Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 °C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

Expression of non-muscle myosin isoforms in rabbit myometrium is estrogen-dependent

The putatative effects of different estrogen levels on the expression of non-muscle myosin isoforms in rabbit myometrium have been investigated using three monoclonal anti-platelet myosin heavy chain (MyHC) antibodies (NM-F6, NM-G2, and NM-A9). Western blotting analysis of proteolytic digests of human platelet actomyosin indicates that these antibodies are specific for three distinct epitopes. Comparative immunofluorescence tests on cultered human fibroblasts with polyclonal sequence-specific anti-MyHCA antibody suggest that the patterns of NM-F6, NM-.G2 and NM-A9, although similar, do not overlap with that of type-A MyHC. Distribution of NM myosin isoforms has been studied in indirect immunofluorescence assays using cryosections of tissues from rabbits at various stages of development, pregnancy, or from ovariectomized, 17#-estradiol-treated ovariectomized, and human chorionic gonadotropin-treated animals. Non-muscle myosin antigenicity is still present in the myometrium when the female becomes sexually competent. The immunoreactivity of non-muscle myosin for NM-F6 is steroid-independent, since it does not change with pregnancy or ovariectomy, but that of NM-G2 is estrogen-dependent; the latter disappears during pregnancy and in ovariectomized animals treated with estradiol, whereas it is expressed in ovariectomized rabbits. Although non-muscle myosin immunoreactivity for NM-A9 is detectable under all the experimental conditions, it can assume different patterns of intracellular distribution in vitro (punctate vs filamentous), depending on culture conditions and the presence of estrogens.     

Distinct and redundant roles of the non-muscle myosin II isoforms and functional domains

We propose that the in vivo functions of NM II (non-muscle myosin II) can be divided between those that depend on the N-terminal globular motor domain and those less dependent on motor activity but more dependent on the C-terminal domain. The former, being more dependent on the kinetic properties of NM II to translocate actin filaments, are less amenable to substitution by different NM II isoforms, whereas the in vivo functions of the latter, which involve the structural properties of NM II to cross-link actin filaments, are more amenable to substitution. In light of this hypothesis, we examine the ability of NM II-A, as well as a motor-compromised form of NM II-B, to replace NM II-B and rescue neuroepithelial cell-cell adhesion defects and hydrocephalus in the brain of NM II-B-depleted mice. We also examine the ability of NM II-B as well as chimaeric forms of NM II (II-A head and II-B tail and vice versa) to substitute for NM II-A in cell-cell adhesions in II-A-ablated mice. However, we also show that certain functions, such as neuronal cell migration in the developing brain and vascularization of the mouse embryo and placenta, specifically require NM II-B and II-A respectively.     

Effect of phosphorylation of myosin light chains on the interaction of myosin minifilaments with F-actin

The effect of phosphorylation of light chains-2 (LC2) of rabbit skeletal muscle myosin on the interaction of myosin minifilaments with F-actin as well as on the actin-stimulated Mg2+-ATPase of minifilaments was studied. It was shown that in the absence of KCl the degree of F-actin-induced stimulation of myosin minifilament Mg2+-ATPase with phosphorylated LC2 exceeds 2-4-fold that with unphosphorylated LC2. Phosphorylation of LC2 considerably increases the rate of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments but exerts only a very weak influence on the affinity of minifilaments for F-actin. After addition of KCl the differences in the actin-stimulated Mg2+-ATPase activity disappear in a great degree; in the presence of 50 mM KCl they do not exceed 50%. It was assumed that the observed specific influence of LC2 phosphorylation on the kinetic parameters of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments is due to unique properties of the minifilaments (e.g., their ability to ordered self-assembly as a result of interaction between the heads of myosin molecules) which reflect their structural peculiarities.     

Smooth muscle-type myosin heavy chain isoforms in bovine smooth muscle and non-muscle tissues

Summary— The distribution of smooth muscle (SM)-type myosin heavy chain isoforms in several bovine muscular and non-muscular (NM) tissues was evaluated by immunofluorescence tests using monoclonal antibodies SM-E7, reactive with 204 (SM1) and 200 (SM2) kDa isoforms, and SM-F11, specific for SM2 isoform. SM-E7 reacted equally with vascular, respiratory and intestinal SM tissues, whereas SM-F11 stained heterogeneously SM cells in the various muscular systems examined and in some peculiar tissues was unreactive (perisinusoidal cells of hepatic lobule, pulmonary interstitial cells and intestinal muscularis mucosae) or uniquely reactive (nerve cells). On the whole, our findings indicate that SM1 and SM2 isoforms are unequally distributed at the cellular level in various SM and NM tissues and support previous results obtained with tissue extracts and electrophoretic procedures.     

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts.

We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.     

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.     

Two mouse cofilin isoforms, muscle-type (MCF) and non-muscle type (NMCF), interact with F-actin with different efficiencies

Two cofilin isoforms, a muscle-type (MCF) and a non-muscle-type (NMCF), are co-expressed in developing mammalian skeletal and cardiac muscles. To clarify how they are involved in the actin filament dynamics during myofibrillogenesis, we examined their localization in muscle tissues and cultured muscle cells using immunocytochemical methods, and their interaction with F-actin in vitro. NMCF was mostly detected in a diffuse pattern in the cytoplasm but MCF was partly localized to the striated structures in myofibrils. The location of chicken cofilin, a homologue of MCF, in the I-bands of myofibrils was determined by an immunocytochemical method. It is suggested that MCF could be associated with actin filaments in muscle cells more efficiently than NMCF. Using purified recombinant MCF and NMCF, their interaction with F-actin was examined in vitro by a cosedimentation assay method. We observed that MCF was precipitated with F-actin more effectively than NMCF. When MCF and NMCF were simultaneously incubated with F-actin, MCF was preferentially associated with F-actin. MCF and NMCF inhibited the interaction of F-actin with tropomyosin, but the former suppressed the actin-tropomyosin interaction more strongly than the latter. These results suggest that MCF interacts with F-actin with higher affinity than NMCF, and although both of them are involved in the regulation of actin assembly in developing myotubes, the two proteins may play somewhat different roles.     

