Search

The spatial and temporal dynamics of forces in cells coordinate essential behaviors like division, polarization, and migration. While intracellular signaling initiates contractile ring assembly during cell division, how mechanical forces coordinate division and their energetic costs remain unclear. Here, we develop an in vitro model where myosin-induced stress drives division-like shape changes in giant unilamellar vesicles (GUVs, liposomes). Myosin activity is controlled by light patterns globally or locally at the equator. Global activation causes slow, shallow cleavage furrows due to a tug-of-war between the equatorial and polar forces. By contrast, local activation leads to faster, deeper, and symmetric division as equatorial forces dominate. Dissociating the actin cortex at the poles is crucial for inducing significant furrowing. During furrowing, actomyosin flows align actin filaments parallel to the division plane, forming a contractile ring-like structure. Mechanical power is not greatest during contraction, but is maximized just before furrowing. This study reveals the quantitative relationship between force patterning and mechanical energy during division-like shape changes, providing insights into cell division mechanics. While biochemical regulation initiates cell division, how mechanical forces ensure successful division remains unclear. Here, the authors develop a biomimetic model of dividing cells to characterize the cytoskeleton's mechanical power prior to division. [ABSTRACT FROM AUTHOR]

Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFPMyh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging. [ABSTRACT FROM AUTHOR]

High-quality proteins from farmed fish, including sea bream, represent a cheaper and healthier alternative to proteins from other animals, especially for people affected by chronic diseases. Aquaculture has also the possibility to move towards more sustainable growth and production. Therefore, the present study was designed to evaluate the impact of a starvation period before slaughtering in comparison to full feeding on quality attributes of sea bream fillet focused on their health promoting values. SDS-PAGE, Western blotting and high-resolution MS/MS analyses of the protein profiles showed that in the fillets from starved fish, only the amount of myosin light chains (MLCs) was markedly reduced at slaughter, suggesting their higher susceptibility to proteolysis. In the full feeding samples, MLCs degradation was evident only during storage at 4 °C. Conversely, skeletal alpha-actin fragmentation was similar regardless of the pre-slaughter feeding system. In vitro gastric digestibility of the myosin complex from starved fish fillets was much higher at pH 4, a condition that may occur in drug-treated patients affected by the gastroesophageal reflux disease (GERD). Using UHPLC–MS/MS, ACE-inhibitor gastric-resistant bioactive peptides potentially coming from MLCs (VF, MF) were found in all the digested samples, with one of these peptides (MF) being present in larger amount in the starved samples. Pre-slaughter starvation, other than decreases the environmental impact of fish aquaculture, is an effective tool in obtaining good-quality sea bream fillets that provide potential health benefits for hypertensive and GERD consumers. [ABSTRACT FROM AUTHOR]

Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human β-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human β-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts. [ABSTRACT FROM AUTHOR]

The recent discovery that myosin has two distinct states in relaxed muscle–disordered relaxed (DRX) and super-relaxed (SRX)–provides another factor to consider in our fundamental understanding of the aging mechanism in skeletal muscle, since myosin is thought to be a potential contributor to dynapenia (age-associated loss of muscle strength independent of atrophy). The primary goal of this study was to determine the effects of age on DRX and SRX states and to examine their sex specificity. We have used quantitative fluorescence microscopy of the fluorescent nucleotide analog 2′/3′-O-(N-methylanthraniloyl) ATP (mantATP) to measure single-nucleotide turnover kinetics of myosin in skinned skeletal muscle fibers under relaxing conditions. We examined changes in DRX and SRX in response to the natural aging process by measuring the turnover of mantATP in skinned fibers isolated from psoas muscle of adult young (3–4 months old) and aged (26–28 months old) C57BL/6 female and male mice. Fluorescence decays were fitted to a multi-exponential decay function to determine both the time constants and mole fractions of fast and slow turnover populations, and significance was analyzed by a t-test. We found that in females, both the DRX and SRX lifetimes of myosin ATP turnover at steady state were shorter in aged muscle fibers compared to young muscle fibers (p ≤ 0.033). However, there was no significant difference in relaxation lifetime of either DRX (p = 0.202) or SRX (p = 0.804) between young and aged male mice. No significant effects were measured on the mole fractions (populations) of these states, as a function of sex or age (females, p = 0.100; males, p = 0.929). The effect of age on the order of myosin heads at rest and their ATPase function is sex specific, affecting only females. These findings provide new insight into the molecular factors and mechanisms that contribute to aging muscle dysfunction in a sex-specific manner. [ABSTRACT FROM AUTHOR]

