scholarly journals Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections

1989 ◽  
Vol 55 (4) ◽  
pp. 713-724 ◽  
Author(s):  
B.L. Trus ◽  
A.C. Steven ◽  
A.W. McDowall ◽  
M. Unser ◽  
J. Dubochet ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Thin Sections ◽  
Myosin Filaments

scholarly journals Connectin filaments in stretched skinned fibers of frog skeletal muscle.

1984 ◽  
Vol 99 (4) ◽  
pp. 1391-1397 ◽  
Author(s):  
K Maruyama ◽  
H Sawada ◽  
S Kimura ◽  
K Ohashi ◽  
H Higuchi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Electron Microscopic ◽  
Thin Filaments ◽  
Thin Sections ◽  
Cytoskeletal Structure ◽  
Present Information ◽  
Sds Page ◽  
Myosin Filaments ◽  
Elastic Protein ◽  
Passive Stretch

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

scholarly journals Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation.

1975 ◽  
Vol 67 (1) ◽  
pp. 93-104 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Structural Features ◽  
Length Frequency ◽  
Single Population ◽  
Skeletal Muscle Myosin ◽  
Distinctive Features ◽  
Myosin Filaments ◽  
The Mean ◽  
Muscle Myosin

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture.

Effect of taxol on the interaction of tubulin with myosin filaments

1984 ◽  
Vol 62 (9) ◽  
pp. 878-884 ◽  
Author(s):  
Toshihiro Fujii ◽  
Tatsuo Suzuki ◽  
Akira Hachimori ◽  
Michiyo Fujii ◽  
Yoshiyuki Kondo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Atpase Activity ◽  
Cross Section ◽  
Molar Ratio ◽  
Tubulin Polymerization ◽  
Myosin Filaments ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The interaction between polymerized tubulin from porcine brain and myosin from rabbit skeletal muscle was examined. The addition of myosin to the solution of tubulin polymerized by taxol resulted in a remarkable increase in turbidity within a few minutes at 37 °C, and a dense and stable precipitate was formed. The maximal molar ratio of tubulin bound to myosin was calculated to be about 4, while the value was about 2 when 6S tubulin was used. Both podophyllotoxin and colchicine suppressed the taxol-dependent increase of the binding of tubulin to myosin, but only when they were preincubated with tubulin prior to addition of taxol. 6S tubulin inhibited with aetin-activated Mg2+-ATPase activity of myosin, and polymerized tubulin inhibited the Mg-ATPase more than 6S tubulin. Dense precipitates of tubulin and myosin were observed by thin-section electron microscopy. Microtubules were observed to be entangled in myosin filaments and single microtubules were occasionally surrounded by five myosin filaments in a cross section, similar to actin–myosin arrays in muscle. After incubation of tubulin with myosin, taxol was able to induce tubulin polymerization in the same way as it polymerized microtubules in the absence of myosin.

scholarly journals Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line.

1988 ◽  
Vol 107 (6) ◽  
pp. 2199-2212 ◽  
Author(s):  
K Wang ◽  
J Wright
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscles ◽  
Smooth Muscles ◽  
Psoas Muscle ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Wide Range ◽  
Determining Factors ◽  
Myosin Filaments ◽  
In Series

Nebulin, a giant myofibrillar protein (600-800 kD) that is abundant (3%) in the sarcomere of a wide range of skeletal muscles, has been proposed as a component of a cytoskeletal matrix that coexists with actin and myosin filaments within the sarcomere. Immunoblot analysis indicates that although polypeptides of similar size are present in cardiac and smooth muscles at low abundance, those proteins show no immunological cross-reactivity with skeletal muscle nebulin. Gel analysis reveals that nebulins in various skeletal muscles of rabbit belong to at least two classes of size variants. A monospecific antibody has been used to localize nebulin by immunoelectron microscopy in a mechanically split rabbit psoas muscle fiber preparation. Labeled split fibers exhibit six pairs of stripes of antibody-imparted transverse densities spaced at 0.1-1.0 micron from the Z line within each sarcomere. These epitopes maintain a fixed distance to the Z line irrespective of sarcomere length and do not exhibit the characteristic elastic stretch-response of titin epitopes within the I band domain. It is proposed that nebulin constitutes a set of inextensible filaments attached at one end to the Z line and that nebulin filaments are in parallel, and not in series, with titin filaments. Thus the skeletal muscle sarcomere may have two sets of nonactomyosin filaments: a set of I segment-linked nebulin filaments and a set of A segment-linked titin filaments. This four-filament sarcomere model raises the possibility that nebulin and titin might act as organizing templates and length-determining factors for actin and myosin respectively.

scholarly journals Carbonic anhydrase III in skeletal muscle fibers: an immunocytochemical and biochemical study.

