The core cylinder in the junctional SR of single intact skeletal muscle fibers of the frog after quick-freezing: Its topography

1983 ◽  
Vol 41 ◽  
pp. 648-649
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
B. Scherer
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Ruthenium Red ◽  
One Dimension ◽  
The Core ◽  
Quick Freezing ◽  
Clear Halo ◽  
Circular Configuration ◽  
Consistent Feature ◽  
Electron Lucent

The "core cylinder" (CC) represents confluent electron lucencies seen in the junctional SR (JSR) when skeletal muscle is fixed in the presence of cations such as ruthenium red. CCs are a consistent feature in quick-frozen muscle fibers (Fig.l), both in the resting state and after various intervals between stimulation and freezing, incl. tetanus (5 sec, 50Hz). The CCs are excentrically located inside the JSR and are separated from the junctional membrane toward the transverse tubule by the coextensive line(CL) and from the rest of the membranous envelope of the JSR by granular material (junctional granules, JG). JGs are also found elsewhere in the free SR. Whereas the JGs fill the JSR completely in many cases, often they are separated from the JSR envelope by a peripheral clear halo except in the region of the CL; in favourable sections they form regular rosettes about the CC (Fig.3). Very frequently, the CC loose their more circular configuration (in one dimension)(Fig.4), with flares connecting the CCs with the peripheral halo at either end of the CL (Fig.3). The junction between the electron-lucent flares and the ends of the CL subtends, approximately, the location of the staggered rows of pits on the E face of the freeze-fractured JSR(Fig.2).

The core cylinder of the JSR of frog skeletal muscle in cryosections

1987 ◽  
Vol 45 ◽  
pp. 414-415
Author(s):  
J. R. Sommer ◽  
T. High ◽  
P. Ingram
Keyword(s):  
Skeletal Muscle ◽  
Carbon Film ◽  
Ruthenium Red ◽  
Frog Skeletal Muscle ◽  
Turbomolecular Pump ◽  
Freeze Dried ◽  
Quick Freezing ◽  
And Function ◽  
Electron Lucent ◽  
Junctional Sarcoplasmic Reticulum

Electron-lucent regions in the junctional sarcoplasmic reticulum (JSR) were first observed in frog skeletal muscle treated with ruthenium red and other exotic cations. Next, they were pointed out in Epon sections of freeze-substituted muscle following quick-freezing both in the presence and absence of cryoartifacts. Whereas their nature and function remain obscure, the so-called “core cylinders” (CCs) are now considered invariant components of the normal anatomy of frog skeletal muscle JSR.Following quick-freezing, part of the single intact frog skeletal muscle fiber was freeze-substituted, embedded in Epon and sectioned (Fig. 1). Cryosections were cut from an immediately adjacent fiber region with a diamond knife specially mounted for that purpose with two free edges (courtesy Diatome-US), on a Reichert Ultracut FC-4D at -150°C, put on the carbon film on 100 mesh nickel grids, freeze-dried in a Balzers FDU 010 fitted with a turbomolecular pump (vac 10-6 torr) against a liquid N2 trap (geometry 14 mm) for 15 hrs with 3 temperature steps, each 3 hrs apart, at -100, -85, and -50°C, respectively (Fig. 2).

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

2000 ◽  
Vol 278 (1) ◽  
pp. C126-C135 ◽  
Author(s):  
Adrian M. Duke ◽  
Derek S. Steele
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Muscle Fibers ◽  
Cyclopiazonic Acid ◽  
Creatine Phosphate ◽  
Ruthenium Red ◽  
Rat Skeletal Muscle ◽  
Atpase Inhibitor ◽  
Skeletal Muscle Fibers ◽  
Efflux Pathway

The effects of Pi on sarcoplasmic reticulum (SR) Ca2+ regulation were studied in mechanically skinned rat skeletal muscle fibers. Brief application of caffeine was used to assess the SR Ca2+ content, and changes in concentration of Ca2+([Ca2+]) within the cytosol were detected with fura 2 fluorescence. Introduction of Pi (1–40 mM) induced a concentration-dependent Ca2+ efflux from the SR. In solutions lacking creatine phosphate (CP), the amplitude of the Pi-induced Ca2+ transient approximately doubled. A similar potentiation of Pi-induced Ca2+ release occurred after inhibition of creatine kinase (CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca2+ release was almost abolished, whereas Pi-induced Ca2+ release was unaffected. However, introduction of the SR Ca2+ ATPase inhibitor cyclopiazonic acid effectively abolished Pi-induced Ca2+ release. These data suggest that Pi induces Ca2+ release from the SR by reversal of the SR Ca2+ pump but not via the SR Ca2+ channel under these conditions. If this occurs in intact skeletal muscle during fatigue, activation of a Ca2+efflux pathway by Pi may contribute to the reported decrease in net Ca2+ uptake and increase in resting [Ca2+].

scholarly journals Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers.

