Homocysteinemia and endothelial damage after methionine load

Cellular titin (c-titin) colocalizes with myosin II in cytoskeletal structures containing actin in vivo and organizes highly ordered myosin bipolar filament arrays in the absence of actin in vitro. We report here that the actin-binding protein alpha-actinin associates with coassemblies of c-titin and myosin through direct interaction with c-titin. These results support the possibility that interaction between the myosin-associated protein c-titin and the actin-associated protein alpha-actinin organizes and stabilizes actin-myosin II cytoskeletal structures in vivo.     

Localization of myosin, actin, alpha-actinin, tropomyosin and vinculin in surface-activated, spreading human platelets

We observed the localization of the contractile proteins myosin, filamentous actin, alpha-actinin, tropomyosin, and vinculin in surface-activated, spreading human platelets using a single fluorescence staining procedure and conventional fluorescence microscopy. Myosin was distributed in a speckled pattern that extended radially from the granulomere. F-actin demonstrated cable-networks. Tropomyosin and alpha-actinin occurred in a punctuate distribution, and vinculin was localized at adhesion sites. Although myosin, F-actin, alpha-actinin, tropomyosin, and vinculin were not studied in resting platelets, our data support the idea that these contractile proteins are reorganized and reassembled in activated platelets during platelet function.     

Study of molecular mechanisms of muscle contraction using polarization fluorometry

The review summarizes results of studies on the conformational changes in contractile proteins during muscle contraction. The studies were carried out by polarized fluorescence technique in the UV and visible light. The revealed were alterations of actin and myosin in muscle fiber, taking place at various stages of contractile cycle. Transition from a weak binding state of actomyosin to a strong one was accompanied by F-actin subunit rearrangements, with C- and N-terminals moving relative to the core of thin filament. Myosin light chains and 20-kDa domain of myosin head moved in the same direction as C- and N-terminal regions of actin. The flexibility of actin filaments increased, whereas that of C- and N-terminal regions decreased sharply. Actin-myosin interaction changed dramatically tropomyosin flexibility and caused displacement of the protein relative to C- and N-terminals of actin. Actin structure "freezing" by glutaraldehyde or phalloidin, actin cleavage by subtilisin, as well as actin alteration in denervational atrophy inhibited markedly the intramolecular movement and isometric tension of muscle contraction. Besides, troponin-, caldesmon-, calponin-, and myosin-systems, regulating muscle contraction, modified actomyosin rearrangements in a Ca(2+)-dependent manner. The role of the movement of polypeptide chains in contractile proteins during muscle contraction is discussed.     

Identification and expression of the SOS response, aidB-like, gene in the marine sponge Geodia cydonium: implication for the phylogenetic relationships of metazoan acyl-CoA dehydrogenases and acyl-CoA oxidases

Lysophosphatidic acid (LPA) is an extracellular signaling molecule that can enter the central nervous system following injury or diseases that disrupt the blood-brain-barrier. Using a combination of time-lapse microscopy, immunocytochemistry, and biochemical techniques, we demonstrate that LPA stimulates profound changes in astrocyte morphology that are due to effects on the actomyosin cytoskeleton. Flat astrocytes in primary culture display prominent actin stress fibers. Treatment with the myosin light chain kinase inhibitor, ML-9, causes stress fiber dissolution and dramatic morphology changes including rounding of the cell body and the formation of processes. LPA can stabilize actin stress fibers and inhibit the morphology changes in ML-9-treated cells. Furthermore, this activity is dependent upon activation of the GTP-binding protein Rho as evidenced by the ability of C3 exoenzyme, a specific inhibitor of Rho, to block the effect. Phosphorylation of the regulatory light (RLC) chain initiates conformational changes in myosin II that result in the formation of myosin filaments and the recruitment of actin into contractile stress fibers. LPA-induced stabilization of stress fibers is accompanied by increases in phosphorylation of the RLC of myosin. Furthermore, astrocytes grown on flexible silicone undergo rapid contraction in response to LPA treatment. The forces generated by these cells manifest themselves as increased wrinkling in the silicone. The observed contraction and accompanying increases in regulatory light chain phosphorylation suggest that LPA-induced signaling cascades in astrocytes regulate actin/myosin interactions.     

