scholarly journals FILAMENT ULTRASTRUCTURE AND ORGANIZATION IN VERTEBRATE SMOOTH MUSCLE

1967 ◽  
Vol 35 (2) ◽  
pp. 303-321 ◽  
Author(s):  
Bernard J. Panner ◽  
Carl R. Honig
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Dense Bodies ◽  
Contraction Mechanism ◽  
Dark Staining ◽  
Technical Factors ◽  
Low Ionic Strength

Using a variety of preparative techniques for electron microscopy, we have obtained evidence for the disposition of actin and myosin in vertebrate smooth muscle. All longitudinal myofilaments seen in sections appear to be actin. Previous reports of two types of longitudinal filaments in sections are accounted for by technical factors, and by differentiated areas of opacity along individual filaments. Dense bodies with actin emerging from both ends have been identified in homogenates, and resemble Z discs from skeletal muscle (Huxley, 1963). In sections, short, dark-staining lateral filaments 15–25 A in diameter link adjacent actin filaments within dense bodies and in membrane dense pataches. They appear homologous with Z-disc filaments. Similar lateral filaments connect actin to plasma membrane. Dense bodies and dense patches, therefore, are attachment points and denote units analogous to sarcomeres. In glycerinated, methacrylate-embedded sections, lateral processes different in length and staining characteristics from lateral filaments in dense bodies exist at intervals along actin filaments. These processes are about 30 A wide and resemble heavy meromyosin from skeletal muscle. They also resemble heads of whole molecules of myosin in negatively stained material from gizzard homogenates. Intact single myosin molecules and dimers have been found, both free and attached to actin, even in media of very low ionic strength. Myosin can, therefore, exist in relatively disaggregated form. Models of the contraction mechanism of smooth muscle are proposed. The unique features are: (1) Myosin exists as small functional units. (2) Movement occurs by interdigitation and sliding of actin filaments.

scholarly journals Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs.

1979 ◽  
Vol 82 (1) ◽  
pp. 212-226 ◽  
Author(s):  
A Spudich ◽  
J A Spudich
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Cell Types ◽  
Optical Diffraction ◽  
Muscle Actin ◽  
Sea Urchin Eggs ◽  
Inner Surface ◽  
Low Ionic Strength ◽  
Helical Parameters

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.

scholarly journals Banding and polarity of actin filaments in interphase and cleaving cells.

1980 ◽  
Vol 86 (2) ◽  
pp. 568-575 ◽  
Author(s):  
J M Sanger ◽  
J W Sanger
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Actin Filaments ◽  
Cell Junctions ◽  
Stress Fibers ◽  
Cleavage Furrow ◽  
Heavy Meromyosin ◽  
Glutaraldehyde Fixation ◽  
Microfilament Bundles ◽  
Uniform Polarity

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.

scholarly journals Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding.

1987 ◽  
Vol 105 (6) ◽  
pp. 3007-3019 ◽  
Author(s):  
K M Trybus ◽  
S Lowey
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Conformational Changes ◽  
Myosin Head ◽  
Smooth Muscle Myosin ◽  
Detectable Effect ◽  
Skeletal Muscle Myosin ◽  
Polar Type ◽  
Low Ionic Strength ◽  
Muscle Myosin

Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.

scholarly journals Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

1982 ◽  
Vol 201 (2) ◽  
pp. 267-278 ◽  
Author(s):  
J Kay ◽  
R F Siemankowski ◽  
L M Siemankowski ◽  
D E Goll
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Light Chain ◽  
Regulatory Light Chain ◽  
Smooth Muscle Myosin ◽  
Heavy Meromyosin ◽  
Bovine Trypsin ◽  
Muscle Myosin

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The ‘regulatory’ light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called ‘regulatory’ light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).

scholarly journals Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

2020 ◽  
Vol 117 (27) ◽  
pp. 15666-15672
Author(s):  
Xiong Liu ◽  
Shi Shu ◽  
Edward D. Korn
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Muscle Contraction ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Smooth Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail–head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin

1992 ◽  
Vol 262 (3) ◽  
pp. C714-C723 ◽  
Author(s):  
E. Homsher ◽  
F. Wang ◽  
J. R. Sellers
Keyword(s):  
Skeletal Muscle ◽  
Ionic Strength ◽  
Actin Filament ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Shortening Velocity ◽  
Weakly Bound ◽  
Factors Affecting ◽  
Cross Bridges

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.

scholarly journals Intermediate-sized filaments of the prekeratin type in myoepithelial cells.

1980 ◽  
Vol 84 (3) ◽  
pp. 633-654 ◽  
Author(s):  
W W Franke ◽  
E Schmid ◽  
C Freudenstein ◽  
B Appelhans ◽  
M Osborn ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Immunofluorescence Microscopy ◽  
Myogenic Differentiation ◽  
Muscle Cells ◽  
Myoepithelial Cells ◽  
Sweat Glands ◽  
Dense Bodies ◽  
Cell Specialization

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.

scholarly journals Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle

1990 ◽  
Vol 272 (2) ◽  
pp. 305-310 ◽  
Author(s):  
S Marston
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Gel Electrophoresis ◽  
Thin Filament ◽  
Critical Concentration ◽  
Actin Monomer ◽  
Heavy Meromyosin ◽  
Thin Filaments

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.

Glomangiomyoma (Glomus Tumor) of the Kidney

2005 ◽  
Vol 129 (9) ◽  
pp. 1172-1174 ◽  
Author(s):  
Noman H. Siddiqui ◽  
Agnieszka Rogalska ◽  
Indu S. Basil
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Glomus Tumor ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Thin Filaments ◽  
Glomus Tumors ◽  
Computed Tomographic ◽  
Dense Bodies ◽  
Computed Tomographic Scan

Abstract We report herein a case of glomus tumor arising in the kidney of a 55-year-old woman, which was found incidentally on a computed tomographic scan. Partial nephrectomy revealed a 2-cm encapsulated mass that was architecturally similar to glomus tumor. Immunohistochemistry showed positivity of tumor cells for vimentin and smooth muscle actin. On electron microscopy, cytoplasmic thin filaments and dense bodies were seen, confirming the smooth muscle nature of the tumor. Glomus tumors arising in visceral organs are rare, and those arising in kidney are exceedingly rare.

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