1986 ◽  
Vol 87 (2) ◽  
pp. 289-303 ◽  
Author(s):  
P Volpe ◽  
G Salviati ◽  
A Chu
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Isometric Force ◽  
Muscle Fibers ◽  
Ruthenium Red ◽  
Scattering Method ◽  
Tension Development ◽  
Maximal Force ◽  
Skeletal Muscle Fibers ◽  
Ca2 Channels

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.

scholarly journals On the connection between the transverse tubules and the plasma membrane in frog semitendinosus skeletal muscle

1975 ◽  
Vol 64 (3) ◽  
pp. 734-740 ◽  
Author(s):  
G Zampighi ◽  
J Vergara ◽  
F Ramon
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Muscle Fibers ◽  
Indirect Evidence ◽  
Electrical Signal ◽  
Ruthenium Red ◽  
Transitional Region ◽  
Transverse Tubular System ◽  
Pinocytotic Vesicles ◽  
And Function

The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis for the inward spread of conduction of the electrical signal that triggers muscle contraction. A historical account of the main steps contributing to the elucidation of the structure and function of the TSS has been presented by Huxley (1971). While the localization of the TSS and its association with the sarcoplasmic reticulum (SR) is well documented; there is still a need further to develop our knowledge of the morphology of the connection between the TSS and the plasma membrane. It is generally believed that the TSS opens directly to the extracellular space and that there is continuity between its membrane and the sarcolemma. However, direct observation of such a connection has been clearly shown only for the myotome of fish (Franzini-Armstrong and Porter, 1964). In other muscle fibers, only indirect evidence of the connection has been provided by experiments showing penetration of extracellular tracers into the TSS. These extracellular markers were also observed inside another membrane-bounded compartment consisting of round profiles named "caveolae" (Yamada, 1955) or "pinocytotic vesicles" (Ashurst, 1969). The present study deals with the communication between the TTS, caveolae, and plasma membrane (Peachey, 1965); Ezerman and Ishikawa, 1967; Schiaffino and Margreth, 1968; and Rayns et al., 1968). A detailed study of the caveolae compartment was undertaken with ruthenium red as an electron-dense tracer. As a result of this study, we propose that in certain species the caveolae compartment represents the transitional region in the connection between the TSS and the sarcolemma.

The SR of skeletal muscle: Its structure after quick-freezing and freeze-etching, following field stimulation of single intact muscle fibers

1984 ◽  
Vol 42 ◽  
pp. 300-301
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
N.R. Wallace
Keyword(s):  
Skeletal Muscle ◽  
Striated Muscle ◽  
Muscle Fibers ◽  
Freeze Fracture ◽  
Electron Gun ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing ◽  
Intact Muscle ◽  
Similar Images ◽  
Freeze Etching

It is known that the P faces of freeze-fractured SR of fixed and cryoprotected striated muscle fibers are studded with particles, whereas the E faces remain smooth, except for two staggered rows of pits in the junctional SR (JSR) which face transverse tubules (junctional pits). Freeze-fracture after quick-freezing of native skeletal muscle provides similar images (1). We have used freeze-etching to look at the SR's structure in single intact skeletal muscle fibers (r.temporaria) without stimulation, following varied post-stimulation intervals, and in tetanus. Single intact skeletal muscle fibers were isolated and quick-frozen as previously reported (2). After quick-freezing, the fibers were transferred to a Balzers 301 device and etched for 3 minutes at -100°C, followed by unidirectional Pt evaporation with an electron gun and carbon coating.

scholarly journals Three-Dimensional Reconstruction of Skeletal Muscle Extracellular Matrix Ultrastructure

2014 ◽  
Vol 20 (6) ◽  
pp. 1835-1840 ◽  
Author(s):  
Allison R. Gillies ◽  
Eric A. Bushong ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Richard L. Lieber
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
High Resolution ◽  
Mononuclear Cells ◽  
Muscle Fibers ◽  
High Resolution Electron Microscopy ◽  
Detailed Examination ◽  
Ruthenium Red ◽  
Variable Pressure

AbstractThe skeletal muscle extracellular matrix (ECM) supports muscle’s passive mechanical function and provides a unique environment for extracellular tissues such as nerves, blood vessels, and a cadre of mononuclear cells. Within muscle ECM, collagen is thought to be the primary load-bearing protein, yet its structure and organization with respect to muscle fibers, tendon, and mononuclear cells is unknown. Detailed examination of extracellular collagen morphology requires high-resolution electron microscopy performed over relatively long distances because multinucleated muscle cells are very long and extend from several millimeters to several centimeters. Unfortunately, there is no tool currently available for high resolution ECM analysis that extends over such distances relevant to muscle fibers. Serial block face scanning electron microscopy is reported here to examine skeletal muscle ECM ultrastructure over hundreds of microns. Ruthenium red staining was implemented to enhance contrast and utilization of variable pressure imaging reduced electron charging artifacts, allowing continuous imaging over a large ECM volume. This approach revealed previously unappreciated perimysial collagen structures that were reconstructed via both manual and semi-automated segmentation methods. Perimysial collagen structures in the ECM may provide a target for clinical therapies aimed at reducing skeletal muscle fibrosis and stiffness.

