Myosin ATPase Research Articles

Interaction of Photochromic Nucleotide Analogue with Nucleotide Required Bio-Molecular Machines

Previously, we have synthesized photochromic ATP analogue Phenylazobenzoyl-iminoethyl-Tri-Phosphate (PABITP) that change its structure reversibly by light irradiation in order to photo-regulate the function of myosin II. PABITP was hydrolyzed by skeletal muscle myosin and induced dissociation of acto-myosin. X-ray small angle solution scattering analysis of myosin interacting with PABITP showed that the radius of gyration values of S1•PABITP(trans) and S1•PABITP(cis) are identical to those of S1•ATP and S1•ADP respectively. The results suggested that the photo-isomerization of the PABITP induce conformational change of myosin head, which may reflect energy transduction. In this study, we examined the possible application of PABITP as a nucleotide analogue to other nucleotide required bio-molecular machines, smooth muscle myosin, myosin V, conventional kinesin, Kif18A, Eg5, tubulin and small G-protein Ras. PABITP was hydrolyzed by these kinesin and myosin ATPase. PABITP induced microtubule gliding on conventional kinesin but not induced actin gliding on skeletal muscle myosin. PABITP induced polymerization of tubulin to microtubules. We also examined the effect of reversible photo-isomerization of PABITP for Ras and actin.

S-Nitrosylation Decreases Ca2+ Sensitivity and Actomyosin ATPase Activity of Contractile Proteins in Cardiac Myofibrils

Increases in muscle nitric-oxide (NO) production can be buffered by reaction with intracellular glutathione, forming S-nitrosoglutathione (GSNO). GSNO has been shown to S-nitrosylate Cys thiols of cardiac contractile proteins in vivo and in vitro, but effects on maximal force, thin-filament Ca2+ sensitivity and actomyosin ATPase activity are unknown. Here, we analyzed the targets of S-nitrosylation in mouse cardiac contractile proteins, and examined the effects of these modifications on function in myocytes and skinned cardiac myofibrils. S-Nitrosylation and denitrosylation were detected using resin-assisted capture (SNO-RAC) and targets for S-nitrosylation were identified by quantitative LC-MS/MS. Isolated cardiomyocytes treated with S-nitrosocysteine (CysNO, 500μM, 10min) showed an increase in total protein-SNO, followed by progressive denitrosylation (30-60min with CysNO). At 10min, CysNO dose-dependently increased S-nitrosylation of specific Cys thiols in myosin heavy chain, actin, TnC, TnI, myosin-binding protein C and other muscle proteins. Myofibril thin-filament Ca2+ sensitivity decreased (P 0.05). Loss of Ca2+ sensitivity was partially reversed by the denitrosation agent, ascorbate. Relaxation kinetics of skinned fibers, as measured by flash photolysis, were also significantly reduced by100μM GSNO (k1,15.33 to 11.68/s; k2 2.33 to 0.87/s; fit to double exponential). Maximal myofibrillar ATPase activity (pCa 5.0) was also dose-dependently inhibited (8, 15, 30%) by 50, 100 and 500μM GSNO, an effect that was reversed by ascorbate. The findings suggest that S-nitrosylation of regulatory Cys thiol(s) can reversibly modulate cardiac muscle contraction and may be able to protect the heart by reducing myosin ATPase demand without affecting the maximal force of contraction.

Coordination of Actin-Activated Phosphate Release and the Myosin Power-Stroke Detected by Transient Time-Resolved FRET

We have used transient kinetics and nanosecond time-resolved FRET to resolve a structural transition in the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with force-transducing rotation of the myosin light chain domain. Previous research in the absence of actin used optical and EPR spectroscopy to demonstrate that the relay helix of Dictyostelium Discoideum myosin II exists in dynamic equilibrium between a bent pre-power-stroke state, stabilized by ATP, and a straight post-power-stroke state that predominates when ADP or no nucleotide is bound. Kinetics studies showed that ATP binding induces a recovery stroke in which this equilibrium is shifted toward the bent state. In the present study, we ask whether actin binding to the myosin.ADP.Pi complex reverses this transition and if so, how actin-activated helix straightening is coordinated with biochemical transitions of the myosin ATPase cycle. We labeled a Cys-lite Dictyostelium myosin motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We measured time-resolved FRET following stopped-flow mixing of myosin (in the presence of ATP) with actin. These experiments allowed us to measure the structural kinetics of the relay helix during the weak-to-strong actin-binding transition. The results clearly show that actin binding straightens the relay helix in the presence of ATP. The helix straightens prior to ADP dissociation and full formation of the strong binding actomyosin interface. Measurements of actin-activated phosphate release demonstrate that dissociation of the hydrolyzed gamma-phosphate is closely coupled to the structural state of the relay helix during the power-stroke.