1988 ◽  
Vol 36 (7) ◽  
pp. 775-782 ◽  
Author(s):  
P Frémont ◽  
P M Charest ◽  
C Côté ◽  
P A Rogers
Keyword(s):  
Skeletal Muscle ◽  
Carbonic Anhydrase ◽  
Vastus Lateralis ◽  
Muscle Fibers ◽  
Water Soluble ◽  
Type I ◽  
Fiber Types ◽  
Thin Sections ◽  
Carbonic Anhydrase Iii ◽  
Skeletal Muscle Fibers

The objectives of the present study were to determine if carbonic anhydrase III (CA III) demonstrated a specific association for any particular organelle or structure of the skeletal muscle cell and to quantify the activity and content of this enzyme in different types of skeletal muscle fibers. Ultrastructural localization of CA III in the soleus (SOL), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL), composed of predominantly type I, IIa, and IIb fibers, respectively, was performed using a high-resolution immunocytochemical technique and antibody specific for CA III on ultra-thin sections of skeletal muscle embedded in the water-soluble medium polyvinyl alcohol (PVA). The results indicated a uniform distribution of CA III within the sarcomere. Mitochondria, nuclei, triads, Z-, and M-bands were not specifically labeled. Immunoblotting of washed myofibril preparations did not show any detectable CA III associated with this structure. In addition to quantification of the immunogold labeling, CA III activity and content were assayed in the post-mitochondrial supernatant of the three muscles. In the SOL, these values were found to be 3.6-7.6 times higher than in the DVL. The SVL showed a labeling intensity slightly higher than background level, while the enzyme activity and content were indistinguishable from background levels. We therefore conclude that CA III is randomly distributed in the cytoplasm of the three muscle fiber types and that the relative CA III content and activity in the three muscles studied is SOL greater than DVL greater than SVL approximately equal to 0.

scholarly journals Bridging structures spanning the junctioning gap at the triad of skeletal muscle.

1979 ◽  
Vol 80 (3) ◽  
pp. 743-750 ◽  
Author(s):  
A V Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Surface Membrane ◽  
Membrane Systems ◽  
Close Coupling ◽  
Thin Sections ◽  
Luminal Membrane ◽  
Terminal Cisterna ◽  
Terminal Cisternae ◽  
Junctional Sarcoplasmic Reticulum

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.

scholarly journals In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells.

1982 ◽  
Vol 92 (2) ◽  
pp. 324-332 ◽  
Author(s):  
M Taniguchi ◽  
H Ishikawa
Keyword(s):  
Skeletal Muscle ◽  
Salt Solution ◽  
Muscle Cells ◽  
High Salt ◽  
Skeletal Muscle Cells ◽  
Thin Filaments ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Glycerinated Muscle

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.

scholarly journals Application of the avidin-biotin system for post-embedding cytochemical demonstration of a biotin-labeled IgG tracer.

1986 ◽  
Vol 34 (4) ◽  
pp. 547-549 ◽  
Author(s):  
R J Seitz ◽  
K Heininger ◽  
G Schwendemann ◽  
K V Toyka
Keyword(s):  
Skeletal Muscle ◽  
Sciatic Nerve ◽  
Blood Vessels ◽  
Tracer Studies ◽  
Thin Sections ◽  
Mouse Skeletal Muscle ◽  
Cytochemical Demonstration ◽  
Mode Of Application ◽  
Embedding Medium

The capacity of the avidin-biotin system for post-embedding cytochemical detection of a biotin-labeled proteinaceous tracer was investigated. By testing modifications of the fixatives, the epoxy embedding medium, and various etching solutions, a procedure was developed to localize specifically the biotinylated tracer on 1-micron thick and thin sections. By use of this technique, a systemically administered IgG tracer was demonstrated after 24 hr throughout the endomysium of mouse skeletal muscle. In the adjoining sciatic nerve, the tracer IgG occurred at the perineurium and within endoneurial blood vessels, although the endoneurium itself was spared because of the presence of the blood-nerve barrier. Because of the small size of the biotin ligand and the non-denaturing method of labeling proteins, our mode of application of the avidin-biotin system appears suitable for tracer studies.

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