Key points: Fast sarcomere‐level mechanics in intact trabeculae, which allows the definition of the mechano‐kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy‐causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm–1) is 2‐ to 3‐fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. Abstract: The mechano‐kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half‐sarcomere both at the twitch force peak (Tp) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+]o), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half‐sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm–1, which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6‐0.8 pN nm–1) and 2‐ to 3‐fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+]o from 1 to 2.5 m m at sarcomere length ∼2.2 μm, is accompanied by an increase of the half‐sarcomere stiffness that is explained by an increase of the fraction of actin‐attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp. Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+]o. The application of fast sarcomere‐level mechanics to intact trabeculae to define the mechano‐kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy‐causing mutations in the myosin motor and testing specific therapeutic interventions. [ABSTRACT FROM AUTHOR]

The motor function of vertebrate myosin-5a is inhibited by its tail in a Ca2+-dependent manner. We previously demonstrated that the calmodulin (CaM) bound to the first isoleucine-glutamine (IQ) motif (IQ1) of myosin-5a is responsible for the Ca2+-dependent regulation of myosin-5a. We have solved the crystal structure of a truncated myosin-5a containing the motor domain and IQ1 (MDIQ1) complexedwith Ca2+-bound CaM (Ca2+-CaM) at 2.5-Å resolution. Comparedwith the structure of the MD-IQ1 complexed with essential light chain (an equivalent of apo-CaM), MD-IQ1/Ca2+-CaM displays large conformational differences in IQ1/CaM and little difference in the motor domain. In the MD-IQ1/Ca2+-CaM structure, the N-lobe and the C-lobe of Ca2+-CaM adopt an open conformation and grip the C-terminal and the N-terminal portions of the IQ1, respectively. Remarkably, the interlobe linker of CaMin IQ1/Ca2+-CaM is in a position opposite that in IQ1/apo-CaM, suggesting that CaM flip-flops relative to the IQ1 during the Ca2+ transition. We demonstrated that CaM continuously associates with the IQ1 during the Ca2+ transition and that the binding of CaM to IQ1 increases Ca2+ affinity and substantially changes the kinetics of the Ca2+ transition, suggesting that the IQ1/CaM complex functions as an intact Ca2+ sensor responding to distinct calcium signals. [ABSTRACT FROM AUTHOR]

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 ( S2) + light meromyosin ( LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+ LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM. [ABSTRACT FROM AUTHOR]

High resolution information about the three-dimensional (3D) structure of myosin filaments has always been hard to obtain. Solving the 3D structure of myosin filaments is very important because mutations in human cardiac muscle myosin and its associated proteins (e.g. titin and myosin binding protein C) are known to be associated with a number of familial human cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand how normal heart muscle works and how it fails, as well as the effects of the known mutations on muscle contractility, it is essential to properly understand myosin filament 3D structure and properties in both healthy and diseased hearts. The aim of this review is firstly to provide a general overview of the 3D structure of myosin thick filaments, as studied so far in both vertebrates and invertebrate striated muscles. Knowledge of this 3D structure is the starting point from which myosin filaments isolated from human cardiomyopathic samples, with known mutations in either myosin or its associated proteins (titin or C-protein), can be studied in detail. This should, in turn, enable us to relate the structure of myosin thick filament to its function and to understanding the disease process. A long term objective of this research would be to assist the design of possible therapeutic solutions to genetic myosin-related human cardiomyopathies. [ABSTRACT FROM AUTHOR]

