N-ethylmaleimide inhibits polymerization of myorod, a contractile protein of thick filaments of molluscan smooth muscles

The effect of N-ethylmaleimide on the polymerization of myorod, a protein of molluscan smooth muscles, which is colocalized with myosin on the surface of paramyosin core of thick filaments and is a product of the alternative splicing of the gene of heavy myosin chains, was studied. It was shown that myorod modified by N-ethylmaleimide completely loses the polymerization ability but acquires the ability to aggregate in the presence of Mg2+. At the same time, treatment of molluscan myosin with N-ethylmaleimide did not affect its polymerization. It was supposed that the effect of N-ethylmaleimide on myorod polymerization is related to the modification of the myorod SH domain containing Cys722.     

Proteolytic substructure of myorod,a thick filament protein of molluscan smooth muscles

Myorod (MR), a new thick filament protein of molluscan smooth muscles, is an alternatively spliced product of the myosin (Mn) heavy chain gene. We studied digestion of MR and Mn from the posterior adductor of Crenomytilus grayanus and the outer portion of adductor of Mizuchopecten (Patinopecten) yessoensis by papain and constructed the proteolytic substructure of MR, that is an analogue to Mn substructure. There are a head domain (analogue of Mn S1) and a rod domain (analogue of Mn rod); the junction between them is split at low ionic strength. The rod, in turn, consists of a neck domain (analogue of Mn S2) and a tail domain (identical to light meromyosin); the junction between them is split at high ionic strength. The localization and possible function of MR are discussed.     

Direction and speed of actin filaments moving along thick filaments isolated from molluscan smooth muscle

The active movement of fluorescence-labeled actin filaments along thick filaments isolated from molluscan smooth muscle was observed. Along a single thick filament, actin filaments moved toward the center of the thick filament at the speed of 1.19 +/- 0.38 microns s-1 (mean +/- SD, n = 42) and detached themselves from it upon reaching the central zone. Movement of actin also occurred in the opposite direction, i.e., away from the center, albeit at a much lower velocity (0.09 +/- 0.07 microns s-1, n = 17). Thus, the thick filament shows functional bipolarity in terms of velocity but does not determine the direction of the movement.     

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.     

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.     

X-ray structure analysis of thin filaments of a molluscan smooth muscle in the living relaxed state.

In the small-angle x-ray diffraction pattern of the living relaxed anterior byssus retractor muscle of Mytilus edulis, the thin filaments showed the following features. The 59.8-A reflection was much stronger and a little farther from the meridian than the 51.9-A reflection, although they are both contributions of the first-order Bessel function and are comparable with each other in the height from the equator. The 381-A reflection, given by the second-order Bessel function, was weaker than the 59.8-A reflection by more than the difference between the peak values of the first- and second-order Bessel functions, and was not so distant radially from the latter as estimated from the amount of peak shift brought about by the alteration of the Bessel order. A model of the thin filament was made on the basis of inverse Fourier transformation of the scattering amplitude, and the above features were explained by the characteristic shape of actin shown in this model. The actin subunits are elongated along the genetic left-hand helix with a pitch of 59.8 A, and are bonded together along the genetic helix in the inner part of the filament.     

Thin filaments of bivalve smooth muscle may contain a calponin-like protein

A novel 40-kDa calponin-like protein (CaP) was detected in thin filaments from catch muscles of the mussel Crenomytilus grayanus. The content of CaP in thin filaments depends on isolation conditions and varies from complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the CaP content in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22°C and optimal values of pH and ionic strength of the extraction solution, all CaP co-sediments with thin filaments. At 2°C it does not interact with thin filaments and remains in the supernatant. Like vertebrate smooth muscle calponin (33 kDa), the mussel CaP is thermostable, inhibits the Mg²⁺-ATPase activity of actomyosin, and can be phosphorylated, which is performed by endogenous (co-isolated) kinases in a Ca²⁺-independent manner. Thus, the C. grayanus CaP is a new member of the family of calponins, the function of which in muscle and nonmuscle cells is still obscure. We suggest that CaP is involved in Ca²⁺-independent regulation of smooth muscle contraction.     

Structure of molluscan thick filaments: a common origin for diverse appearances.

