FIGURES 11-13 - uploaded by Avril V Somlyo
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Arrows indicate the periodic longitudinally aligned particles within the TC. Note the scalloping of the junctional SR membrane in Fig. 11 and the adherence of the particles to the membrane. Methods B, B, and C, respectively. Fig. 11, x 100,000; Fig. 12, x 84,000; Fig. 13, x 135,000.
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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 globul...
Citations
... Indeed, in freeze-fracture images of skeletal muscle, the entire SR membrane, except for the junctional face membrane facing the T tubules, is occupied by a dense, uniformly distributed carpet of particles representing the Ca 2+ ATPase (Fig. 5 A). Extensions of the protein over the SR surface were first detected by tannic acid mordating (Somlyo, 1979;Chu et al., 1988). The two heroes of the ATPase story are Giuseppe Inesi and Cikoshi Toyoshima. ...
The concept of excitation–contraction coupling is almost as old as Journal of General Physiology . It was understood as early as the 1940s that a series of stereotyped events is responsible for the rapid contraction response of muscle fibers to an initial electrical event at the surface. These early developments, now lost in what seems to be the far past for most young investigators, have provided an endless source of experimental approaches. In this Milestone in Physiology, I describe in detail the experiments and concepts that introduced and established the field of excitation–contraction coupling in skeletal muscle. More recent advances are presented in an abbreviated form, as readers are likely to be familiar with recent work in the field.
... A substantial gap separates the T membrane and the adjacent SR membrane, a gap accessible to solutes in the sarcoplasmic solution [Eisenberg,Mathias,and Gilai (30); Franzini-Armstrong (41)]. The gap between T system and SR is spanned by well-delineated structures called pillars [Eisenberg and Gilai (29); Eisenberg, Mathias, and Gilai (30); Somlyo (96); Eisenberg and Eisenberg (28a)] and amorphous material called feet [reviewed in Franzini-Armstrong (42)]. The lumen of the SR is inaccessible to large molecules in the bathing solution that diffuse easily into the lumen of the T system. ...
The sections in this article are:
Methods and Techniques
Microelectrode Techniques
Analysis of Sinusoidal Data
Impedance Analysis With Fourier Techniques
Instrumentation Noise
Manipulation of Impedance Data
Theory and Curve Fitting
Electrical Models of the T System
Necessity for Morphometry
Results of Impedance Measurements
Impedance Measurements of Normal Frog Fibers
Other Preparations of Skeletal Muscle
Impedance Measurements of Muscle Fibers in Various Conditions
Comparison With Other Results
Discussion
Impedance Measurements of Nonlinearities
Other Methods
... Small periodically repeating densities traverse the gap. They are described variably as`dimples' (Kelly, 1969), `SR feet' (Franzini-Armstrong, 1970), `bridges' (Somlyo, 1979), `pillars' (Eisenberg and Eisenberg, 1982) or`bridging junctional processes' (Forbes and Sperelakis, 1980). Now they are to be understood as the cytoplasmic components of the ryanodine receptors (Block et al., 1988). ...
... It is not clear how the dierent arrangement of the material in the gaps relates to the recent morphological concept of the structure of the SR feet (cf. Franzini- Armstrong, 1973; Somlyo, 1979; Franzini-Armstrong and Nunzi, 1983; Schmalbruch, 1985). ...
Serial sections through motor end plate regions of mouse muscle fibres demonstrated junctions between the subsynaptic folds and the rough sarcoplasmic reticulum of the sole plate nuclei. The shape of these structures resembles that of the well-known peripheral couplings, diads and triads of muscle fibres. However, the location of the new junctions between the surface membrane and the sole plate nuclei at a large distance from myofibrils, indicates a different function. The connection with the rough sarcoplasmic reticulum possibly influence the regulation of fibre protein metabolism, for example, gene expression of acetylcholine receptor synthesis.
... 10,40,41 However, on close examination, the bridging structures are different, in that the spacing of feet between bridging structures is greater and the density is less than in striated muscles. 42,43 Considering the localization of RyRs to terminal cisternae, it may seem surprising that in smooth muscle RyRs are present on both central and peripheral SR. However, this need not imply similar excitationcontraction coupling mechanisms and raises the possibility of different mechanisms gating the RyRs in peripheral SR and central SR, respectively. ...