A modified SDS-PAGE system has been employed to resolve polymorphic myosin heavy chain (MyHC) isoforms in different muscle types of three freshwater teleosts displaying different modes of respiration, adaptive features and life styles. Investigated species include accessory air-breather Channa punctata along with exclusive aquatic breather major carps Labeo rohita and Catla catla. All the selected species show specificity and expressivity of at least three MyHC isoforms, one each in red, head and pectoral muscles. Chymotryptic peptide maps unambiguously support substructural individuality of each MyHC isoforms with the type-specific dispersal of chymotryptic cleavage sites. Specific Ca- and Mg-ATPase activities of natural actomyosin (NAM) of lateral line red muscle of C. punctata were low and less sensitive to pH, but sensitive to KCl concentrations between 0.05 and 0.15 M. In comparison, the specific enzymatic activities of NAM of red muscle from the carps ( L. rohita and C. catla) were substantially high with prominent peaks at pH 7.5 and near insensitivity to 0.05-0.15 M KCl, while C. punctata had shown a different response at these molarities. Thus, the data favor a correlation between breathing modes and life style and the differences in pH or ionic strength sensitivities of ATPases. Unique profiles of peptide maps and the dispersal patterns of hydrophobic residues (cleavage sites of chymotrypsin) in MyHC of different muscle types further reflect individuality of their evolutionary histories. [ABSTRACT FROM AUTHOR]

ABSTRACT The physiological and biological properties of skeletal muscle in crustacea have not been well understood compared with those of vertebrates. The present study focused on myosin, the major protein in skeletal muscle, from shrimps. In our previous study, two full-length genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle of kuruma Marsupenaeus japonicus, black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps, and named as MHCa and MHCb. In this study, we renamed these as MHC1 and MHC2, respectively, due to the presence of various isoforms newly identified. Partial MHC sequences were identified from pleopod muscle of these shrimps. Two MHCs, named MHC3 and MHC4, were identified from pleopod muscle of kuruma shrimp, whereas two MHCs, named MHC4 and MHC5, were cloned from Pacific white shrimp pleopod. MHC3 was cloned only from black tiger shrimp pleopod. Partial MHC sequences from zoea, mysis, and postlarvae of black tiger and Pacific white shrimps were also determined. The phylogenetic tree demonstrated that most MHCs from pleopod muscle and larval MHCs formed clades with MHC1 and MHC2, respectively. These MHCs were considered to be of fast type, since MHC1 and MHC2 are fast-type MHCs according to our previous study. MHC5 obtained from pleopod muscle of Pacific white shrimp in this study was monophyletic with American lobster Homarus americanus S2 slow tonic MHC previously reported, indicating that MHC5 from Pacific white shrimp is of slow type. J. Exp. Zool. 319A:268-276, 2013. © 2013 © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The physiology and biochemistry of skeletal muscles in shrimps have been poorly understood compared with those from vertebrates. The present study was conducted focusing on myosin, the major protein in skeletal muscle, from adult specimens of black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps. Two genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle and defined as MHCa and MHCb according to our previous study on kuruma shrimp Marsupenaeus japonicus. Random cloning demonstrated that the MHCb gene ( MHCb) was expressed more abundantly than MHCa. The full-length cDNA clones of MHCa and MHCb from black tiger shrimp consisted of 5,926 and 5,914 bp, respectively, which encoded 1,914 and 1,909 amino acids, respectively, whereas those from Pacific white shrimp consisted of 5,923 and 5,908 bp, respectively, which encoded 1,913 and 1,909 amino acids, respectively. Both MHCa and MHCb were considered to be fast muscle type due to their strict localization in fast muscle. The amino acid identities between MHCa and MHCb of black tiger shrimp were 77%, 60%, and 73% in the regions of subfragment-1 (S1), subfragment-2 (S2) and light meromyosin (LMM), respectively, with 71% in total, whereas those of Pacific white shrimp were 78%, 60%, and 73% in the regions of S1, S2, and LMM, respectively, with 72% in total. In situ hybridization and northern blot analysis using different regions from abdominal muscle demonstrated different localizations of MHCa and MHCb transcripts in this muscle, suggesting their distinct physiological functions. J. Exp. Zool. 317A:608-621, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To elucidate the structure of the network of the water retention mechanism of fish-meat gel, the microscopic relationship between the water content and viscoelasticity was investigated, and the specific surface area ( S) where the water molecules are adsorbed was also measured. The gel structures of fish-meat, actomyosin (AM), and myosin (M) were investigated. In the fish-meat gel, as the water content increased, the number of network chains ( ν) decreased and the molecular weight between the cross-linking points ( M) increased. At 10 % salinity and approximately 79 % moisture, ν decreased to a minimum and M began to increase remarkably. In addition, when the moisture increased to 78 %, S increased about 1.5 times and the elasticity decreased significantly, indicating that this level of water content is the limit for sufficiently forming a gel structure in fish-meat gel. In the AM and M gels, M and S were similar to those observed in the fish-meat gels. When the moisture content increased, they also increased, regardless of the salt concentration. This result suggests that the size of the network was enlarged or that the network chains were cleaved. [ABSTRACT FROM AUTHOR]