The regulatory effects of tropomyosin and troponin-I on the interaction of myosin loop regions with F-actin

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088-5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac beta-myosin, presumed to be sites of interaction with F-actin (residues 554-584, 622-646, and 633-660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134-147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.     

Binding of myosin subfragment-1 to F-actin.

During a part of the hydrolytic cycle, myosin head (S1) carries no nucleotide and binds strongly to an actin filament forming a rigor bond. At saturating concentration of S1 in rigor, S1 is well known to form 1:1 complex with actin. However, we have provided evidence that under certain conditions S1 could also form a complex with 2 actin monomers in a filament (Andreev, O.A. & Borejdo, J. (1991) Biochem. Biophys. Res. Comm. 177, 350-356). This view was recently challenged by Carlier & Didry (Carlier, M-F. & Didry, D. (1992) Biochem. Biophys. Res. Comm. 183, 970-974) who interpreted our data by suggesting that F-actin underwent a simple depolymerization and implied that, when only actin in the F-form was scored, the real stoichiometry in our experiments was 1:1. We show here that under conditions of our experiments less than 8% of actin was depolymerized. Moreover, we have repeated the experiments in the presence of phalloidin and show that under these conditions too, when S1 was added slowly to a fixed concentration of F-actin, it formed a different complex with F-actin than when it was added quickly. This confirms our original conclusion that S1 can bind actin in two different ways and shows that depolymerization of F-actin is not responsible for this finding.     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

Regulation of non-muscle myosin structure and function

In vertebrate and invertebrate nonmuscle myosins, light- and heavy-chain phosphorylation regulate myosin assembly into filaments, and interaction with actin. Vertebrate non-muscle myosins can exist in vitro in three main states, either ‘folded’ (assembly-blocked) or ‘extended’ (assembly-competent) monomers, and filaments. Light-chain phosphorylation regulates the ‘dynamic equilibrium’ between these states. The ability of the myosin to undergo changes in conformation and state of assembly may be an important mechanism in regulating the organization of the cytoskeleton and cell motility.     

The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.

C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.     

NH2-terminal calcium-binding domain of human complement C1s- mediates the interaction of C1r- with C1q

The assembly of C1, the first component of human complement, involves interactions between various domains of each of its three subcomponents, C1q, C1r, and C1s. The isolation, assignment of function, and structural characterization of the individual domains of C1r and C1s are critical for a thorough understanding of this complex assembly. The present study describes a 27-kDa plasmin-generated fragment derived from the NH2-terminal half of the heavy A chain of C1s-, the activated form of C1s. This fragment, C1s-alpha, was shown in the presence of Ca2+ to mimic the ability of whole C1s- to self-associate, bind to C1r-, and facilitate the binding of C1r to C1q. These results directly prove that the Ca2(+)-binding sites of C1s as well as all of the determinants necessary for binding of C1s- to C1r- and C1q are located in the NH2-terminal 27-kDa alpha region of the A chain.     

Interaction between myosin and F-actin. Correlation with actin-binding sites on subfragment-1

F-Actin bindings to subfragment-1 (S-1) and S-1 after limited proteolysis by trypsin (S-1t) were studied in the absence and presence of ATP by means of ultracentrifugation. No significant difference in the affinities for F-actin was observed between S-1 and S-1t in the absence of ATP. In contrast, the affinity for F-actin in the presence of ATP was decreased about 50 times by the limited proteolysis of the S-1 heavy chain. The S-1 whose SH1 and SH2 groups were cross-linked by N,N'-p-phenylenedimaleimide bound F-actin weakly. The affinity for F-actin was similar to that of unmodified S-1 in the presence of ATP and was also decreased markedly by limited proteolysis of the cross-linked S-1. Reciprocals of the dissociation constant of acto-S-1 complex decreased markedly with increase of ionic strength in the presence of ATP, but decreased only slightly at the rigor state. All these results are consistent with our proposal that S-1 has two different actin binding sites, as reported previously (Katoh, T., Imae, S., & Morita, F. (1984) J. Biochem. 95, 447-454). The mechanism of activation of S-1 ATPase by F-actin is discussed.     

Cooperative interaction between developmentally regulated troponin T and tropomyosin isoforms in the absence of F-actin

Troponin T (TnT) is the tropomyosin (Tm) binding subunit of the troponin complex that mediates the Ca(2+) regulation of actomyosin interaction in striated muscles. Troponin T isoform diversity is marked by a developmentally regulated acidic to basic switch that may modulate muscle contractility. We previously reported that transgenic expression of fast skeletal muscle TnT altered the cooperativity of cardiac muscle. In the present study, we have demonstrated that the binding of acidic TnT to troponin I is weaker than that of basic TnT. However, affinity chromatography experiments showed that Tm bound to acidic TnT with a greater affinity than to basic TnT, consistent with the significantly higher maximal binding of acidic TnT to Tm in solid phase binding assays. Competition and co-immunoprecipitation experiments demonstrated that the binding of TnT to Tm was cooperative in the absence of F-actin. The cooperativity between TnT molecules for Tm binding can be initiated by the conserved COOH-terminal T2 fragment of TnT. This indicates that the interaction of TnT with Tm induces a conformational change in Tm, promoting interaction of TnT with adjacent Tm dimers. This finding suggests a role for TnT and its acidic and basic isoforms in the cooperative release of the inhibition of striated muscle actomyosin interaction.     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.