Removal Of Myosin Research Articles

Electron microscopic examination of podosomes induced by phorbol 12, 13 dibutyrate on the surface of A7r5 cells

The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation. However, ML-7, an inhibitor of the kinase activity of MLCK, failed to inhibit podosome formation. Based on our previous report (Thatcher et al. J.Pharm.Sci. 116 116–127, 2011), we outlined the important role of the actin-binding activity of MLCK in producing smaller podosomes.

Myosin Kinase of Molluscan Smooth Muscle. Regulation by Binding of Calcium to the Substrate and Inhibition of Myorod and Twitchin Phosphorylation by Myosin

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin. These supramolecular complexes and filaments assembled from purified myosin contained an endogenous protein kinase that phosphorylated myosin and myorod. Significantly, the activity of this novel myosin-associated (MA) kinase was inhibited at calcium concentrations of >0.1 microM. After partial purification of the kinase, we established that the inhibition resulted from binding of calcium to the substrate (myosin) and not from the binding to the enzyme (kinase). No inhibition was observed when myorod was used as a substrate, although the latter is identical to the rod portion of myosin lacking the head domains. Phosphorylation sites of myorod were identified, three at the C-terminal tip and three at the N-terminal domain. In the presence of calcium, addition of myosin to the TW-MR complex resulted in inhibition of this phosphorylation, while in the absence of myosin, this inhibition was negligible. Added myosin also inhibited phosphorylation of twitchin by PKA-like kinase, the latter also present in the complex. The opposite was true with the TW-MY-MR complex; that is, phosphorylation of myosin was inhibited by twitchin and/or myorod. Thus, in parallel to the well-established direct activation by calcium, molluscan catch muscle myosin also regulated its own phosphorylation. Therefore, in addition to the established phosphorylation of twitchin by PKA-like kinase, phosphorylation of myosin and myorod by myosin-associated kinase appears to play an important role in the development of the catch state.

Immunocytochemical studies using a monoclonal antibody to bovine cardiac titin on intact and extracted myofibrils.

A monoclonal antibody specific to bovine cardiac titin has been identified. The antibody recognizes a common antigenic site in striated muscles of several species. In relaxed myofibrils, specific staining at the A-I junction resulted in a doublet of fluorescent bands within a sarcomere. The distance between the doublets in successive sarcomeres varied according to the degree of myofibrillar contraction. Staining on formamide-extracted myofibrils has confirmed that this epitope is located near the outer edges of isolated A bands. Selective extraction of myofibrillar proteins resulted in different staining patterns. Disrupting the structural integrity of the M-line or the A-band centre caused a significant amount of titin to translocate toward the Z-line region. In contrast, shortening of the A-band by removal of myosin from the ends of the thick filaments resulted in anti-titin staining moving closer to the M-line region. Several conclusions can be drawn from this study: (a) two aligned groups of titin molecules are placed symmetrically to the M-line in a sarcomere; (b) titin may attach directly or via intermediary protein(s) to sites near the M-line and Z-line such that the protein is under tension and (c) removal of proteins from either region results in titin staining in the opposite region. However, the edges of the A-band give some hindrance to collapse of the titin toward the M-line.

Connectin, an elastic protein of muscle. Identification of "titin" with connectin.

When whole muscle fibers or myofibrils of rabbit and chicken skeletal muscles are directly solubilized in hot SDS solution, a very high molecular protein called titin can be isolated by gel filtration (Wang et al. 1979). Connectin, an elastic protein of muscle (Maruyama et al. 1977), can be isolated by a similar method from thoroughly extracted muscle residues. Studies of electrophoretic mobility on 2-3% polyacrylamide gel electrophoresis, amino acid composition, and localization in myofibrils determined by the indirect immunofluorescence technique showed that titin and connectin are identical. Connectin was found to be unstable in SDS solution on storage for a few days at room temperature; the doublet band of connectin on SDS gel electrophoresis became diffuse and eventually disappeared. Connectin was concentrated around the A-I junction region of a myofibril, although it was present in an entire sarcomere except for the Z lines. On removal of myosin, the A-I junction was still fluorescent, when treated with fluorescent antibody against connectin. In the KI-extracted myofibril, materials accumulated on both sides of the Z lines were strongly stained, and there were fluorescent filaments between the neighboring Z lines, but the Z lines were not stained at all.

