Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.     

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.     

Indentations in the terminal cisternae of amphibian and mammalian skeletal muscle fibers

Indentations (hillocks and dimples) in the terminal cisternae of mammalian and amphibian skeletal muscle fibers were studied using freeze-fracture and serial thin-section techniques. The structures were seen in all muscles and had a regular separation from each other and from the T-tubule. Indentations were smaller than fenestrations and formed concavities in, but not macromolecular pores through, the terminal cisternae. The average numbers of indentations in rat muscles (measured along the length of the terminal cisternae, within 150 nm of the triadic junction) varied from 0.9 per micrometer in soleus fibers to 9.6 per micrometer in posterior cricoarytenoid fibers. The average numbers in amphibian sartorius fibers varied from 1.6 to 3.6 per micrometer in muscles from different species. The regular alignment of the indentations along the triad, as well as a close correlation between their numbers and the contractile properties of the muscle, suggest that they function in contractile activation and may represent sites of calcium release from the terminal cisternae.     

Particle Assemblies in the Plasma Membrane of Tetrahymena: Relationship to Cell Surface Topography and Cellular Morphogenesis1

During a freeze-fracture electron microscopical study of the plasma membrane of Tetrahymena, several different types of organized particle assemblies were observed. Three of these were found only on the protoplasmic face and were localized in the anterior-ventral region of the cell. These consisted of plate-like arrays composed of 4–25 triplet rows of small 3–4 nm particles; long, paired linear arrays localized at the tops of cortical ridges and composed of 7–8 nm particles; and elongated tetragonal arrays located in the grooves between ridges and composed of approximately 10 nm particles. The distribution of these arrays is consistent with roles in cellular morphogenesis, chemoreception, or cell-cell pairing during conjugation. In addition, a unique particle track associated with the cytoproct (anal pore) was observed in the external face of the plasma membrane. Furthermore, the protoplasmic face of the plasma membrane is characterized by a high density of particles organized into localized microarrays, consisting of small paracrystals or strings, which exhibit a loose higher-order patterning most evident toward the anterior end of the cell. Particle distributions on the protoplasmic face do not appear to be significantly altered by conditions that cause clumping of alveolar membrane particles. Taken together, these observations are consistent with the idea that the proteins of the plasma membrane are highly ordered and relatively immobile and that the structure of the plasma membrane is regionally differentiated.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

Bridging structures spanning the junctioning gap at the triad of skeletal muscle

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.     

Distribution of calcium ATPase in the sarcoplasmic reticulum of fast- and slow-twitch muscles determined with monoclonal antibodies

Summary Four monoclonal antibodies against the calcium ATPase in sarcoplasmic reticulum (SR) of rabbit fast-twitch skeletal muscle were characterized using SDS-PAGE, Western blots and immunofluorescence. The ultrastructural distribution of the antigens was determined using post-embedding immunolabeling. The antibodies recognized the calcium ATPase in the SR but not in transverse (T-) tubule or plasma membranes. The antibody, D12, had the same binding affinity for the calcium ATPase from fast-twitch (rabbit sternomastoid) and slow-twitch (rabbit soleus) fibers and the affinity fell by 30% after fixation for electron microscopy in both types of muscle fiber. Ultrastructural studies revealed that the density of D12 antibody binding to the terminal cisternae membrane of extensor digitorum longus (edl) and sternomastoid fibers was on average seven times greater than in the slow-twitch soleus and semimembranosus fibers. Since the affinity of the ATPase for the antibody was the same in SR from fast- and slow-twitch muscles, the concentration of calcium ATPase in the terminal cisternae membrane of fast-twitch fibers was seven times greater than in slow-twitch fibers. This conclusion was supported by the fact that the concentration of calcium ATPase in light SR membranes was six times greater in SR from fast-twitch fibers than in SR from slow-twitch fibers. The results provide strong evidence that the different calcium accumulation rates in mammalian fast- and slow-twitch muscles are due to different concentrations of calcium ATPase molecules in the SR membrane.     

Ultrastructural organisation and the sites of calcium localisation in the lamprey striated muscle

The present paper examines the ultrastructure of the sarcoplasmic recitulum (SR) and the T system in the striated muscle of the lamprey. The pyroantimonate method was used to visualise the sites of intracellular calcium localisation. Characteristic for the muscle studied are the presence of numerous intricately shaped invaginations on the surface membrane of muscle fibres and peripheral contacts between SR cisternae and the sarcolemma. In addition to calcium localised in the terminal cisternae of SR and N-bands of the I-disk, as typical of vertebrate muscles, a great amount of calcium is present in the subsarcolemmal region, corresponding to the area of invaginations, and in longitudinal elements of SR.     

Observations on freeze-fractured membranes of a Trypanosome

Pure preparations of Trypanosoma brucei, free from plasma and cellular components were isolated from rat blood, and concentrated into loose pellets by low-speed centrifugation. Pellets were either processed for thin sectioning as a control for general morphology, or glycerol-treated after glutaraldehyde fixation for preparation of freeze-fracture replicas. Concentration of cells of 50,000–100,000/mm² of sectioned or fractured surface facilitated identification of fracture faces of the cell body, invaginated flagellar pocket and flagellum. Particle distribution and A and B faces of these regions of the cell are described. A collar of B face particles occurs around the neck of the flagellar pocket, possibly associated with a junction controlling ingress of ingested materials to coated vesicles formed along the membrane defining the pocket. A and B faces of the flagellum and adjoining surface of the cell body have shown that the only intra-membrane specialization corresponding to the miniature ‘maculae adherentes’ described previously in thin sections is probably an uninterrupted series of small clusters (3–6) of 80 Å particles on the A face of the flagellar membrane. It is proposed that these arrays represent attachment points for strands linking the axoneme and paraxial rod to the flagellar surface, and are not directly concerned with the physical adhesion of the flagellum to the cell body surface—a linkage that appears to be established within the extracellular gap between these apposed surfaces of the cell. The potential use of freeze-etching in further study of the external antigens of the infective cell is discussed.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.     