CGMP-dependent Protein Kinase Iβ Regulates Breast Cancer Cell Migration and Invasion via a Novel Interaction with the Actin/Myosin-associated Protein Caldesmon

The two isoforms of type I cGMP-dependent protein kinase (PKGIα and PKGIβ) differ in their first ∼100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein-protein interactions. Using the PKGIβ dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGIβ-specific binding protein. PKGIβ phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGIβ and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGIβ enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGIβ in human breast cancer cells, suggesting that PKGIβ is a potential target for breast cancer treatment.

How Muscle Exploits the Steady State to Regulate Contraction

Activation of muscle requires myosin binding. The active state of muscle is stabilized by high affinity rigor bonds at equilibrium but, the steady-state catalytic cycle of myosin ATPase decreases the lifetime of the myosin interaction by several orders of magnitude. Low myosin affinity could be a justification for including Ca2+ binding states in a model of activation. Our work supports a simple model for steady state activation, namely, the regulatory protein tropomyosin (Tm) exists in just two states, ground (C) and excited (M), separated by an energy barrier which can be overcome by interaction with myosin. The elongated shape of Tm allows for multiple potential myosin binding partners (Ui). During its strong binding phase of the catalytic cycle, any one myosin (U1) combines with C to form M by an equilibrium pathway. At time, t=0, M probability, PM=1. Although U1 most likely decays by a non-equilibrium rate (i.e., ATP binding), the lifetime of PM is constant. For t>0, before M decays to C, interaction with Ui in the strong binding phase can restore PM=1. We show that an analytical function (M function) is fully derived from the combination of equilibrium and steady state pathways and the additional opportunities (second chances) to restore PM=1 depend on the rate at which Ui transitions into the strong binding phase. When Ca2+ is introduced as a mass action regulator of the U or C supply, the M function takes the general form of the Hill equation, which is widely employed for fitting the steady state response of muscle to Ca2+. We show by simulations that stochastic events predicted by a second chance mechanism are consistent with solutions to the M function. Our results justify kinetic experiments that focus specifically on M.

1P141 アクチン重合、ミオシンATP加水分解活性化に対するTyr143変異の効果(10.筋肉,ポスター,日本生物物理学会年会第51回(2013年度))

1P141 アクチン重合、ミオシンATP加水分解活性化に対するTyr143変異の効果(10.筋肉,ポスター,日本生物物理学会年会第51回(2013年度))

Activation and Inhibition of F-Actin and Cardiac Thin Filaments by the N-Terminal Domains of Cardiac Myosin Binding Protein C

Myosin binding protein-C (MyBPC) has been known for over thirty years to inhibit actomyosin ATP hydrolysis and more recently has been shown to affect the calcium sensitivity of force in muscle fibers. The various domains of MyBPC have previously been shown to have complex interactions with other myofibrillar proteins. The C-terminal domains have been shown to bind to the thick filament and the N-terminal domains interact with the S2 region of myosin and with f-actin. The number of mutations in cardiac MyBPC (cMyBPC) are the second most prevalent of sarcomeric proteins in producing cardiomyopathies. We have used steady state kinetics to study the molecular mechanism by which the soluble N-terminal C0C1, C1C2 and C0C2 domains of mouse and human cMyBPC affect the activation of myosin ATP hydrolysis by f-actin and native porcine thin filaments.The N-terminal domains of cMyBPC inhibit the activation of the steady state ATPase myosin by f-actin. However, in the presence of native cardiac thin filaments.the N-terminal domains C1C2 and C0C2 of mouse and human cMyBPC produce biphasic effects on the steady state ATP hydrolysis of cardiac myosin-S1. That is, ATPase is activated at low ratios of cMyBPC-N-terminal domain to thin filament and is inhibited by higher ratios similar to the effects observed with f-actin. These data suggest that low ratios of cMyBPC N-terminal domains activate thin filaments in a mechanism similar to that of rigor myosin-S1 but higher ratios inhibit the ATPase rate by competing with myosin-S1-ADP-Pi binding to actin and thin filaments. Effects also appear species-dependent since the C0C1 domains of human cMyBPC produce similar effects as C1C2 and C0C2 on ATPase in the presence of thin filaments, but mouse C0C1 does not produce significant activation.

