Smooth muscle-like Ca2+-regulation of actin-myosin interaction in adult jellyfish striated muscle.

Cnidaria is an animal phylum, whose members probably have the most ancestral musculature. We prepared and characterized, for the first time to our knowledge, native actomyosin from the striated myoepithelium of the adult moon jelly Aurelia sp. The actomyosin contained myosin, paramyosin-like protein, Ser/Thr-kinase, actin, and two isoforms of tropomyosin, but not troponin, which is known to activate contraction dependent on intracellular Ca2+ signaling in almost all striated muscles of bilaterians. Notably, the myosin comprised striated muscle-type heavy chain and smooth muscle-type regulatory light chains. In the presence of Ca2+, the Mg-ATPase activity of actomyosin was stimulated and Ser21 of the regulatory light chain was concomitantly phosphorylated by the addition of calmodulin and myosin light chain kinase prepared from chicken smooth muscle. Collectively, these results suggest that, similar to smooth muscle, the contraction of jellyfish striated muscle is regulated by Ca2+-dependent phosphorylation of the myosin light chain.

Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins.

Metastasis is responsible for most cancer-related deaths, but the current clinical treatments are not effective. Recently, gold nanoparticles (AuNPs) were discovered to inhibit cancer cell migration and prevent metastasis. Rationally designed AuNPs could greatly benefit their antimigration property, but the molecular mechanisms need to be explored. Cytoskeletons are cell structural proteins that closely relate to migration, and surface receptor integrins play critical roles in controlling the organization of cytoskeletons. Herein, we developed a strategy to inhibit cancer cell migration by targeting integrins, using Arg-Gly-Asp (RGD) peptide-functionalized gold nanorods. To enhance the effect, AuNRs were further activated with 808-nm near-infrared (NIR) light to generate heat for photothermal therapy (PPTT), where the temperature was adjusted not to affect the cell viability/proliferation. Our results demonstrate changes in cell morphology, observed as cytoskeleton protrusions-i.e., lamellipodia and filopodia-were reduced after treatment. The Western blot analysis indicates the downstream effectors of integrin were attracted toward the antimigration direction. Proteomics results indicated broad perturbations in four signaling pathways, Rho GTPases, actin, microtubule, and kinases-related pathways, which are the downstream regulators of integrins. Due to the dominant role of integrins in controlling cytoskeleton, focal adhesion, actomyosin contraction, and actin and microtubule assembly have been disrupted by targeting integrins. PPTT further enhanced the remodeling of cytoskeletal proteins and decreased migration. In summary, the ability of targeting AuNRs to cancer cell integrins and the introduction of PPTT stimulated broad regulation on the cytoskeleton, which provides the evidence for a potential medical application for controlling cancer metastasis.

Nanowire-imposed geometrical control in studies of actomyosin motor function.

Recently, molecular motor gliding assays with actin and myosin from muscle have been realized on semiconductor nanowires coated with Al2O3. This opens for unique nanotechnological applications and novel fundamental studies of actomyosin motor function. Here, we provide a comparison of myosin-driven actin filament motility on Al2O3 to both nitrocellulose and trimethylchlorosilane derivatized surfaces. We also show that actomyosin motility on the less than 200 nm wide tips of arrays of Al2O3-coated nanowires can be used to control the number, and density, of myosin-actin attachment points. Results obtained using nanowire arrays with different inter-wire spacing are consistent with the idea that the actin filament sliding velocity is determined both by the total number and the average density of attached myosin heads along the actin filament. Further, the results are consistent with buckling of long myosin-free segments of the filaments as a factor underlying reduced velocity. On the other hand, the findings do not support a mechanistic role in decreasing velocity, of increased nearest neighbor distance between available myosin heads. Our results open up for more advanced studies that may use nanowire-based structures for fundamental investigations of molecular motors, including the possibility to create a nanowire-templated bottom-up assembly of 3D, muscle-like structures.

The green tea polyphenol (-)-epigallocatechin-3-gallate inhibits magnesium binding to the C-domain of cardiac troponin C.