Chymotrypsin digestion and Circular dichroism (CD) spectroscopy were used to study the structural stability of myosin rods prepared from silver carp in summer and winter. Differences in the thermal stability were clearly demonstrated by the chymotryptic digestion patterns and CD data obtained at pH 7.5 in 0.5 M KCl for myosin rods in both seasons, indicating the structural properties differed seasonally. In winter, the myosin rods were more susceptible to be cleaved into short (40 kDa) subfragments when digested at high temperature (30 °C). The major peaks in unfolding profiles showed that transition temperatures for the rods were 35 °C in winter, and 36 and 41 °C in summer. Rod subfragments (S-2 and LMM) were isolated and further analyzed by CD. In both winter and summer, the LMM fragments showed an unfolding transition at around 35.5 °C, but the S-2 fragments showed large seasonal difference; in winter, they showed a major unfolding peak around 35 °C and a minor peak at 40.5 °C, while in summer, they unfolded mostly at 44.5 °C, which was 8 °C higher than that for the LMM fragments. Short (40 kDa) S-2 subfragments isolated from winter rods gave a single unfolding transition at 41 °C. These results suggest that differences in the thermal stability of myosin rods between winter and summer were due to differences in the stability of S-2 region. The aim of this study was to detect the differences in the structure thermal stability of myosin rods between summer and winter, which is a very important portion for gel formation. The information will be very useful for producing surimi from freshwater fish species in different seasons. [ABSTRACT FROM AUTHOR]

Microvascular barrier dysfunction is implicated in the initiation and progression of inflammation, posttraumatic complications, sepsis, ischaemia–reperfusion injury, atherosclerosis, and diabetes. Under physiological conditions, a precise equilibrium between endothelial cell–cell adhesion and actin–myosin-based centripetal tension tightly controls the semi-permeability of microvascular barriers. Myosin light chain kinase (MLCK) plays an important role in maintaining the equilibrium by phosphorylating myosin light chain (MLC), thereby inducing actomyosin contractility and weakening endothelial cell–cell adhesion. MLCK is activated by numerous physiological factors and inflammatory or angiogenic mediators, causing vascular hyperpermeability. In this review, we discuss experimental evidence supporting the crucial role of MLCK in the hyperpermeability response to key cell signalling events during inflammation. At the cellular level, in vitro studies of cultured endothelial monolayers treated with MLCK inhibitors or transfected with specific inhibiting peptides have demonstrated that induction of endothelial MLCK activity is necessary for hyperpermeability. Ex vivo studies of live microvessels, enabled by development of the isolated, perfused venule method, support the importance of MLCK in endothelial permeability regulation in an environment that more closely resembles in vivo tissues. Finally, the role of MLCK in vascular hyperpermeability has been confirmed with in vivo studies of animal disease models and the use of transgenic MLCK210 knockout mice. These approaches provide a more complete view of the role of MLCK in vascular barrier dysfunction. [ABSTRACT FROM PUBLISHER]

