scholarly journals DIFFERENTIATION OF THE SARCOPLASMIC RETICULUM AND T SYSTEM IN DEVELOPING CHICK SKELETAL MUSCLE IN VITRO

1967 ◽  
Vol 35 (2) ◽  
pp. 405-420 ◽  
Author(s):  
Elizabeth B. Ezerman ◽  
Harunori Ishikawa
Keyword(s):  
Skeletal Muscle ◽  
Electron Microscope ◽  
Sarcoplasmic Reticulum ◽  
Osmium Tetroxide ◽  
Muscle Cells ◽  
Simultaneous Occurrence ◽  
Chick Embryos ◽  
Developing Muscle ◽  
T System

The electron microscope was used to investigate the first 10 days of differentiation of the SR and the T system in skeletal muscle cultured from the breast muscle of 11-day chick embryos. The T-system tubules could be clearly distinguished from the SR in developing muscle cells fixed with glutaraldehyde and osmium tetroxide. Ferritin diffusion confirmed this finding: the ferritin particles were found only in the tubules identified as T system. The proliferation of both membranous systems seemed to start almost simultaneously at the earliest myotube stage. Observations suggested that the new SR membranes developed from the rough-surfaced ER as tubular projections. The SR tubules connected with one another to form a network around the myofibril. The T-system tubules were formed by invagination of the sarcolemma. The early extension of the T system by branching and budding was seen only in subsarcolemmal regions. Subsequently the T-system tubules could be seen deep within the muscle cells. Immediately after invaginating, the T-system tubule formed, along its course, specialized connections with the SR or ER: triadic structures showing various degrees of differentiation. The simultaneous occurrence of myofibril formation and membrane proliferation is considered to be important in understanding the coordinated events resulting in the differentiated myotube.

scholarly journals THE FINE STRUCTURE OF EMBRYONIC CHICK SKELETAL MUSCLE CELLS DIFFERENTIATED IN VITRO

1967 ◽  
Vol 35 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Y. Shimada ◽  
D. A. Fischman ◽  
A. A. Moscona
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Fine Structure ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Chick Embryos ◽  
Embryonic Chick ◽  
Developing Muscle

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.

scholarly journals Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles

2003 ◽  
Vol 160 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Paola Bagnato ◽  
Virigina Barone ◽  
Emiliana Giacomello ◽  
Daniela Rossi ◽  
Vincenzo Sorrentino
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Physiological Activity ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Structural Function ◽  
Striated Muscles

Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of ∼800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

scholarly journals The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle

2020 ◽  
Vol 153 (1) ◽  
Author(s):  
Aldo Meizoso-Huesca ◽  
Bradley S. Launikonis
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Indirect Effects ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Repetitive Stimulation ◽  
Ec Coupling ◽  
Dependent Inhibition ◽  
T System

BTP2 is an inhibitor of the Ca2+ channel Orai1, which mediates store-operated Ca2+ entry (SOCE). Despite having been extensively used in skeletal muscle, the effects of this inhibitor on Ca2+ handling in muscle cells have not been described. To address this question, we used intra- and extracellular application of BTP2 in mechanically skinned fibers and developed a localized modulator application approach, which provided in-preparation reference and test fiber sections to enhance detection of the effect of Ca2+ handling modulators. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of resting extracellular Ca2+ flux. Increasing concentrations of BTP2 caused a shift from inducing accumulation of Ca2+ in the t-system due to Orai1 blocking to reducing the resting [Ca2+] in the sealed t-system. This effect was not observed in the absence of functional ryanodine receptors (RYRs), suggesting that higher concentrations of BTP2 impair RYR function. Additionally, we found that BTP2 impaired action potential–induced Ca2+ release from the sarcoplasmic reticulum during repetitive stimulation without compromising the fiber Ca2+ content. BTP2 was found to have an effect on RYR-mediated Ca2+ release, suggesting that RYR is the point of BTP2-induced inhibition during cycles of EC coupling. The effects of BTP2 on the RYR Ca2+ leak and release were abolished by pre-exposure to saponin, indicating that the effects of BTP2 on the RYR are not direct and require a functional t-system. Our results demonstrate the presence of a SOCE channels–mediated basal Ca2+ influx in healthy muscle fibers and indicate that BTP2 has multiple effects on Ca2+ handling, including indirect effects on the activity of the RYR.

scholarly journals Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2.

1991 ◽  
Vol 115 (5) ◽  
pp. 1345-1356 ◽  
Author(s):  
B E Flucher ◽  
J L Phillips ◽  
J A Powell
Keyword(s):  
Muscle Cells ◽  
Distribution Patterns ◽  
Receptor Alpha ◽  
Alpha Subunits ◽  
Nuclear Domain ◽  
T Tubules ◽  
Immunofluorescence Labeling ◽  
T System ◽  
Triad Junctions

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.

In Vitro Effects of Soy Phytoestrogens on Rat L6 Skeletal Muscle Cells

2005 ◽  
Vol 8 (3) ◽  
pp. 327-331 ◽  
Author(s):  
K.L. Jones ◽  
J. Harty ◽  
M.J. Roeder ◽  
T.A. Winters ◽  
W.J. Banz
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
In Vitro Effects ◽  
L6 Skeletal Muscle Cells

Characterization of β-adrenoceptors on rat skeletal muscle cells grown in vitro

1990 ◽  
Vol 40 (5) ◽  
pp. 1043-1048 ◽  
Author(s):  
Marie-Helene Disatnik ◽  
Sanford R. Sampson ◽  
Asher Shainberg
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Rat Skeletal Muscle

scholarly journals Biosynthesis of titin in cultured skeletal muscle cells.

1989 ◽  
Vol 109 (5) ◽  
pp. 2189-2195 ◽  
Author(s):  
W B Isaacs ◽  
I S Kim ◽  
A Struve ◽  
A B Fulton
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Significant Progress ◽  
Large Protein ◽  
Time Period ◽  
And Function ◽  
Triton X ◽  
Developing Muscle ◽  
Synchronized Cultures

Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with [35S]methionine and immunoprecipitation with an anti-titin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases greater than 4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases greater than 12-fold; (b) newly synthesized titin has a t1/2 of approximately 70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes.

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