Extraction and Functional Reformation of Thick Filaments in Chemically Skinned Molluscan Catch Muscle Fibers

A method for the almost complete extraction of myosin from smooth muscle fibers of the anterior byssal retractor muscle (ABRM) of Mytilus edulis was developed, and functional reformation of thick filaments in the fibers was achieved. Complete removal of myosin from the glycerol-extracted ABRM fibers with a solution containing 600 mM KCl, 5 mM MgCl2, and 5 mM ATP was difficult. However, successive treatments of the ABRM fibers with glycerol and saponin made the plasma membrane permeable to Mg-ATP and myosin. The extraction of myosin completely eliminated the tension induced by the addition of Mg-ATP. Partial recovery of tension development was observed by irrigation of myosin into fibers from which myosin had been extracted. Similar results were obtained using rabbit myosin instead of ABRM myosin. Addition of heavy meromyosin, on the other hand, had a suppressive effect on the tension development, as is the case in glycerinated rabbit psoas muscle fibers.

The existence of an insoluble Z disc scaffold in chicken skeletal muscle

Extraction of glycerinated chicken skeletal muscle with 0.6 M potassium iodide leaves a framework of insoluble components within each muscle fiber. This framework is composed primarily of planes of in-register Z discs that have been thickened by the accumulation of material on both sides of each disc during extraction. Membrane vesicles, presumably remnants of the T system, remain surrounding the Z discs. When the framework is sheared in a blender, it is preferentially cleaved between Z planes, resulting in the formation of large sheets of interconnected, closely packed Z discs in a honeycomb-like array. Cleavage occurs in regions formerly occupied by the A bands, which have been weakened by the removal of myosin. The existence and stability of these planar Z disc arrays demonstrate the presence and strength of connections between adjacent myofibrils. SDS-polyacrylamide gel electrophoresis reveals that this framework consists primarily of actin and desmin, with lesser amounts of a few proteins including α-actinin, myosin and tropomyosin. Z disc sheets and KI-extracted myofibrils provide a distinct face-on view and side view, respectively, of the Z disc. In indirect immunofluorescence, these two views have revealed that desmin is present at the periphery of each Z disc, forming a network of proteinaceous collars within the Z plane. α-Actinin is localized within each disc, giving a face-on fluorescence pattern that is complementary to that of desmin. Actin is present throughout the thickened Z plane, while myosin and tropomyosin exist only in the insoluble residue that coalesces on both faces of each disc. We conclude that desmin, perhaps in conjunction with actin, is responsible for interlinking Z discs of adjacent myofibrils, and may thus serve as a mechanical and structural integrator of muscle fibers. Its hydrophobic nature and coincident distribution with the T system suggest that it may also be responsible for mediating filament-membrane interactions and anchoring the triad to the Z disc. Its collar-like distribution suggests that it may aid in maintaining the structural integrity of the Z disc and the actin filaments inserted into it.

Fine structure of connectin nets in cardiac myofibrils.

A net-like structure extending through Z lines of myofibrils in frog cardiac muscle was clearly demonstrated under an electron microscope after the removal of myosin and actin. The diameter of the very thin filaments forming the nets was approximately 2 nm; this width was thinner than that of the actin filament. The net structure is ascribed to connectin, an elastic protein of muscle.

The role of connectin in elastic properties of insect flight muscle

1. 1. The removal of myosin from flbrillar flight muscle fibre of the waterbug Lethocerus resulted in a great drop in the tension produced by passive stretch, while a considerable amount of tension developed in similarly treated flight muscle fibres of the locust. 2. 2. Connectin, which functions as a parallel elastic component of muscle, was obtained from both the waterbug and locust muscles. The content in the waterbug was less than in the locust and they both contained connectin in amounts less than vertebrate skeletal muscle. 3. 3. The role of connectin in the passive elasticity of insect flight muscles is discussed in relation to the function of connecting material which links myosin filaments to Z-lines. 4. 4. An unusually high content of phenylalanine was observed in SDS†-insoluble proteins from insect flight muscle.