Rho‐kinase mediates diphosphorylation of myosin regulatory light chain in cultured uterine, but not vascular smooth muscle cells

Phosphorylation of myosin regulatory light chain (RLC) triggers contraction in smooth muscle myocytes. Dephosphorylation of phosphorylated RLC (pRLC) is mediated by myosin RLC phosphatase (MLCP), which is negatively regulated by rho-associated kinase (ROK). We have compared basal and stimulated concentrations of pRLC in myocytes from human coronary artery (hVM), which has a tonic contractile pattern to myocytes from human uterus (hUM), which has a phasic contractile pattern. Our studies reveal fundamental differences between hVM and hUM regarding the mechanisms regulating phosphorylation RLC. Whereas hVM responded to stimulation by phosphorylation of RLC at S19, hUM responded by forming diphosphorylated RLC (at T18 and S19; ppRLC), which, compared to pRLC, causes two to threefold greater activation of myosin ATPase that provides energy to power the contraction. Importantly, the conversion of pRLC to ppRLC is mediated by ROK. In hUM, MLCP has high activity for ppRLC and this is inhibited by ROK through phosphorylation of the substrate targeting subunit (MYPT1) at T853. Inhibitors of ROK significantly reduce contractility in both hVM and hUM. We demonstrated that inhibition of ppRLC in phasic myocytes (hUM) is 100-fold more sensitive to ROK inhibitors than is pRLC in tonic myocytes (hVM). We speculate that these differences in phosphorylation of RLC might reflect evolution of different contractile patterns to perform distinct physiological functions. Furthermore, our data suggest that low concentrations of ROK inhibitors might inhibit uterine contractions with minimal effects on vascular tone, thus posing a novel strategy for prevention or treatment of conditions such as preterm birth.

Mn2+–Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance

Pulsed electron paramagnetic resonance at the microwave K(a) band (~30 GHz) was used to study the coordination of adenosine nucleotides to Mn(2+) at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electron-nuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mn·nucleotide complexes. Therefore, to directly detect binding of the Mn·nucleotide to myosin, we used nonhydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electron-electron double resonance to detect spin probe-manganese dipolar interaction. We found that under substoichiometric conditions, both Mn·AMPPNP and Mn·ADP·AlF(4) form a complex with myosin, and Mn·ADP does not form such a complex. This correlates well with the biological dissociation of Mg·ADP from myosin after the hydrolysis of ATP. The analysis of (31)P ENDOR spectra reveals that in Mn·AMPPNP, Mn·ATP, and Mn·ADP at myosin or in solution, the nucleotide is coordinated to Mn(2+) by two phosphate groups, whereas in Mn·ADP·AlF(4), only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of Mn·ADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ions is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metal-protein binding when such binding does not provide reliable spectroscopic signatures.

Determination of Muscle Fiber Type in Rodents

Skeletal muscles consist of muscle fibers that can differ in both composition and functional characteristics. These three types of muscle fibers, broadly categorized as slow, fast IIa, and fast IIb muscle fibers, express characteristic myosin heavy chain proteins and have different metabolic and enzymatic activities, which can be used as surrogate markers to identify the different fiber types. Pathological changes affecting the muscle, such as denervation, muscle disuse, and atrophy not only manifest on a functional level, but also as marked changes in the composition of muscle fiber type of individual muscles. In this unit we describe three methods for histological identification of slow/type I, fast fatigue resistant/type IIa, and fast fatigable/type IIb fibers by staining for either myosin ATPases or oxidative enzyme capacity (succinate dehydrogenase, SDH)-or, alternatively, immunostaining for specific myosin heavy chain isoforms in muscles of mouse hindlimbs. Curr. Protoc. Mouse Biol. 2:231-243 © 2012 by John Wiley & Sons, Inc.

In ovoleptin administration accelerates post‐hatch muscle growth and changes myofibre characteristics, gene expression and enzymes activity in broiler chickens

