Properties of filament-bound myosin light chain kinase. (1/3815)

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.  (+info)

RhoA activity is required for fibronectin assembly and counteracts beta1B integrin inhibitory effect in FRT epithelial cells. (2/3815)

FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells. Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous beta1A integrin and is inhibited by the expression of the dominant-negative beta1B subunit. We tested the hypotheses that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract beta1B integrin inhibitory effect. FRT-beta1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme. Both treatments inhibited fibronectin assembly and caused loss of actin microfilaments and adhesion plaques. On the other hand, FRT-beta1B cells were transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E. coli cytotoxic necrotizing factor 1, which directly activates RhoA. Either treatment restored microfilament and adhesion plaque assembly and promoted fibronectin fibril organization. A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-beta1B cells with TGF-beta. Our data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of beta1B integrin.  (+info)

Filament assembly from profilin-actin. (3/3815)

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 microM under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 microM under physiological conditions. The PAcov polymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.  (+info)

Origin of contractile dysfunction in heart failure: calcium cycling versus myofilaments. (4/3815)

BACKGROUND: Chronic congestive heart failure is a common, often lethal disorder of cardiac contractility. The fundamental pathophysiology of the contractile failure remains unclear, the focus being on abnormal Ca2+ cycling despite emerging evidence for depressed myofilament function. METHODS AND RESULTS: We measured intracellular Ca2+ concentration ([Ca2+]i) and contractile force in intact ventricular muscle from SHHF rats with spontaneous heart failure and from age-matched controls. At physiological concentrations of extracellular Ca2+ ([Ca2+]o), [Ca2+]i transients were equal in amplitude in the 2 groups, but [Ca2+]i peaked later in SHHF muscles. Twitch force peaked slowly and was equivalent or modestly decreased in amplitude relative to controls. Steady-state analysis revealed a much greater (53%) depression of maximal Ca2+-activated force in SHHF muscles, which, had other factors been equal, would have produced an equivalent suppression of twitch force. Phase-plane analysis reveals that the slowing of Ca2+ cycling prolongs the time available for Ca2+ to activate the myofilaments in failing muscle, partially compensating for the marked dysfunction of the contractile machinery. CONCLUSIONS: Our results indicate that myofilament activation is severely blunted in heart failure, but concomitant changes in [Ca2+]i kinetics minimize the contractile depression. These results challenge prevailing concepts regarding the pathophysiology of heart failure: the myofilaments emerge as central players, whereas changes in Ca2+ cycling are reinterpreted as compensatory rather than causative.  (+info)

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvbeta1.1 subunits modulated by G protein beta gamma subunits. (5/3815)

Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.  (+info)

Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers. (6/3815)

The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesion-mediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN-/-) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis. FN-/- fibroblasts plated on the cell-binding domain of fibronectin or on antibodies directed against mouse beta1 integrin chains attach but fail to spread and do not form focal adhesions or actin stress fibers. When such cells are treated with antibodies directed against the ectodomain of mouse syndecan-4, they spread fully and assemble focal adhesions and actin stress fibers indistinguishable from those seen in cells plated on intact fibronectin. These results identify syndecan-4 as a heparan sulfate proteoglycan involved in the assembly process. The antibody-stimulated assembly of focal adhesions and actin stress fibers in cells plated on the cell-binding domain of fibronectin can be blocked with C3 exotransferase, an inhibitor of the small GTP-binding protein Rho. Treatment of cells with lysophosphatidic acid, which activates Rho, results in full spreading and assembly of focal adhesions and actin stress fibers in fibroblasts plated on the cell-binding domain of fibronectin. We conclude that syndecan-4 and integrins can act cooperatively in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. We conclude further that these joint signals are regulated in a Rho-dependent manner.  (+info)

Radiation induced endothelial cell retraction in vitro: correlation with acute pulmonary edema. (7/3815)

We determined the effects of low dose radiation (<200 cGy) on the cell-cell integrity of confluent monolayers of pulmonary microvascular endothelial cells (PMEC). We observed dose- and time-dependent reversible radiation induced injuries to PMEC monolayers characterized by retraction (loss of cell-cell contact) mediated by cytoskeletal F-actin reorganization. Radiation induced reorganization of F-actin microfilament stress fibers was observed > or =30 minutes post irradiation and correlated positively with loss of cell-cell integrity. Cells of irradiated monolayers recovered to form contact inhibited monolayers > or =24 hours post irradiation; concomitantly, the depolymerized microfilaments organized to their pre-irradiated state as microfilament stress fibers arrayed parallel to the boundaries of adjacent contact-inhibited cells. Previous studies by other investigators have measured slight but significant increases in mouse lung wet weight >1 day post thoracic or whole body radiation (> or =500 cGy). Little or no data is available concerning time intervals <1 day post irradiation, possibly because of the presumption that edema is mediated, at least in part, by endothelial cell death or irreversible loss of barrier permeability functions which may only arise 1 day post irradiation. However, our in vitro data suggest that loss of endothelial barrier function may occur rapidly and at low dose levels (< or =200 cGy). Therefore, we determined radiation effects on lung wet weight and observed significant increases in wet weight (standardized per dry weight or per mouse weight) in < or =5 hours post thoracic exposure to 50 200 cGy x-radiation. We suggest that a single fraction of radiation even at low dose levels used in radiotherapy, may induce pulmonary edema by a reversible loss of endothelial cell-cell integrity and permeability barrier function.  (+info)

Locomotory behaviour of epitheliocytes and fibroblasts on metallic grids. (8/3815)

Behaviour of epitheliocytes and fibroblasts on special discontinuous substrata (metallic grids with square openings of 45x45 microm2) was examined in order to compare the ability of these cells to spread in two mutually perpendicular directions and to stretch over the void spaces. Two cell types with typical fibroblastic morphology, the AGO 1523 line of human foreskin fibroblasts and secondary cultures of mouse embryo fibroblasts, and three cell types with typical epithelial morphology, primary mouse hepatocytes, the IAR-2 line of rat liver cells and the MDCK line of canine kidney epithelial cells (clone 20) were used. We also examined the epitheliocytes (MDCK cells, clone 20) transformed to fibroblast-like morphology by treatment with hepatocyte growth factor/scatter factor (HGF/SF). Time-lapse video microscopy, scanning electron microscopy and immunofluorescence microscopy were used to examine cell reorganizations at various stages of spreading. It was found that early stages of spreading of fibroblasts and epitheliocytes were similar: the cell spread along two bars, perpendicular to each other (bar and crossbar), with the formation of a small triangular lamellar cytoplasm stretched over the opening. Later central parts of the bodies of the fibroblasts retracted from the bars so that the cells remained attached only by their polar lamellae. Successive expansions and partial retractions of these lamellae led to elongation of the cell body crossing several openings of the grid. Epitheliocytes, in contrast to fibroblasts, at the late stages of spreading did not retract their bodies and did not contract polar lamellae. As a result, their central lamellae stretched progressively over the openings. As a result of the treatment of MDCK epitheliocytes with HGF/SF the behaviour of the cells on the grids became similar to that of fibroblasts. It is suggested that these distinct spreading patterns of epitheliocytes and fibroblasts are due to the type-specific differences in the actin-myosin cortex. Experiments with microtubule-specific drugs, colcemid and taxol, indicate that the organization of this cortex is under microtubular control.  (+info)

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