Morphology of isolated triads.

Abstract

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freezefracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend -100 ,~ from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.The triad is composed of two different membrane systems, the transverse tubule and the terminal cisternae of sarcoplasmic reticulum, in junctional association (2-4). During excitationcontraction coupling in the muscle fiber, the signal for Ca ++ release traverses this junction from t-tubule to terminal cisternae (5). As early as 1962 (6), periodic electron-dense structures were noted bridging the gap between the two types of muscle membranes. Franzini-Armstrong (7, 8) reported that these junctional "feet" (4) were arranged in repeating diamondshaped units attached to one another at the corners. The major portion of each foot seemed to be more securely attached to the terminal cisternal portion of the triad (4,7,8). A variety of interpretations have been presented that suggest roles for the intragap material in excitation-contraction coupling (9-14).In the companion paper (1), we described the isolation of purified triad structures from rabbit skeletal muscle. The present study deals with a description of the morphology of isolated triads and evaluates the susceptibility of triad structure to composition of the media, osmotic changes, and handling. MATERIALS AND METHODSOsmium tetroxide and ghitaraldehyde (vacuum distilled in glass at low temperature and the distillate treated with a special grade of high s...

Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum.

“…The JFM as isolated has a number of features that distinguish it from the calcium pump membrane. Morphologically, the membrane has characteristic feet structures oriented asymmetrically, and uniformly spaced, comparable to their arrangement in situ (14,29,33). Our standard preparation of JFM-CC (using 0.5% Triton and 1 mM CaC12) and JFM have a phospholipid content of 0.11 lamol P/mg protein and 0.16 gmol/mg protein, respectively (Table I).…”

Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying, rotary-shadowing study.

“…This treatment helps to separate T tubules from SR (26), but under prolonged use it results in the disappearance of feet (13). It was shown that the degradation of bridging structures is progressive (8), and by using a brief KCI treatment we preserved feet on our heavy SR vesicles, even though some were lost. We also obtained heavy SR vesicles from guinea pig muscle without using any KCI treatment, and in this preparation the feet had the same shape as in KCI washed vesicles, but were more numerous and more frequently maintained the original alignment in long rows.…”

“…The "heavy" SR fraction contains vesicles whose origin from the lateral sacs of the triad (terminal cisternae) is deduced on the basis of electron-dense contents with CaE+-binding properties (12). Under the appropriate isolation conditions, heavy SR vesicles retain structures identifiable as feet on the portion of their surface (junctional SR) which was originally participating in triadic junctions (8,13,14). We have developed a procedure for exposing surface details in isolated membrane vesicles that faithfully preserves three-dimensional relationships.…”

Minor sarcoplasmic reticulum membrane components that modulate excitation–contraction coupling in striated muscles

“…Depolarization of the plasma membrane of skeletal muscle leads to release of Ca 2+ from the SR resulting in muscle contraction, by a process known as excitation–contraction coupling (Schneider & Chandler, 1972; Melzer et al 1995; Berchtold et al 2000). Excitation–contraction coupling (EC coupling) occurs at the triad, a structure composed of the two membrane compartments, transverse tubules containing the voltage sensing dihydropyridine receptor (DHPR, an L‐type Ca 2+ channel) and terminal cisternae on which ryanodine receptor (RyR) Ca 2+ release channels are localized (Mitchell et al 1983; Rios & Pizarro, 1991). The disposition of DHPRs and RyRs on their respective membranes is highly ordered and each DHPR faces alternate rows of RyR tetramers (Fig.…”