scholarly journals Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting.

1982 ◽  
Vol 93 (3) ◽  
pp. 533-542 ◽  
Author(s):  
J P Brunschwig ◽  
N Brandt ◽  
A H Caswell ◽  
D S Lukeman
Keyword(s):  
Skeletal Muscle ◽  
Tannic Acid ◽  
Dense Matter ◽  
Transverse Tubules ◽  
Electron Dense Material ◽  
Junctional Region ◽  
Intact Muscle ◽  
Characteristic Features ◽  
Triton X ◽  
Terminal Cisternae

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.

scholarly journals The Intracellular Site of Calcium Activation of Contraction in Frog Skeletal Muscle

1970 ◽  
Vol 55 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Saul Winegrad
Keyword(s):  
Skeletal Muscle ◽  
Room Temperature ◽  
Half Time ◽  
Frog Skeletal Muscle ◽  
Calcium Efflux ◽  
Transverse Tubules ◽  
Calcium Activation ◽  
Thin Filaments ◽  
Almost All ◽  
Terminal Cisternae

Radioautography has been used to localize 45Ca in isotopically labeled frog skeletal muscle fibers which had been quickly frozen during a maintained tetanus, a declining tetanus, or during the period immediately following a tetanus or a contracture. During a tetanus almost all of the myofibrillar 45Ca is localized in the region of the sarcomere occupied by the thin filaments. The amount varies with the tension being developed by the muscle. The movement of calcium within the reticulum from the tubular portion to the terminal cisternae during the posttetanic period has a half-time of about 9 sec at room temperature and a Q10 of about 1.7. Repolarization is not necessary for this movement. Evidence is given to support the notion that most calcium efflux from the cell occurs from the terminal cisternae into the transverse tubules.

scholarly journals Identification and extraction of proteins that compose the triad junction of skeletal muscle.

1984 ◽  
Vol 99 (3) ◽  
pp. 929-939 ◽  
Author(s):  
A H Caswell ◽  
J P Brunschwig
Keyword(s):  
Freeze Fracture ◽  
Membrane Fragment ◽  
Transverse Tubules ◽  
Low Profile ◽  
Junction Formation ◽  
Nonionic Detergent ◽  
Selective Retention ◽  
Triad Junction ◽  
Triton X ◽  
Terminal Cisternae

Treatment of both transverse tubules and terminal cisternae with a combination of Triton X-100 and hypertonic K cacodylate causes dissolution of nonjunctional proteins and selective retention of membrane fragments which are capable of junction formation. Treatment of vesicles with Triton X-100 and either KCl or K gluconate causes complete dissolution of all components. Therefore K cacodylate exerts a specific preservative action on the junctional material. The membrane fragment from treatment of transverse tubules with Triton X-100 + cacodylate contains a protein of Mr = 80,000 in SDS gel electrophoresis as the predominant protein while lipid composition is enriched in cholesterol. The membrane fragment retains in electron microscopy the trilaminar appearance of the intact vesicles. Freeze fracture of transverse tubule fragments reveals a high density of low-profile, intercalated particles, which frequently form strings or occasional small arrays. The fragments from Triton X-100 plus cacodylate treatment of terminal cisternae include the protein of Mr = 80,000 as well as the spanning protein of the triad, calsequestrin, and some minor proteins. The fragments are almost devoid of lipid and display an amorphous morphology suggesting membrane disruption. The ability of the transverse tubular fragment, which contains predominantly the Mr = 80,000 protein, to form junctions with terminal cisternae fragments suggests that it plays a role in anchoring the membrane to the junctional processes of the triad. The junctional proteins may be solubilized in a combination of nonionic detergent and hypertonic NaCl. Subsequent molecular sieve chromatography gives an enriched preparation of the spanning protein. This protein has subunits of Mr = 300,000, 270,000 and 140,000 and migrates in the gel as a protein of Mr = 1.2 X 10(6) indicating a polymeric structure.

scholarly journals Sizes of components in frog skeletal muscle measured by methods of stereology.