Cardiac muscle contraction is activated via the single Ca(2+)-binding site (site II) in the N-domain of troponin C (cTnC). The two Ca(2+)/Mg(2+) binding sites in the C-domain of cTnC (sites III and IV) have been considered to play a purely structural role in anchoring cTnC to the thin filament. However, several recent discoveries suggest a possible role of this domain in contractile regulation. The green tea polyphenol (-)-epigallocatechin 3-gallate (EGCg), which binds specifically to the C-domain of cTnC, reduces cardiac myofilament Ca(2+) sensitivity along with maximum force and acto-myosin ATPase activity. We have determined the effect of EGCg on Ca(2+) and Mg(2+) binding to the C-domain of cTnC. In the absence of Mg(2+) there was no significant effect of EGCg on the Ca(2+)-cTnC affinity. Surprisingly, in the presence of Mg(2+) EGCg caused an increase in Ca(2+) affinity for sites III and IV of cTnC. However, in the absence of Ca(2+) the addition of EGCg caused a significant reduction in Mg(2+)-cTnC affinity. This reduction is presumably responsible for the increase in Ca(2+)-cTnC affinity produced by EGCg in the presence of Mg(2+). We propose that the inhibitory effect of EGCg on myofilament Ca(2+) activation may be related to an enhanced Ca(2+)-Mg(2+)exchange at sites III and IV of cTnC, which might reduce the myosin crossbridge dependent component of thin filament activation.

Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function.

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

Hypersensitivity of myofilament response to Ca2+ in association with maladaptation of estrogen-deficient heart under diabetes complication.

The amelioration of cardioprotective effect of estrogen in diabetes suggests potential interactive action of estrogen and insulin on myofilament activation. We compared Ca2+-dependent Mg2+-ATPase activity of isolated myofibrillar preparations from hearts of sham and 10-wk ovariectomized rats with or without simultaneous 8 wk-induction of diabetes and from diabetic-ovariectomized rats with estrogen and/or insulin supplementation. Similar magnitude of suppressed maximum myofibrillar ATPase activity was demonstrated in ovariectomized, diabetic, and diabetic-ovariectomized rat hearts. Such suppressed activity and the relative suppression in alpha-myosin heavy chain level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca2+ hypersensitivity detected only in the ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rats, which was restored upon estrogen supplementation. Binding kinetics of beta1-adrenergic receptors and immunoblots of beta1-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of the myofilament response to Ca2+. The amount of beta1-adrenoceptors significantly increased concomitant with Ca2+ hypersensitivity of the myofilament, without differences in the receptor binding affinity among the groups. In contrast, changes in HSP72 paralleled that of maximum myofibrillar ATPase activity. These results indicate that hypersensitivity of cardiac myofilament to Ca2+ is specifically induced in ovariectomized rats even under diabetes complication and that alterations in the expression of beta1-adrenoceptors may, in part, play a mechanistic role underlying the cardioprotective effects of estrogen that act together with Ca2+ hypersensitivity of the myofilament in determining the gender difference in cardiac activation.

High-level production of functional muscle alpha-tropomyosin in Pichia pastoris.

Although numerous studies have reported the production of skeletal muscle alpha-tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg(2+)-ATPase in the absence of troponin. On the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha-tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha-tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg(2+)-ATPase.

Actomyosin: law and order in motility.

The crystal structures of smooth muscle and scallop striated muscle myosin have both been completed in the past 18 months. Structural studies of unconventional myosins, in particular the stunning discovery that myosin VI moves backwards on actin, are starting to have deep impact on the field and have induced new ways of thinking about actin-based motility. Sophisticated genetic, biochemical and biophysical studies were used to test and refine hypotheses of the molecular mechanism of motility that were developed in the past. Although all these studies confirmed some aspects of these hypotheses, they also raised many new unresolved questions. Much of the evidence points to the importance of the actin-myosin binding process and an associated disorder-to-order transition.

The kinetic mechanism of myosin V.

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590-593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.

Quantitative fitting of atomic models into observed densities derived by electron microscopy.

