Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluoresceinlabeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fasttwitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" ($1) and "rod" portions of myosin, as well as the alkali-and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to $1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Nonuniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted 10 J. CELL BIOLOGY 9 The Rockefeller University Press 9

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm) . The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondria) content, and width of the Z line . In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact . Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical . The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse . In the white fiber long, flat axonal terminals present considerable axoplasmic surface . Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area . Axoplasmic and sarcoplasmic vesicles are numerous . Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules . The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds . In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules .

A comparative investigation of the mammalian diaphragm has revealed a correlation between certain cytological aspects of red and white muscle fibers and functional activity. This skeletal muscle presents the advantage of a similar and constant function among the mammals, but its functional activity varies in a quantitative manner. Both the rate of breathing (and hence the rate of contraction of the diaphragm) and metabolic activity are known to be inversely related to body size; and this study has demonstrated a relationship between cytological characteristics of the diaphragm and body size of the animal. Small fibers rich in mltochondria (red fibers) are characteristic of small mammals, which have high metabolic activity and fast breathing rates; and large fibers with relatively low mitochondrial content predominate in large mammals, which have lower metabolic activity and slower breathing rates. In mammals with body size intermediate between these two groups (including the laboratory rat), the diaphragm consists of varying mixtures of fiber types. In general, the mitochondrial content of diaphragm fibers is inversely related to body size. It appears, then, that the red fiber reflects a high degree of metabolic activity or a relatively high rate of contraction within the range exhibited by this muscle.