Akitsugu Saito

[4] Isolation of sarcoplasmic reticulum fractions referable to longitudinal tubules and functional terminal cisternae from rabbit skeletal muscle

Publisher Summary This chapter focuses on the isolation of sarcoplasmic reticulum fractions referable to longitudinal tubules and junctional terminal cistemae from rabbit skeletal muscle. The sarcoplasmic reticulum (SR) of fast skeletal muscle fibers is a sleeve-like network surrounding the myofibrils that controls the cytoplasmic calcium concentration and—thereby, regulates muscle contraction and relaxation. The membranous system consists of mainly two regions: (1) Tubular longitudinal elements and (2) bulbous terminal cisternae. The latter is junctionally associated with the tranverse tubule via bridging structures referred to as “feet.” Recently, the preparation of a highly enriched SR fraction of junctional terminal cisternae with morphologically intact feet structures has been reported. The isolation procedures for small-land large-scale preparations are described in the chapter. The small-scale preparation uses swinging bucket rotors, while the large-scale preparation uses zonal rotors. SR microsomes are first isolated by homogenization of muscle and differential centrifugation. Fast skeletal muscle from the hind legs of three rabbits is used. The selection of predominantly white muscle and homogenization of ground muscle are carried out as described above for the small-scale preparation to the supernatant (SUP) step.

Ultrastructural changes in muscle and motor end-plate of the dystrophic mouse

A comparative study of dystrophic mice (C57BL/6J-dy2J) and normal littermates, 6 to 9 months old, has revealed numerous ultrastructural changes in the dystrophic soleus. Vacuoles, swollen mitochondria, vesicular aggregates, membranous bodies, Z-line degradation, localized hypercontraction, myofibrillar disorientation, and focal necrosis were common. We report here new observations. Discontinuous, dense bands (240 A wide) were observed rarely between inner and outer membranes in some nuclei. Closely associated with infoldings of nuclear membrane were paracrystalline, vesicular structures, actin-like filaments, and autophagic vacuoles. With the degeneration of the nuclear membrane, actin-like filaments were observed within the nucleus. The primary synaptic clefts were widened and contained osmophilic-dense granules (diameter 0.2 to 0.7 micrometer). There was loss of secondary synaptic folds. Discontinuity of pre- and postsynaptic membranes could be observed, though rarely. Lysosome-like dense bodies were present among myofilaments. The myeloid bodies and autophagic vacuoles that were associated with nuclei, sarcoplasmic reticulum, and mitochondria gave a positive acid phosphatase reaction. Such degeneration changes were not observed in the normal soleus.

Intramitochondrial tubules in adrenal glands of rat.

Tubular inclusions have been observed in the matrix of mitochondria of rat adrenal cortex. They appear in groups varying from 15 to 40 tubules. The intramitochondrial tubules (IMT) are approximately 100 A wide and can be 0.5 μ long. The wall of IMT is electron opaque, not trilaminar, and is approximately 25 A thick. IMT has been observed in close proximity to the cristael membrane suggestive of apparent contact.

Glycogen-membrane complexes in denervated human skeletal muscle.

We describe an unusual intracellular complex of glycogen with smooth, cisternal, cytoplasmic membranes (glycogen-membrane complexes, GMC) in denervated human skeletal muscle. Glycogen particles were always intimately associated with these structures, although the staining intensity varied markedly with different fixation conditions. This may account for previous investigations of similar structures in which an association with glycogen was not recognized. Electron micrographs of tannic acid-enhanced specimens, and of freeze-fracture replicas, showed similarities between the GMCs and the terminal cisternae of the sarcoplasmic reticulum: (i) GMCs resembled the terminal cisternae of the sarcoplasmic reticulum in the size and asymmetric distribution of intramembranous particles seen in freeze-fracture replicas; (ii) tannic acid-enhanced thin sections of GMCs showed intense staining of the cisternal contents and irregular staining of the cytoplasmic leaflet, similar to the appearance of the sarcoplasmic reticulum terminal cisternae; and (iii) triad-like junctions were seen between the glycogen-membrane complexes and elements of the transverse tubule system.