Ultrastructural and immunohistochemical studies of Wharton's jelly umbilical cord cells

In order to determine the significance of Wharton's jelly, the characteristics of these cells were examined by means of electron microscopy and immunohistochemistry. These cells possessed ultrastructural characteristics of both fibroblasts and smooth muscle cells, indicating that they are modified, rather than typical fibroblasts. Immunohistochemically those 'myofibroblasts' stained positive for actin, non-muscle myosin, vimentin and desmin. Staining for muscle myosin was negative, supporting the ultrastructural findings. As our results indicate that these cells can function in both fibrogenesis and cell contraction, we speculate that they may contribute to the elasticity of Wharton's jelly, by synthesizing collagen fibers, and participate in the regulation of umbilical blood flow by virtue of their contractile properties.     

Distribution of attachment events relative to actin binding sites as evidenced in a bidirectional actomyosin interaction model

Optical trapping is one of the most evolving technologies that measures biophysical quantities and provides insights into some of the fundamental questions in the study of molecular motor proteins such as myosin. Several laboratories have successfully used this technique to observe and score nanometre-size displacements produced by myosin on interacting with actin. We have studied the distribution of attachment events for two myosin molecules with different orientations interacting with an actin filament within the framework of a Langevin-type bidirectional mathematical model. When myosin is detached from actin, our model predicts Brownian displacements centred at 0 +/- 8 nm (mean +/- SD, n = 251,058). When attached, the time-averaged displacements of the actin filament system produced step sizes with peaks of 8 +/- 6 nm (mean +/- SD, n = 22,174) (forward displacements) and -8 +/- 6 nm (mean +/- SD, n = 26,769) (reverse displacements). We infer from our results that the population distribution of attachment events is strongly dependent on (i) the magnitude of the Brownian displacements, (ii) the location of the actin binding sites relative to the myosin molecules, (iii) the orientation of the myosin molecules, and (iv) the relative kinetics (rate constants) for the forward and reverse displacement events.     

Structural invariance of constitutively active and inactive mutants of acanthamoeba myosin IC bound to F-actin in the rigor and ADP-bound states

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)-AMIA, AMIB, and AMIC-are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin's actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-A resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31 degrees on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998) J. Cell Biol. 141, 155-162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.     

Gender differences in correlates of disablement among the elderly in Egypt

Abnormal mechanical stress on pulmonary structures is associated with increased airway resistance and impaired gas exchange as a result of increased airway smooth muscle (ASM) deposition. Using an in vitro system with cultured ASM cells, we have demonstrated that cyclic deformational strain increases ASM cellular myosin and myosin light chain kinase. To determine if these contractile protein increases were accompanied by ultrastructural changes in cells indicating phenotypic modulation, cells subjected to strain were compared to cells grown under static conditions by transmission electron microscopy (TEM) and fluorescent staining. The strained ASM cells oriented perpendicular to the strain direction were more elongated and contained more actin stress fibers than identical cells grown under physically static conditions. The stress fiber bundles were thicker and reorganized parallel to the long axis of the cell. Marked increases in the numbers and lengths of focal adhesions between the cell membrane and the substratum were found by both TEM and immunostaining for talin. Mechanical strain thus increases organization of cytoskeletal elements in cultured ASM cells. Similar effects in vivo may serve to promote the expression of the contractile phenotype of cultured ASM cells independent of other in vivo factors and alter cell contractility. Increased organization of cytoskeletal elements might also increase the efficiency of signal transduction from the extracellular matrix into the cell interior.     

Observations on rat Sertoli ectoplasmic ('junctional') specializations in their association with germ cells of the rat testis

The ectoplasmic ('junctional') specialization, a subsurface modification of the Sertoli cell that is often seen facing germ cells, was studied in relation to the development and maturation of these germ cells. This structure is composed of subsurface bundles of filaments and more deeply placed endoplasmic reticulum. The data indicate that these subsurface modifications of Sertoli cells are reutilized in a cyclic fashion, being transferred from their position facing late spermatids to one opposing less mature germ cells. Ectoplasmic specializations appeared to function mechanically in grasping the heads of the spermatids which are undergoing the elongation and maturation phases of spermiogenesis rather than in actually attaching Sertoli cells to these germ cells. It is postulated that the ectoplasmic specialization imparts rigidity to that area of the Sertoli cell that surrounds the head region of the germ cell, forming a recess and a mantle by which the germ cell may be moved toward the base or toward the surface of the seminiferous epithelium. The observed linkage of microtubules to the cisternae of the complex provided a morphological basis for the changes in the cytoarchietecture of the Sertoli cell, which must accompany these movements.     