Morphology of isolated triads

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 freeze-fracture 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 approximately 100 A 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.     

Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus , and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and 'truncated cone' regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle-free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.     

A freeze-fracture study on the differentiation of Golgi and plasma membranes in plant cells

Dictyosomes, Golgi vesicles, and plasma membranes were investigated after freeze-fracture in cells from growing root tips of cress (Lepidium sativum L.), that are distinguishable by different cellulose content of the cell wall, into (i) meristematic cells during early formation of the cell plate, (ii) statocytes of the root cap, and (iii) cortex cells of the differentiation zone. The results of this study show that the number of intramembrane particles (imps) is high in dictyosome cisternae, but low in membranes of budding or dictyosome-derived vesicles. Imps are disperse in the vesicle membranes of meristematic cells (i), but are often grouped into clusters in vesicle membranes of statocytes (ii), and of cortex cells (iii). For the number of particle aggregates in vesicle membranes, the following relation holds: (i) < (ii) < (iii). The number of particles on both fracture faces (PF and EF) of the plasma membrane differs widely between the cell types investigated. There are approximately 250, 1400, and 3100 imps microns-2 on the PF and 50, 500, and 300 on the EF of (i), (ii), and (iii), respectively. The structural complexity of the plasma membrane as judged by the degree of particle aggregations on the PF and the number of cellulose microfibrils in the cell wall show the same relationship: (i) < (ii) < (iii). Thus, the strong correlation between the distribution of imps in vesicle membranes, the structural complexity of the plasma membrane, and the content of cellulose microfibrils indicate that selection of imps during vesicle formation at dictyosome cisternae is an integral component of biogenesis and structural differentiation of plant plasma membranes.     

A freeze-fracture and concanavalin A-binding study of the membrane of cleaving Xenopus embryos.

The freeze-fracture appearance and concanavalin A-binding capacity of the plasma membrane of cells of the cleaving Xenopus embryo have been examined up to the 16-cell stage. It was found that membrane on the outer surface of the embryo, which faces the vitelline membrane and is remote from cleavage furrows, and membrane in the shallow regions of the furrow possessed a high population of intramembranous particles on the PF-face (1171 per mum2). The EF-face of these membranes showed a lower particle population (245 per mum2). By contrast, membrane deep in the furrow and bounding the blastocoel did not display a face with high particle numbers. Both faces of this membrane, which is newly exposed as the furrow grows, were relatively poorly supplied with particles (93 per mum2). Therefore it appears that, in this tissue, newly added membrane possesses fewer intramembranous particles than the pre-existing membrane. Concanavalin A, as detected cytochemically using peroxidase and haemocyanin techniques, bound extensively to both particle-rich and particle-poor membrane. Thus there was no correlation between intramembranous particle frequency and degree of concanavalin A binding.     

Freeze-etched membrane faces and photosynthetic activity in normal and mutantTradescantia chloroplasts

Summary Membrane structure and photosynthetic activity was investigated in normal and mutant plastids ofTradescantia albiflora cv.aureo-vittata. In the stacked membrane regions (the macrograna) of mutant plastids, the B fracture faces lack both 170 Å particles and photosystem II (PS II) activity. The C face has the normal 110 Å particles, and photosystem I (PS I) activity is also similar to that in normal chloroplasts. In dilated macrograna the particle size on the C face significantly decreases, and as progressive plastid destruction occurs so PS I activity also disappears. It has been concluded that the integrity of B face particles is related to PS II activity, rather than for membrane stacking. A similar correlation seems to be valid for C face particles and PS I activity.     

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Summary Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorius muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SR. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca⁺⁺-free or contained Mn⁺⁺ or La⁺⁺⁺; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 × hypertonic with sucrose (known to produce swelling of T tubules and (TC). Since Mn⁺⁺ and La⁺⁺⁺ are known to depress Ca⁺⁺ influx, these results suggest that washout of Ca⁺⁺ from the TC, and penetration of La⁺⁺⁺ or Mn⁺⁺ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca⁺⁺ influx into the myoplasm.Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.     

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Summary Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorius muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SR. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca⁺⁺-free or contained Mn⁺⁺ or La⁺⁺⁺; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 × hypertonic with sucrose (known to produce swelling of T tubules and (TC). Since Mn⁺⁺ and La⁺⁺⁺ are known to depress Ca⁺⁺ influx, these results suggest that washout of Ca⁺⁺ from the TC, and penetration of La⁺⁺⁺ or Mn⁺⁺ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca⁺⁺ influx into the myoplasm.Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.     

Membrane structure of nonactivated and activated human blood platelets as revealed by freeze-fracture: evidence for particle redistribution during platelet contraction

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.     

Structural and cytochemical differentiation of membrane elements of the apical membrane of amphibian urinary bladder epithelial cells. A label fracture study

It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.