Beta-actinin, a regulatory protein of muscle. Purification, characterization and function.

beta-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. beta-Actinin consisted of two subunits, beta I and betaII, with chain weights of 37,000 and 34,000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. beta-Actinin had the following effects on actin: (1) inhibition of reassociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) acceleration of polymerization of G-actin. All these actions of beta-actinin can be explained in terms of action as an "ending factor". Experimental evidence favored the view that beta-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of polymerization. Fluorescence-labeled anti-beta-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that beta-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that beta-actinin functions as an ending factor for actin filaments.

New elastic protein from muscle

IT has long been assumed that an elastic component other than extracellular collagen fibres is present in muscle fibres to explain their elastic properties, especially during passive stretch1,2 The presence of such an elastic component has been demonstrated in skinned fibres of frog skeletal muscle3,4. Furthermore, it has been observed that myofibrils after the removal of myosin are reversibly extended, indicating their structural continuity5. The chemical entity responsible for the intracellular elasticity of muscle has, however, remained obscure. When rabbit myofibrils were thoroughly extracted with salt solutions such as 0.6 M KI, it was noticed that remaining Z lines still maintained their continuity, although nothing could be seen between the adjacent Z lines under a phase microscope (compare ref. 6). Starting from this observation, we have been able to isolate an elastic protein from myofibrils which is clearly distinguishable from elastin or collagen.

Myosin-free ghosts of single fibers and an attempt to re-form myosin filaments in the ghost fibers.

With the final aim of replacing myosin in a single muscle fiber, a technique for removing myosin almost completely from single fibers was developed and an attempt to "re-form" thick filaments in the myosin-free ghosts of single fibers was made. Complete removal of myosin from single glycerol-treated rabbit psoas fibers with Hasselbach-Schneider solution was difficult. However, when skinned glycerol-treated fibers were used and 1% (v/v) Triton X-100 was added to the Hasselbach-Schneider solution, almost complete removal of myosin was possible. The myosin-free ghosts of skinned single fibers were very fragile but retained the overall structure. In the ghost fibers, Z-membranes and thin filaments remained. The ghost fibers, after irrigation with myosin, underwent contraction upon addition of Mg-ATP. In the myosin-irrigated fibers, thick filaments were re-formed in lengths from one Z-membrane to the other Z-membrane of a sarcomere, running parallel to the thin filaments. The packing of these two filaments was not good. The isometric tension developed by the irrigated fibers upon addition of mg-ATP was about 10% of the tension developed by untreated fibers. The weak tension developed by irrigated fibers is probably due to the irregular packing of the thick and thin filaments in the fibers. The ghost fibers also contracted, though only slightly, upon addition of Mg-ATP after irrigation with heavy meromyosin.

Histology of highly-stretched beef muscle. II. Further evidence on the location and nature of gap filaments

The response of beef muscle fibers to gross overstretch varied. In most fibers the A-filaments dislocated and stretched, and then a gap opened between the A- and I-bands. In a small proportion of fibers a wide gap opened between A- and I-bands while the A-band remained intact. These anomalous fibers appear to dislocate under extreme stretch, since transitions from such sarcomeres to the dislocated type were sometimes seen along a fiber. Thus there appear to be two routes to the same final state. A few fibers appeared to have gone into rigor before stretching, since cross-bridging of actin and myosin filaments survived, while thick filaments fractured. Extraction of myosin and actin from stretched fibers left the gap filaments running from an overlap in the center of the sarcomere through the Z-line, supporting the view that each gap filament was present as a core to the thick filament, emerging at one end only. Fibers released after stretch always sprang back, even after removal of myosin and actin. The order in “springbacks” varied greatly with the circumstances but the gap filaments appear to provide the restoring force in every case. A model is postulated in which each gap filament forms a core to two thick filaments, each in adjacent sarcomeres, linking them through the Zline and forming an elastic component in muscle, centered on the Z-line.