Right-handed rotation of an actin filament in an in vitro motile system

Muscle contraction occurs by mutual sliding between thick (myosin) and thin (actin) filaments. But the physical and chemical properties of the sliding force are not clear; even the precise direction of sliding force generated at each cross-bridge is not known. We report here the use of a recently developed in vitro motile assay system to show supercoiling of an actin filament in which the front part of the filament was fixed to a glass surface through cross-linked heavy-meromyosin and the rear part was able to slide on a track of heavy-meromyosin. A left-handed single turn of superhelix formed just before supercoiling, suggesting that the sliding force has a right-handed torque component that induces the right-handed rotation of an actin filament around its long axis. The presence of the torque component in the sliding force will explain several properties of the contractile system of muscle.     

Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures

The mouse Nedd5 gene encodes a 41.5-kD GTPase similar to the Saccharomyces and Drosophila septins essential for cytokinesis. Nedd5 accumulates near the contractile ring from anaphase through telophase, and finally condenses into the midbody. Microinjection of anti-Nedd5 antibody interferes with cytokinesis, giving rise to binucleated cells. In interphase and postmitotic cells, Nedd5 localizes to fibrous or granular structures depending on the growth state of the cell. The Nedd5-containing fibers are disrupted by microinjection of GTPgammaS and by Nedd5 mutants lacking GTP-binding activity, implying that GTP hydrolysis is required for its assembly. The Nedd5-containing fibers also appear to physically contact actin bundles and focal adhesion complexes and are disrupted by cytochalasin D, C3 exoenzyme, and serum starvation, suggesting a functional interaction with the actin-based cytoskeletal systems in interphase cells.     

Egg shells of mallophagans and anoplurans (Insecta: Phthiraptera): morphogenesis of specialized regions and the relation to F-actin cytoskeleton of follicular cells

The egg shells of investigated phthirapterans consist of three basic elements: an anterior operculum, a main egg shell and a posterior hydropyle. In some species these elements show further regional specializations: bristles and projections that facilitate attachment to feathers of the host, micropyles and aeropylar openings. All of the egg shell specializations are formed by distinct subpopulations of follicular cells. Staining with rhodamine-conjugated phalloidin has revealed that these subpopulations significantly differ in the distribution of microfilaments (F-actin). In this respect four morphological categories of the follicular cells have been distinguished: (1) cells devoid of processes and microvilli, with basal arrays of microfilaments, responsible for the secretion of a flat chorion; (2) cells devoid of processes and microvilli, separated by intercellular spaces, with basal arrays of microfilaments, responsible for the secretion of attachment structures; (3) cells equipped with actin-containing processes, responsible for the formation of micropyles or aeropyles, and (4) cells equipped with bundles of microvilli, responsible for the formation of hydropyles.     

Assembly mechanism of Dictyostelium myosin II: regulation by K+, Mg2+, and actin filaments

Regulated assembly of myosin II in Dictyostelium discoideum amoebae partially controls the orderly formation of contractile structures during cytokinesis and cell migration. Kinetic and structural analyses show that Dictyostelium myosin II assembles by a sequential process of slow nucleation and controlled growth that differs in rate and mechanism from other conventional myosins. Nuclei form by an ordered progression from myosin monomers to parallel dimers to 0.43 microns long antiparallel tetramers. Lateral addition of dimers to bipolar tetramers completes the assembly of short (0.45 microns) blunt-ended thick filaments. Myosin heads are not staggered along the length of tapered thick filaments as in skeletal muscle, nor are bipolar minifilaments formed as in Acanthamoeba. The overall assembly reaction incorporating both nucleation and growth could be kinetically characterized by a second-order rate constant (kobs,N+G) of 1.85 x 10(4) M-1 s-1. Individual rate constants obtained for nucleation, kobs,N = 4.5 x 10(3) M-1 s-1, and growth, kobs,G = 2.5 x 10(4) M-1 s-1, showed Dictyostelium myosin II to be the slowest assembling myosin analyzed to date. Nucleation and growth stages were independently regulated by Mg2+, K+, and actin filaments. Increasing concentrations of K+ from 50 to 150 mM specifically inhibited lateral growth of dimers off nuclei. Intracellular concentrations of Mg2+ (1 mM) accelerated nucleation but maintained distinct nucleation and growth phase kinetics. Networks of actin filaments also accelerated the nucleation stage of assembly, mechanistically accounting for spontaneous formation of actomyosin contractile fibers via myosin assembly (Mahajan et al., 1989). The distinct assembly mechanism and regulation utilized by Dictyostelium myosin II demonstrates that myosins from smooth muscle, striated muscle, and two types of amoebae form unique thick filaments by different pathways.     