To evaluate the effect of maternal leptin on muscle growth, we injected 0 μg (control, CON), 0.5 μg (low leptin dose, LL) or 5.0 μg (high leptin dose, HL) of recombinant murine leptin dissolved in 100 μl of PBS into the albumen of broiler eggs prior to incubation. The newly hatched chicks were all raised under the same conditions until 21 days of age (D21), when body weight was measured and samples of gastrocnemius muscle were collected and weighed. Myosin ATPase staining was applied to identify myofibre types and measure the cross-sectional area (CSA) of myofibres. Real-time PCR was performed to quantify leptin receptor (LEPR), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor (GHR) and myostatin (MSTN) mRNA expression in the gastrocnemius muscle. The activity of calpains (CAPNs) in the gastrocnemius muscle was measured using a quantitative fluorescence detection kit. Male chickens treated with both high and low doses of leptin had significantly higher (p < 0.05) body weight on D21. The high leptin significantly increased the CSA (p < 0.05) of gastrocnemius muscle in male chickens, which coincided with a 93% increase (p < 0.05) in IGF-1 mRNA expression. Likewise, the LL dose increased the weight of gastrocnemius muscle in male chickens (p < 0.05), which was accompanied by a 41% down-regulation (p < 0.05) of MSTN mRNA expression and a decreased activity of CAPNs. However, all these changes were not observed in female chickens. The proportion of myofibre types did not altered. No significant change was detected for LEPR and GHR mRNA expression. These results indicate that in ovo leptin treatment affects skeletal muscle growth in chickens in a dose-dependent and sex-specific manner. The altered expression of IGF-1, MSTN mRNA and activity of CAPNs in skeletal muscle may be responsible for such effects.

The velocity of cardiac sarcomere shortening: mechanisms and implications.

A classical paper published by Michael Barany almost 50years ago demonstrated a tight correlation between the mechanical parameter of maximal velocity of shortening and the biochemical parameter of myosin ATPase activity in a wide spectrum of species. Here, we review the determinants of muscle dynamics by mechanical load and the relation between sarcomere shortening velocity and cross-bridge dynamics in rat myocardium containing a range of fast and slow myosin. Observations from molecular level to mechanics of the intact human heart suggest that cardiac actin-myosin kinetic properties are matched so as to optimize myocardial strain rate and allow for the maximum rate of hydraulic energy output observed during ejection in the whole ventricle.

Disease causing mutations of troponin alter regulated actin state distributions

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin-tropomyosin-troponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50% of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Tension cost correlates with mechanical and biochemical parameters in different myocardial contractility conditions

OBJECTIVES:Tension cost, the ratio of myosin ATPase activity to tension, reflects the economy of tension development in the myocardium. To evaluate the mechanical advantage represented by the tension cost, we studied papillary muscle contractility and the activity of myosin ATPase in the left ventricles in normal and pathophysiological conditions.METHODS:Experimental protocols were performed using rat left ventricles from: (1) streptozotocin-induced diabetic and control Wistar rats; (2) N-nitro-L-arginine methyl ester (L-NAME) hypertensive and untreated Wistar rats; (3) deoxycorticosterone acetate (DOCA) salt-treated, nephrectomized and salt- and DOCA-treated rats; (4) spontaneous hypertensive rats (SHR) and Wistar Kyoto (WKY) rats; (5) rats with myocardial infarction and sham-operated rats. The isometric force, tetanic tension, and the activity of myosin ATPase were measured.RESULTS:The results obtained from infarcted, diabetic, and deoxycorticosterone acetate-salt-treated rats showed reductions in twitch and tetanic tension compared to the control and sham-operated groups. Twitch and tetanic tension increased in the N-nitro-L-arginine methyl ester-treated rats compared with the Wistar rats. Myosin ATPase activity was depressed in the infarcted, diabetic, and deoxycorticosterone acetate salt-treated rats compared with control and sham-operated rats and was increased in N-nitro-L-arginine methyl ester-treated rats. These parameters did not differ between SHR and WKY rats. In the studied conditions (e.g., post-myocardial infarction, deoxycorticosterone acetate salt-induced hypertension, chronic N-nitro-L-arginine methyl ester treatment, and streptozotocin-induced diabetes), a positive correlation between force or plateau tetanic tension and myosin ATPase activity was observed.CONCLUSION:Our results suggest that the myocardium adapts to force generation by increasing or reducing the tension cost to maintain myocardial contractility with a better mechanical advantage.

Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice

Aerobic exercise training (AET) is an important mechanical stimulus that modulates skeletal muscle protein turnover, leading to structural rearrangement. Since the ubiquitin-proteasome system (UPS) and calpain system are major proteolytic pathways involved in protein turnover, we aimed to investigate the effects of intensity-controlled AET on the skeletal muscle UPS and calpain system and their association to training-induced structural adaptations. Long-lasting effects of AET were studied in C57BL/6J mice after 2 or 8 wk of AET. Plantaris cross-sectional area (CSA) and capillarization were assessed by myosin ATPase staining. mRNA and protein expression levels of main components of the UPS and calpain system were evaluated in plantaris by real-time PCR and Western immunoblotting, respectively. No proteolytic system activation was observed after 2 wk of AET. Eight weeks of AET resulted in improved running capacity, plantaris capillarization, and CSA. Muscle RING finger-1 mRNA expression was increased in 8-wk-trained mice. Accordingly, elevated 26S proteasome activity was observed in the 8-wk-trained group, without accumulation of ubiquitinated or carbonylated proteins. In addition, calpain abundance was increased by 8 wk of AET, whereas no difference was observed in its endogenous inhibitor calpastatin. Taken together, our findings indicate that skeletal muscle enhancements, as evidenced by increased running capacity, plantaris capillarization, and CSA, occurred in spite of the upregulated UPS and calpain system, suggesting that overactivation of skeletal muscle proteolytic systems is not restricted to atrophying states. Our data provide evidence for the contribution of the UPS and calpain system to metabolic turnover of myofibrillar proteins and skeletal muscle adaptations to AET.

A novel actin binding site of myosin required for effective muscle contraction

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Collagen Organization Critical Role in Wound Contraction

Open wound closure by wound contraction produces a healed defect made up mostly of dermis. Generating thicker collagen fibers condenses granulation tissue, which pulls surrounding skin into the defect. What is the mechanism for open wound contraction? Is it through the generation of contractile force using sustained myosin ATPase, thus causing cell contraction or by rapid myosin ATPase that condenses collagen fibrils into fibers? The mechanism for wound contraction is not often debated after the discovery of the myofibroblast. Myofibroblasts are the major cell phenotype in maturing granulation tissue. It is concluded, not quite accurately, that myofibroblasts are responsible for wound contraction. As wound contraction progresses, polarized light microscopy reveals birefringence patterns associated with ever-increasing thickening of collagen fibers. Collagen fibers thicken by eliminating water between fibrils. Wound contraction requires collagen synthesis and granulation tissue compaction. Both myofibroblasts and fibroblasts synthesize collagen, but fibroblasts, not myofibroblasts, compact collagen. Free-floating fibroblast-populated collagen lattices (FPCL) contract by rapid myosin ATPase, thus resulting in thicker collagen fibers by elongated fibroblasts. The release of an attached FPCL, using sustained myosin ATPase, produces rapid lattice contraction, now populated with contracted myofibroblasts in the absence of thick collagen fibers. In vivo and in vitro studies show that rapid myosin ATPase is the motor for wound contraction. Myofibroblasts maintain steady mechano-tension through sustained myosin ATPase, which generates cell contraction forces that fail to produce thicker collagen fibers. The hypothesis is that cytoplasmic microfilaments pull collagen fibrils over the fibroblast's plasma membrane surface, bringing collagen fibrils in closer contact with one another. The self-assembly nature of collagen fixes collagen fibrils in regular arrays generating thicker collagen fibers. Wound contraction progresses through fibroblasts generating thicker collagen fibers, using tractional forces; rather than by myofibroblasts utilizing cell contraction forces.

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA–ROCK–myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.

Manganese Cation Reports on the Number of Coordinated Phosphates from the Nucleotide at the Active Site in Myosin ATPase

ATP is an important cofactor for myosin ATPase. ATP binding initiates myosin conformation change (recovery stroke), priming it for force generation. In the presence of a divalent cation (usually Mg2+), myosin hydrolyzes ATP to ADP and phosphate, and their subsequent release presumably triggers the power stroke in actomyosin. What is the role of ATP hydrolysis in myosin? What is the relation between the kinetics of ATP hydrolysis and those of myosin conformation changes? To address these problems, we propose to use pulsed electron paramagnetic resonance (EPR) techniques and study the coordination environment of the divalent cation complexed with a nucleotide at the myosin active site. To enable the EPR investigations, Mn2+ can be used instead of Mg2+ without interruption of the myosin ATPase activity. In this preliminary work, we studied the myosin.Mn complexes with non-hydrolyzable nucleotide analogs, AMPPNP (triphosphate) and ADP.AlF4 (diphosphate), as well as Mn.nucleotide complexes without myosin. Mn2+ binding to myosin was monitored by pulsed electron-electron double resonance (ELDOR), which detected the magnetic dipole interaction between Mn2+ and a nitroxide spin probe attached to the A639C myosin mutant. The number of coordinated phosphates in myosin.Mn.AMPPNP complex determined by 31P pulsed electron-nuclear double resonance (ENDOR) was twice that detected for the myosin.Mn.ADP.AlF4 complex. These results demonstrate the potential of pulsed EPR for distinguishing between the myosin.ATP and myosin.ADP biochemical states and study the role of ATP hydrolysis in myosin structural kinetics.