The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide-stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca2+-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca2+-release channels and origins of Ca2+ sparks, Ca2+ oscillations, and Ca2+ waves.
... The electrical excitatory signal is thought to be communicated from the T-tubule to the SR at junctional locations where large protein structures span the narrow gap separating the two membranes (Peachey and Franzini-Armstrong, 1983). These structures have been variously termed "feet" (Franzini-Armstrong, 1970), "bridges" (Somlyo, 1979), "pillars" (Eisenberg and Eisenberg, 1982), or "spanning proteins" (Caswell and Brandt, 1989), and are now believed to be identical with the ligand-gated, ryanodine-sensitive "Ca 2+ release" channel of SR (for reviews see Fleischer and Inui, 1989;; but see also Zaidi, Lagenaur, Hilker, Xiong, Abramson, and Salama, 1989). The specific mechanism of signal transduction from the T-tubule to SR, commonly referred to as excitation-contraction (E-C) coupling, remains to be fully defined, however. ...
The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)-conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.
... The latter is the Ca 2÷ channel concentrated in the membrane of the muscle sarcoplasmic reticulum terminal cisternae, where it accounts for the discrete particles bridging the triad gap, the so-called junctional feet. In conventional thin sections, feet appear as regularly spaced bridges (Somlyo, 1979;Franzini-Armstrong and Nunzi, 1983), similar to those observed in between our stacked cisternae. Based on this similarity and on the recently reported geometry of the InsP3R ( Maeda et al., 1990), the possibility should be considered that the regularly spaced bridges of the stacks are composed by receptor molecules, possibly arranged in register in the adjacent membranes. ...
The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.
... The latter is the Ca 2÷ channel concentrated in the membrane of the muscle sarcoplasmic reticulum terminal cisternae, where it accounts for the discrete particles bridging the triad gap, the so-called junctional feet. In conventional thin sections, feet appear as regularly spaced bridges (Somlyo, 1979;Franzini-Armstrong and Nunzi, 1983), similar to those observed in between our stacked cisternae. Based on this similarity and on the recently reported geometry of the InsP3R (Maeda et al., 1990), the possibility should be considered that the regularly spaced bridges of the stacks are composed by receptor molecules, possibly arranged in register in the adjacent membranes. ...
The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.
... s. P. Langton and K. Sanders (unpublished). membrane similar to that seen in cardiac muscle and in triadic junctions between terminal SR 46 1 cisternae and T-tubules of skeletal muscle. That is, the SR tubules frequently establish structurally similar close couplings with the plasma membrane (e.g., Somner and Johnson, 1980;Franzini-Armstrong, 1970;A. V. Somlyo, 1979). The SR of smooth muscle also makes close contacts with the caveolae (Fig. 8) and gap junctions of the plasma membrane (Garfield and Somlyo, 1985). ...
... At the sites of contact with the plasma membrane, the 12-to 20-nm space separating the two membranes contains periodically spaced electron-opaque "bridging" structures (A. P. Somlyo et al., 1971;A. V. Somlyo, 1979;A. P. Somlyo, 1985). These structures have been studied in detail in quick-frozen, deep-etched samples of smooth muscle, where they appear as single strands linking the two membranes (see Fig. 4b of A. V. Somlyo and Franzini-Armstrong, 1985). Their function remains unknown, although A. V. Somlyo and Franzini-Armstrong (1985) speculate th ...
The concept that a well-developed appreciation of structure is an essential prerequisite to an understanding of function permeates research into all physical processes but is especially true for the biological sciences. The electron microscopic and x-ray diffraction studies conducted on the myometrium during the past 25 years have provided important insights into the physiology of this organ. This chapter reviews our understanding of the ultrastructure of the myometrium. Particular emphasis is placed on the development and functional significance of gap junctions between myometrial cells, a topic not covered in previous editions of this book because of its relatively recent development. We believe that the observation that gap junctions appear in large numbers between myometrial cells during the onset and progression of labor (Garfield et al., 1977) was a major step forward in our understanding of the circumstances that control parturition. We also provide a detailed description of the structure of the contractile apparatus and its relationship to the plasma membrane and cytoskeleton. Several important observations have been made in this area since the last edition of this book.