1975 ◽  
Vol 66 (1) ◽  
pp. 31-45 ◽  
Author(s):  
B A Mobley ◽  
B R Eisenberg
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Surface Area ◽  
Volume Ratio ◽  
Sartorius Muscle ◽  
Morphological Parameters ◽  
Frog Skeletal Muscle ◽  
Fiber Volume ◽  
Transverse Tubules ◽  
Terminal Cisternae

Stereological techniques of point and intersection counting were used to measure morphological parameters from light and electron micrographs of frog skeletal muscle. Results for sartorius muscle are as follows: myofibrils comprise 83% of fiber volume; their surface to volume ratio is 3.8 mum-1. Mitochondria comprise 1.6% of fiber volume. Transverse tubules comprise 0.32% of fiber volume, and their surface area per volume of fiber is 0.22 mum-1. Terminal cisternae of the sarcoplasmic reticulum comprise 4.1% of fiber volume; their surface area per volume of fiber is 0.54 mum-1. Longitudinal sarcoplasmic reticullum comprises 5.0% of fiber volume, and its surface area per volume of fiber is 1.48 mum-1. Longitudinal bridges between terminal cisternae on either side of a Z disk were observed infrequently; they make up only 0.035% of fiber volume and their surface area per volume of fiber is 0.009 mum-1. T-SR junction occurs over 67% of the surface of transverse tubules and over 27% of the surface of terminal cisternae. The surface to volume ratio of the caveolae is 48 mum-1; caveolae may increase the sarcolemmal surface area by 47%. Essentially the same results were obtained from semitendinosus fibers.

scholarly journals Cell fractionation studies indicate that dystrophin is a protein of surface membranes of skeletal muscle

1989 ◽  
Vol 258 (3) ◽  
pp. 837-841 ◽  
Author(s):  
G Salviati ◽  
R Betto ◽  
S Ceoldo ◽  
E Biasia ◽  
E Bonilla ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cholesterol Content ◽  
Binding Activity ◽  
Cell Fractionation ◽  
Ouabain Binding ◽  
Low Concentrations ◽  
Peripheral Membrane Protein ◽  
T Tubules ◽  
Triton X ◽  
Terminal Cisternae

We studied the subcellular localization of dystrophin in rabbit skeletal muscle. In Western-blot analysis of membrane preparations, dystrophin was associated with the sarcolemmal fraction, as indicated by cholesterol content and co-purification with ouabain-binding activity and beta-adrenergic receptor. Dystrophin was also found with junctional T-tubules, but not with ‘free’ T-tubules, longitudinal portions or terminal cisternae of the sarcoplasmic reticulum. Dystrophin was not solubilized by high salt solutions, but it was solubilized by low concentrations of detergents (Triton X-100 and deoxycholate), suggesting that it is a peripheral membrane protein.

Transport pathway, maturation, and targetting of the vesicular stomatitis virus glycoprotein in skeletal muscle fibers

1996 ◽  
Vol 109 (6) ◽  
pp. 1585-1596
Author(s):  
P. Rahkila ◽  
A. Alakangas ◽  
K. Vaananen ◽  
K. Metsikko
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Vesicular Stomatitis Virus ◽  
Muscle Fibers ◽  
Viral Glycoprotein ◽  
Transverse Tubules ◽  
Skeletal Muscle Fibers ◽  
Vesicular Stomatitis ◽  
Terminal Cisternae

We have infected isolated skeletal muscle fibers with the vesicular stomatitis virus or the mutant tsO45, whose glycoprotein is blocked in the endoplasmic reticulum at 39 degrees C. Immunofluorescence analysis for the viral glycoprotein indicated that the fibers were infected over their entire length at a virus dose of 10(9)/ml. When we infected the myofibers with the tsO45 mutant at 39 degrees C, the viral glycoprotein appeared to be localised to the terminal cisternae of the sarcoplasmic reticulum. Upon shifting the cultures to the permissive temperature, 32 degrees C, in the presence of dinitrophenol, which blocks vesicular transport, the viral glycoprotein proceeded to completely fill the sarcoplasmic reticulum. Thus, both the endoplasmic reticulum located at the terminal cisternae of the sarcoplasmic reticulum, and the entire endoplasmic and sarcoplasmic reticulum appeared to be continuous. Shifting the culture temperature from 39 degrees C to 20 degrees C, resulted in prominent perinuclear staining throughout the fibers, accompanied by the appearance of distinct bright dots between the nuclei. Electron microscopic immunoperoxidase labeling indicated that these bright structures represented the Golgi apparatus. When either the tsO45-infected or wild-type virus-infected fibers were incubated at 32 degrees C, the viral glycoprotein showed a staining pattern that consisted of double rows of punctate fluorescence. Immunogold labeling showed that the viral glycoprotein was present in both the transverse tubules as well as the endoplasmic/sarcoplasmic reticulum endomembranes. In addition, extensive viral budding was observed in the transverse tubules. Metabolic labeling experiments revealed that only half of the glycoprotein was processed in the Golgi, and this processed form had become incorporated into the budding viral particles. Thus, the processed viral glycoprotein was targeted to the transverse tubules. The other half of the glycoprotein remained endoglycosidase H-sensitive, suggesting its retention in the endoplasmic/sarcoplasmic reticulum endomembranes.