Thick filaments from the smooth adductor muscles of the oysters Ostrea edulis and Crassostrea angulata have been examined in the electron microscope after negative staining. The two well-known patterns of stain (whose origin and relation have been uncertain), one a series of transverse narrow lines at intervals of 144 Å along the filament axis and the other a regular two-dimensional arrangement of stained spots (Bear &; Selby, 1956), are found to be mutually interconvertible by rotating the grid around the filament axis. This is interpreted to mean that the spots are the projections of stained regions running through the filament in a common direction. Only when looking along this direction will the net pattern be seen with maximum clarity and sharpness. On rotation of the filament round its axis, the spots broaden transversely to the axis, overlapping and ultimately only the axial periodicity will remain. The structure is therefore not helical, but resembles a crystal lattice, although no period can be discerned normal to the net plane.The addition of 10 mm-EDTA to all solutions used in the filament preparation (except the stain), especially when ammonium molybdate is the stain employed, removes many puzzling appearances (probably caused by positive staining) which render the interpretation difficult. The appearance of the negatively stained filament can be related to the stain patterns in negatively stained paramyosin paracrystals (Cohen et al., 1971).     

Molecular organization of paramyosin in the core of molluscan thick filaments

An arrangement of paramyosin molecules in the polar part of molluscan thick filaments is proposed which accounts for the X-ray diffraction pattern of the smooth adductor muscle (other than the part ascribed to actin) and for the appearance of separated filaments in the electron microscope. The proposed structure is based on the PI arrangement of Cohen et al. (1971), and contains sets of parallel, equidistant molecules with successive molecules displaced along the molecular axis by 72 nm, which we call PI sheets. Every molecule belongs to two PI sheets which are nearly perpendicular. This array is not propagated throughout the filament, but is sheared periodically in the direction of the molecular (filament) axis by 2/5 X 72 nm. The shear occurs along parallel equidistant planes which are inclined to the PI sheets. The analysis of the X-ray data has been made possible by concentrating on those patterns from filaments in which the two sets of PI sheets appear to be mutually perpendicular, a condition brought about by bathing the muscle in aqueous acetone. In one set, there are four intermolecular spaces between shear planes (this appears to be true at least for the smooth adductors of Ostrea edulis, Crassostrea angulata and Mercenaria mercenaria). In the other set, the number varies with species and probably lies between eight and ten in the first two and appears to be six in the last named species. The known paracrystalline nature of paramyosin filaments suggests that this number, though dominant in one species, is not exactly constant.     

Filamin isoforms in molluscan smooth muscle

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.     

Thin filaments of molluscan catch muscles contain a calponin-like protein

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.     

Polarity of myofilaments in molluscan smooth muscle

Summary Myofilaments were isolated by gently homogenizing smooth muscle cells isolated from the pedal retractor muscle (PRM) of Mytilus edulis, and observed by electron microscopy. The thick filaments isolated in the presence of ATP (10–20 mM) had projections of myosin heads except near their centre (central bare zone). After extraction of myosin, the paramyosin core of the thick filaments showed a Bear-Selby net or a striated pattern with a main periodicity of 14.5 nm. Both the Bear-Selby net and the striated patterns had a polarity that reversed at the centre of the filament where the patterns were obscured. The thin filaments were attached to dense bodies. Decoration of the thin filaments with heavy meromyosin showed that they have opposite polarity on opposing sides of the dense body. The results indicate that the thick filaments are bipolar and also that the dense bodies are functionally analogous to the Z-disk of the striated muscle.     

Sequential disassembly of vertebrate muscle thick filaments

Native thick filaments from rabbit psoas muscle have been sequentially dissolved by incremental rises in salt concentration. Three quite separate stages of depolymerization can be detected; these presumably reflect constraints imposed on the disassembly process by variations in the packing of myosin and by the presence of other thick filament proteins.     

Intermediate filaments in smooth muscle

The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle. cytoskeleton; force development; vimentin; desmin     

Structure of Limulus telson muscle thick filaments

Computer analysis of electron micrographs of negatively stained thick filaments isolated from the telson levator muscle of the horseshoe crab (Limulus polyphemus) has shown that they have a four-stranded helical structure. The repeating units along each helix have a bent extended shape (measuring approximately 20 nm × 8 nm × 8 nm) and are inclined at an angle of about 30 ° to the helical path. At the resolution of this study, it was difficult to establish the exact size of the surface subunits, but our results are probably more consistent with each unit representing the two heads of a single myosin molecule rather than larger aggregates.