... Thin section electron microscopy has provided images suggestive of such a connection, by showing that the internal proteins form a dense band, or plate, parallel to the junctional SR (jSR) membrane and joined to it across an intervening space by small particles or granules (Johnson and' Sommer, 1967;Sommer and Johnson, 1979;Walker et al., 1971). This socalled coextensive density is particularly visible in cardiac muscle (Johnson and Sommer, 1967;Waugh and Sommer, 1974), and in rapid-frozen skeletal muscle (Somlyo, 1979;Nassar et al., 1986) and has been described in skeletal muscle fixed during K contractures (Eisenberg and Gilai, 1979) and in isolated SR fractions . ...
... Fine, barely visible lines join the rows of grains and the jSR membrane, facing the T tubules (T). Preservation obtained with freeze-substitution after an initial fixation in glutaraldehyde is better than with standard osmium postfixation (Figs. 2 and 3), and is comparable to that shown in rapid-frozen tissue (Somlyo, 1979;Nassar et al., 1986). This is confirmed by the close similarity of the freeze-substituted and deep-etched structures (see below). ...
... Arrows point to small aggregates within the SR lumen but not in the terminal cisternae. T tubules (T) run horizontally in Fig. 11 and vertically in Fig. 12. ages agree quite well with those derived from rapid-frozen muscles, not exposed to chemical fixation (Somlyo, 1979;Nassar et al., 1986), except that in the latter the translucent strip may be located more centrally in the terminal cisternae. Fig. 13 summarizes current understanding of triadic junction, combining structural and biochemical identification of junctional components from enriched fractions (Meissner, 1975;Lau et al., 1977;Campbell et al., 1980;Mitchell et al., 1983;Caswell and Brunschwig, 1984;Saito et al., 1984;Costello et al., 1986). ...
We have examined the structure of calsequestrin in three-dimensional images from deep-etched rotary-replicated freeze fractures of skeletal muscle fibers. We selected a fast-acting muscle because the sarcoplasmic reticulum has an orderly disposition and is rich in internal membranes. Calsequestrin forms a network in the center of the terminal cisternae and is anchored to the sarcoplasmic reticulum membrane, with preference for the junctional portion. The anchorage is responsible for maintaining calsequestrin in the region of the sarcoplasmic reticulum close to the calcium-release channels, and it corroborates the finding that calsequestrin and the spanning protein of the junctional feet may interact with each other in the junctional membrane. Anchoring filaments may be composed of a protein other than calsequestrin.
... Considerable morphological information is available regarding the triad junction, and it is clear that although the two membranes are separated by a gap of •100 ~t, the T-tubule and SR are physically joined and in communication by virtue of bridging structures that span the junctional space. These structures have been referred to variously as feet (L3), bridges (21), pillars (11), and spanning protein (SP) (7). Biochemically, very little information is available about the junctional constituents. ...
... The diamond pattern images (14,20) as well as our own thin section images suggest that 100 A units may be observed. The bridges have also been described to have an electron-translucent core in thin section of intact muscle (21). This conforms with our observation in thin section that the SP is electron translucent in the center. ...
A monoclonal antibody has been developed against the putative junctional protein or spanning protein (SP) from skeletal muscle triads. By immuno-affinity chromatography, we have purified this protein. The native protein has a molecular mass of 630-800 kD, as determined by gel filtration and rate zonal centrifugation. Within the limits of the methods used, the basic unit of the SP appears to be a dimer. In electron micrographs, it is shown to exhibit a circular profile with a diameter of approximately 100 A. In thin section analysis, the protein is frequently observed as parallel tracks of electron-dense particles bordering a translucent core. We suggest that the basic unit of the junctional structure is a dimer of 300-kD subunits and that four such entities constitute the intact SP. The purified protein has been used to develop polyclonal antibodies. By immunoelectron microscopy using immunogold probes, the SP has been localized to the junctional gap of the triad. By attaching the SP to an affinity resin, three proteins have been identified as forming associations with the SP. The Mrs of the proteins are 150, 62, and 38 kD; the 62-kD protein is calsequestrin.