Side bridge geometry after quick-freezing of stimulated and unstimulated frog skeletal muscle fibers

1983 ◽  
Vol 41 ◽  
pp. 464-465
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
S. Walker
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Muscle Fibers ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Skeletal Muscle ◽  
Specimen Holder ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing ◽  
The Impact

Quick-freezing allows the structural analysis of timed perturbations of morphology. We are presenting preliminary results concerning the feasibility of studying directly the side bridge geometry of actin-myosin interactions within the time course of a twitch in single intact frog skeletal muscle fibers, both by freeze-substitution and freeze-fracture after quick-freezing, and following various time intervals between stimulation and impact of the fibers on a liquid He-cooled copper block.Materials and Methods. The quick-freezing device was a "Slammer"(Polaron) for which the electronics had been redesigned; they are capable, in combination with a Grass S48 stimulator, of any stimulation interval between 0 and 1 sec prior to freezing, including tetanus. The actual elapsed time between stimulation and freezing is recorded with a digital clock. Single intact tendonto- tendon frog skeletal muscle fibers (semitendinosus of r. temporaria) or toe muscle bundles (r.pipiens) were isolated by sharp dissection and placed between coextensive Pt stimulation wires on blackened 2% agarose, the height of which on the specimen holder was adjusted appropriately with respect to a spacer ring both, to calibrate the impact time and to prevent smashing of the fibers.

NAD(P)H fluorescence imaging of mitochondrial metabolism in contracting Xenopus skeletal muscle fibers: effect of oxygen availability

2005 ◽  
Vol 98 (4) ◽  
pp. 1420-1426 ◽  
Author(s):  
Michael C. Hogan ◽  
Creed M. Stary ◽  
Robert S. Balaban ◽  
Christian A. Combs
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Peripheral Region ◽  
Oxygen Availability ◽  
Fluorescence Signal ◽  
The Core ◽  
Skeletal Muscle Fibers ◽  
Work Level ◽  
Effect Of Oxygen ◽  
Contraction Cycle

The blue autofluorescence (351 nm excitation, 450 nm emission) of single skeletal muscle fibers from Xenopus was characterized to be originating from mitochondrial NAD(P)H on the basis of morphological and functional correlations. This fluorescence signal was used to estimate the oxygen availability to isolated single Xenopus muscle fibers during work level transitions by confocal microscopy. Fibers were stimulated to generate two contractile periods that were only different in the Po2 of the solution perfusing the single fibers (Po2 of 30 or 0–2 Torr; pH = 7.2). During contractions, mean cellular NAD(P)H increased significantly from rest in the low Po2 condition with the core (inner 10%) increasing to a greater extent than the periphery (outer 10%). After the cessation of work, NAD(P)H decreased in a manner consistent with oxygen tensions sufficient to oxidize the surplus NAD(P)H. In contrast, NAD(P)H decreased significantly with work in 30 Torr Po2. However, the rate of NAD(P)H oxidation was slower and significantly increased with the cessation of work in the core of the fiber compared with the peripheral region, consistent with a remaining limitation in oxygen availability. These results suggest that the blue autofluorescence signal in Xenopus skeletal muscle fibers is from mitochondrial NAD(P)H and that the rate of NAD(P)H oxidation within the cell is influenced by extracellular Po2 even at high extracellular Po2 during the contraction cycle. These results also demonstrate that although oxygen availability influences the rate of NAD(P)H oxidation, it does not prevent NAD(P)H from being oxidized through the process of oxidative phosphorylation at the onset of contractions.

Last second twitch-response test of single intact skeletal muscle fibers prior to quick-freezing after various stimulation intervals

1983 ◽  
Vol 41 ◽  
pp. 526-527
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
I. Taylor
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Morphological Changes ◽  
Muscle Fibers ◽  
Visual Observation ◽  
Time Interval ◽  
Time Intervals ◽  
Twitch Response ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing

Conventional chemical fixation of muscle fibers is not suited to disclose morphological changes occurring with a time course of only a few msec, e.g.during excitation-contraction-coupling. We have taken advantage of a quick-freeze method to be able to study single intact frog skeletal muscle fibers (r.temporaria) after various time intervals following electrical stimulation. The electronics were designed to permit any time interval from 0 to more than 1 sec between stimulation and impact of the specimen on a liquid He-cooled copper block. It is important to monitor the twitch-response to stimulation. Given the geometry of the freezing device ("Slammer", Polaron), and the fact that the device does not operate vibration-free, it is quite difficult to design and built an effective monitor able to record the actual twitch-response following the stimulation of an isolated single muscle fiber during its descent prior to freezing. We have built a simple device that allows visual observation of the twitch-response within a few seconds prior to the definitive stimulation during the specimen drop.

Sign in / Sign up

Export Citation Format

Share Document