The Z band in skinned fibers of a slow skeletal muscle

1990 ◽  
Vol 48 (3) ◽  
pp. 460-461
Author(s):  
Margaret A. Goldstein ◽  
John P. Schroeter
Keyword(s):  
Skeletal Muscle ◽  
Skinned Fibers ◽  
Muscle Properties ◽  
Slow Skeletal Muscle ◽  
Band Form ◽  
Intact Muscle ◽  
Band Spacing ◽  
Triton X

We have shown that Z bands in rat soleus muscles exhibit two structural states. The small square (ss) form is seen in relaxed intact muscle and the basketweave (bw) form is seen in maximally activated muscle (tetanized and in rigor). The Z spacing measured in the bw form is 20% larger than that in the ss form. The dimensions of the two Z band lattice forms in these muscle states are independent of A band spacing. Skinned fibers have been used to assess various muscle properties and expansion of the A band has been observed in some cases. We have examined the effects of chemical skinning on Z band form and dimension and A band spacing in relaxed muscle.Rat soleus muscles were dissected and tied to applicator sticks and removed to a solution of 1% Triton X-100 in lOmM PIPES buffer with 5mM EGTA and lOmM ATP overnight at 4°C, then rinsed with the PIPES buffer with EGTA and ATP only for 4 hours.

scholarly journals Calcium and magnesium contents and volume of the terminal cisternae in caffeine-treated skeletal muscle.

1984 ◽  
Vol 99 (2) ◽  
pp. 558-568 ◽  
Author(s):  
T Yoshioka ◽  
A P Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Common Channel ◽  
Electron Probe Analysis ◽  
Caffeine Contracture ◽  
P Content ◽  
Ca Uptake ◽  
Intact Muscle ◽  
Mg Content ◽  
Terminal Cisternae

(a) The effects of caffeine on the composition and volume of the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in frog skeletal muscle were determined with rapid freezing, electron microscopy, and electron probe analysis. (b) Caffeine (5 mM) released approximately 65% of the Ca content of the TC in 1 min and 84% after 3 min. The release of Ca from the TC was associated with a highly significant increase in its Mg content. This increase in Mg was not reduced by valinomycin. There was also a small increase in the K content of the TC at 1 min, although not after 3 min of caffeine contracture. (c) On the basis of the increase in Mg content during caffeine contracture and during tetanus (Somlyo, A. V., H. Gonzalez-Serratos, H. Shuman, G. McClellan, and A. P. Somlyo, 1981, J. Cell Biol., 90:577-594), we suggest that both mechanisms of Ca release are associated with an increase in the Ca and Mg permeability of the SR membranes, the two ions possibly moving through a common channel. (d) There was a significant increase in the P content of the TC during caffeine contracture, while in tetanized muscle (see reference above) there was no increase in the P content of the TC. (e) Mitochondrial Ca content was significantly increased (at 1 and at 3 min) during caffeine contracture. Valinomycin (5 microM) blocked this mitochondrial Ca uptake. (f) The sustained Ca release caused by caffeine in situ contrasts with the transient Ca release observed in studies of fragmented SR preparations, and could be explained by mediation of the caffeine-induced Ca release by a second messenger produced more readily in intact muscle than in isolated SR. (g) The TC were not swollen in rapidly frozen, caffeine-treated muscles, in contrast to the swelling of the TC observed in conventionally fixed, caffeine-treated preparation, the latter finding being in agreement with previous studies. (h) The fractional volume of the TC in rapidly frozen control (resting) frog semitendinosus muscles (approximately 2.1%) was less than the volume (approximately 2.5%) after glutaraldehyde-osmium fixation.

Sign in / Sign up

Export Citation Format

Share Document