A new methodology for fitting atomic models into density distributions is described. This approach is based on a global density correlation analysis that can be optionally supplemented by biochemical as well as biophysical data. The procedure is completely general and enables an objective evaluation of the resulting docking in the light of available biochemical and biophysical information as well as density correlation alone. In this paper we describe the implementation of the algorithm and its application to two biological systems. In both cases the procedure provided an interface model on the atomic level and located parts of the structure that were missing in the atomic model but present in the electron-microscopic construct. It also detected and quantified conformational changes in actomyosin complexes.

Distinct troponin T genes are expressed in embryonic/larval tail striated muscle and adult body wall smooth muscle of ascidian.

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the sessile adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actomyosin Mg2+-ATPase activity in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only <60% identity with each other. Northern blotting and whole mount in situ hybridization revealed that these isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.

Tissue-type transglutaminase from red sea bream (Pagrus major). Sequence analysis of the cDNA and functional expression in Escherichia coli.

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from a cDNA library prepared from the liver of red sea bream (Pagrus major). The cDNA sequence had an open reading frame coding for a protein of 695 amino acids and showed 43% identity to the sequence of guinea pig liver TGase, revealing a relatively low overall similarity. However, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two species, and was also identical to the corresponding regions of human and bovine endothelial cell TGases. In addition, the critical residues (His332 and Asp355) thought to form the catalytic-center triad together with Cys272, were found in the highly conserved region. The red sea bream TGase had an extension of 11 amino acids in the C-terminal region and some differences in the N-terminal region when compared with guinea pig TGase. From the cloned cDNA, a semi-synthetic TGase gene suitable for overexpression in Escherichia coli was constructed (pTTG2-22). At a reduced temperature (28 degrees C), E. coli cells transformed with pTTG2-22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombinant red sea bream TGase induced gelation of actomyosin solutions, accompanied by a significant increase of epsilon-(gamma-glutamyl)lysine bonds, which are predominantly derived from the cross-linking of myosin heavy chains. These results indicate that this fish TGase should be useful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials.

Explaining load dependence of ventricular contractile properties with a model of excitation-contraction coupling.

A theory is present which accounts for a very broad range of ventricular properties that have been noted in recent experiments. The theory is based upon a four-state biochemical scheme that accounts for the dynamic interaction between calcium, actin and myosin which includes a calcium-free force generating complex between actin and myosin. This original scheme was supplemented by incorporating two additional basic properties of cardiac muscle: length dependence of calcium binding affinity and load dependence of force generation. The biochemical scheme was used to provide the force-length-time properties of cardiac muscle which were used to construct a ventricle via a spherical geometry. In addition to being able to accurately interrelate previously measured calcium and muscle force transients, this theory was able to account for many fundamental aspects of ventricular performance including: a realistic contractility dependent curvilinearity of the end-systolic pressure-volume relationship: enhancement of contractile strength on ejecting compared to isovolumic beats; improved contractile efficiency on ejecting as compared to isovolumic beats; appropriate load-dependent changes in time to peak pressure, time constant of relaxation and duration of contraction on isovolumic and ejecting beats; realistic estimated time course of tension-dependent heat generation. The explanation for these phenomena were explored within the context of the theory and presented in detail.

Isolation, purification and partial characterization of tropomyosin and troponin subunits from the lobster tail muscle.