Length-dependence of actin-myosin interaction in skinned cardiac muscle fibers in rigor

It has been suggested that the length dependence of myofilament Ca2+ sensitivity and of Ca2+ binding to troponin C, observed over the ascending limb of the cardiac force-length curve, is based on variation in the number of interacting cross-bridges. This interaction would be reduced at short sarcomere length as a consequence of double overlap of oppositely polarized actin filaments and increased lateral separation of actin and myosin filaments. Based on current evidence, it is not clear to what extent the actin-myosin interaction is hindered at sarcomere lengths where Ca2+ sensitivity is reduced. We have used two biochemical assays to assess cross-bridge attachment in rigor muscle at sarcomere lengths corresponding to the ascending limb of the cardiac force-length curve. These are based on (1) the inhibition of K+-activated myosin ATPase by the complexation of actin with myosin, and (2) the enhancement of Ca2+ binding to troponin C by rigor bridge attachment to actin. Measurements were made with skinned fibers from bovine ventricle. As a check on our method, measurements were also made with skinned rabbit psoas muscle fibers. With both muscle types, a reduction in sarcomere length along the ascending limb of the force-length curve was associated with an increase in K+-activated ATPase activity and a reduction in Ca2+ binding to the regulatory sites of troponin C. These results indicate that actin-myosin interaction is significantly reduced at short sarcomere length.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Mechanics of active contraction in cardiac muscle: Part I--Constitutive relations for fiber stress that describe deactivation

Constitutive relations for active fiber stress in cardiac muscle are proposed and parameters are found that allow these relations to fit experimental data from the literature, including the tension redeveloped following rapid deactivating length perturbations. Contraction is driven by a length-independent free calcium transient. The number of actin sites available to react with myosin is determined from the total number of actin sites (available and inhibited), free calcium and the length history-dependent association and dissociation rates of two Ca2+ ions and troponin as governed by a first-order, classical kinetics, differential equation. Finally, the relationship between active tension and the number of available actin sites is described by a general cross-bridge model. Bridges attach in a single configuration at a constant rate, the force within each cross-bridge varies linearly with position, and the rate constant of bridge detachment depends both on position and time after onset of contraction. In Part II, these constitutive relations for active stress are incorporated in a continuum mechanics model of the left ventricle that predicted end-systolic transmural strain distributions as observed experimentally.     

Contents of erythorbic acid in the tissues of guinea pigs intraperitoneally administered erythorbic acid

The peripheral distribution of Sertoli cell F-actin, a cytoskeletal protein found in Sertoli cell ectoplasmic specializations, is associated with enhanced spermatid binding to the Sertoli cell and, as such, serves as a functional marker for its acquisition of binding competency. Previous studies suggest that the peripheral distribution of actin is dependent on follicle-stimulating hormone (FSH). To investigate the developmental pattern of Sertoli cell actin distribution in relation to peripubertal FSH and testosterone levels, we examined epithelial sheets from 2-8 week-old-rats. Tissues were processed for light microscopy and for the visualization of rhodamine-labeled F-actin. At 2 weeks, actin staining was diffuse throughout most of the Sertoli cells and was similar to that observed in binding-incompetent Sertoli cells. By 4 weeks, actin distribution was peripheral, acquiring the same staining pattern as observed in binding-competent Sertoli cells. Serum levels of FSH peaked at 4 weeks and declined to adult levels thereafter. Testosterone levels did not increase significantly until 6 weeks. Results show that Sertoli cell actin undergoes peripheral reorganization concurrent with the peripubertal peak of FSH but prior to the peripubertal rise of testosterone. The study demonstrates a temporal correlation between the peripubertal FSH rise and the actin redistribution in Sertoli cells that is consistent with an induction of this redistribution by FSH. These results suggest that FSH induces binding competency in Sertoli cells.     

ATPase activity of myosin in hair bundles of the bullfrog's sacculus

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.