxley, H. E., Nature 111, 530 (1953). 



3. — Symposia Soc. Exptl. Biol. 9, 228 (1954). 



4. Hodge, A. J., Huxley, H. E., and Spiro, D., /. Exptl. 



Med. 99, 201 (1954). 



5. Huxley, A. F. and Niedergerke, R., Nature 173, 971 



(1954). 



6. Huxley, H. E. and Hanson, J., Nature 173, 973 (1954). 



The Ultrastructure of Skeletal Muscle Myofilaments 

 F. S. Sjostrand and Ebba Andersson 



The Laboratory for Biological Ultrastructure Research of the Department of Anatomy, 



Karolinska Institutet, Stockholm 



This study aims at a detailed analysis of the struc- 

 tural organization of the individual myofilaments of 

 skeletal muscle at various states of shortening of the 

 sarcomere. 



Leg muscles and abdominal muscles from frog, 

 intercostal muscles and abdominal muscles from 

 mouse, were fixed in situ in the living anesthetized 

 animal or rapidly after decapitation of the ex- 

 perimental animal. The frog muscle tissue was 

 cooled by keeping the living frogs in a cold room at 

 ~3 to 5 C before dissecting free the muscle tis- 

 sue. In most cases, a minute bundle of muscle fibers 

 were dissected free in situ by means of fine glass 

 needles and the rest of the muscle tissue removed. 

 The excitability of the muscle tissue to direct electric 

 stimulus was checked before the fixation. At the 

 lowest temperatures, the excitability was greatly 

 reduced. Contracted muscle tissue was obtained by 

 continuous electric stimulation to tetanic contrac- 

 tion during the first 15 minutes of fixation. 



A series of experiments were performed, in which 



the muscle tissue in situ was subjected to extraction 

 with a 1 : 1 glycerin-water solution and the standard 

 solutions for myosin and myosin-actin extraction. 



1 % isotonic solutions of osmium tetroxide buf- 

 fered to various pH were used as well as buflfered 

 formalin solutions (10 "o). The l^ hours' fixation 

 was performed at 1 to -2'C. Dehydration with 

 ethyl or iso-propyl alcohol and embedding in 

 methacrylate. For high resolution pictures, the 

 ultrathin sections were mounted on metallized 

 formvar nets and analyzed in an RCA EMU 2c 

 electron microscope with double objective without 

 objective aperture. The electron optical magnifica- 

 tions were 40,000-60,000 times. 



In the extensive material of skeletal muscle tissue 

 that has been analyzed, only one type of myofila- 

 ments has been observed (8). The myofilaments 

 run through the whole length of the sarcmoere 

 without interruption and continue through the Z- 

 line (fig. 1). The diameter of the myofilament is 

 diff'erent in the A- and the I-bands, which partly 