In a search for an invertebrate muscle from which the muscle regulatory proteins could be obtained in a great quantity and at high homogeneity, the regulatory proteins, tropomyosin (Tm) and three subunits of troponin (Tn), have been isolated from the lobster tail muscle, purified and partially characterized. The calcium-sensitive ATPase of lobster myofibril was restored when purified lobster Tm and lobster Tn were added to actin. Quantitative SDS-polyacrylamide gel electrophoresis showed that the lobster muscle contains actin, Tm, Tn with a molar ratio 7:1:1 and that lobster Tn consists of three subunits, one of each I, C and T. Each subunit was identified according to its effect on the acto-S1 ATPase rate. The isomer composition in each fraction of purified Tn subunit and in Tm are different from the rabbit skeletal muscle proteins; Tm consists of a single species of polypeptide of M(r) 38,000; the TnT fraction appears to be homogeneous with M(r) 43,000; the TnI fraction contains five isomers, all showing similar isoelectric pH, differing in M(r) in the range from 28,000 to 31,000; two TnC fractions contain three isomers in total with a range of M(r) from 18,500 to 19,000. Further study of the lobster Tm elucidated that digestion by carboxypeptidase A gave rise to a homogeneous preparation of truncated and non-polymerizable Tm which is devoid of 11 residues at the C-terminus of the molecule. The C-terminal amino acid sequence of 11 residues is homologous to the thoracic isomer generated from Drosophila melanogaster Tm-I gene. The present study indicated that, despite heterogeneities owing to the occurrence of isomers, the lobster regulatory proteins serve as an invertebrate source of the proteins for structural and biophysical studies, alternative to vertebrate counterparts.

Structural study of gizzard caldesmon and its interaction with actin. Binding involves residues of actin also recognised by myosin subfragment 1.

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1-44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389-397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20-41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483-578) derived from the N-terminal portion of a 35-kDa proteolytic cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin-filament regulation in smooth and striated muscle.

[The Ca2+ reactivity and the subunit composition of myocardial actomyosin during the pyromecaine and pyrroxan treatment of experimental ischemia]. / Ca2+-reaktivnost' i sub''edinichnyi sostav aktomiozina miokarda pri lechenii éksperimental'noi ishemii piromekainom i pirroksanom.

The treatment with pyromecaine and pyrroxan for 5 days was found to prevent disturbances of the physicochemical properties and the composition of the components of actomyosin complex of the rat heart left ventricle caused by experimental myocardial ischemia. Pyrroxan is able to change also the gene expression thereby leading to the appearance of a new isoform of myocardial myosin reminding by its characteristics the isoform of myosin of the rapidly contracting skeletal muscles.

High-molecular-weight serine proteinase from lobster muscle that degrades myofibrillar proteins.

A latent alkaline serine proteinase (ASP) has been extracted from the soluble fraction of lobster claw and abdominal muscles. The enzyme, which was irreversibly activated 30- to 40-fold by brief (2-3 min) heating at 60 degrees C, had an optimal caseinolytic activity at pH 7.75. Its molecular weight was estimated to be 740,000 by gel filtration chromatography. Serine protease inhibitors (diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, benzamidine, and chloromethyl ketones) suppressed ASP activity 22 to 70%. In addition, sulfhydryl-blocking reagents and hemin inhibited activity 69 to 100%; leupeptin and E-64, however, did not. Pepstatin A, ethylenediaminetetraacetate, and adenosine triphosphate were without effect. These results suggest that the lobster ASP is a serine proteinase that contains one or more sulfhydryl groups essential for catalysis. ASP was stimulated by dithiothreitol and inhibited by CaCl2 and oleic and linoleic acids. The enzyme was partially activated by low concentrations of sodium dodecyl sulfate; 0.05% produced activities 13% of that of preparations heated at 60 degrees C. Neither poly-L-lysine, urea, dimethylsulfoxide, oleic acid, linoleic acid, nor N-ethylmaleimide activated the enzyme. The ASP degraded most myofibrillar proteins, but showed a preferential hydrolysis of paramyosin, troponin-I and -C, and myosin alpha light chain.

[Effect of cardiac glycosides on myocardial contractile proteins]. / Vliianie serdechnykh glikozidov na kontraktil'nye belki miokarda.

By using the method of the measurement of the degree of actomyosin superprecipitation of the rabbit cardiomyocytes it was shown that isolanide (0.1 mg/kg), adoniside (0.1 ml/kg), strophanthin K (0.05 mg/kg) and corglycon (0.15 mg/kg) prevented the allergic process-induced disturbance of ATP energy utilization by myocardial contractile proteins.

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution.

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.

Reaction of two heads of gizzard myosin with ATP.

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)