In order to elucidate the molecular mechanism of transglutaminase-mediated myosin crosslinking, a fluorescent monodansylcadaverine (MDC) was incorporated into carp Cyprinus carpio myosin and the reactive Gln residues were analyzed by cyanogen bromide cleavage. The fluorescence was predominantly detected in a 10.5 kDa BrCN fragment, assumed to be located in subfragment 2 of the myosin heavy chain. Furthermore, lysyl endopeptidase digestion of the 10.5 kDa fragment revealed that MDC was specifically incorporated into the 520th Gln residue of the subfragment 2 domain. When meat paste prepared from frozen walleye pollack ( Theragra chalcogramma) surimi was incubated with MDC, the fluorescence was mostly observed in a 16 kDa BrCN fragment and also slightly detected in other three bands. By digesting the 16 kDa fragment with lysyl endopeptidase, it was elucidated that MDC was incorporated specifically into Gln-520 of myosin subfragment 2, as also detected in carp. This domain around Gln-520 is likely to be a critical region for the formation of myosin heavy chain dimers that both fish species have in common. In walleye pollack, other reactive Gln residues are presumed to exist at the C-terminus of the light meromyosin. This slight difference may have a significant effect on the capacity of myosin to form tetramers or even larger multimers. [ABSTRACT FROM AUTHOR]

The complete cDNA sequences encoding predominant types of myosin heavy chain (MYH) in the fast skeletal muscle were determined for brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso, which are used as materials for preparing high-quality surimi-based products. The cDNA consisted of 5973 and 5987 bp, respectively, and both encompassed an open reading frame encoding a polypeptide of 1936 amino acid residues. Brushtooth and wanieso lizardfish MYH showed the amino acid sequence identity of 92–93% to white croaker MYH, which was higher than that of 90% to walleye pollack MYH. The putative binding sites for ATP, actin, and regulatory and essential light chains in the subfragment-1 region of brushtooth lizardfish MYH exhibited a high identity with white croaker counterparts as well as the sequences of subfragment-2 and light meromyosin. In contrast, phylogenetic tree, constructed by the neighbor-joining method based on mitochondrial 16S rRNA gene, revealed that the two lizardfish species formed a cluster with walleye pollack, which was paraphyletic with white croaker. Therefore, a good reputation for lizardfish and white croaker to have a high thermal-gel forming ability seemed to be reflected by MYH rather than biological similarity as revealed by the mitochondrial 16S rRNA gene. [ABSTRACT FROM AUTHOR]

Biochemical and conformational changes of purified sardine myosin were investigated at various pHs. The purity of myosin, as determined by SDS-PAGE, was approximately 94.6%. One major band at 205 kDa, corresponding to myosin heavy chain, and 3 light chains at 31, 24, and 23 kDa were observed on the SDS-PAGE gel. The greatest myosin protein solubility was observed at pH 7 and remained constant up to pH 11. Sardine myosin showed no solubility at pHs 2.5 to 5.0. Three endothermic peaks were obtained for samples prepared at pHs 7 and 10, while no peaks were shown for pH 2 samples, indicating chemical denaturation of myosin occurred before thermal treatment. The greatest Ca2+-ATPase activity was observed at pH 7, while no activity was observed between pHs 2 and 5 and at pH 11. Total sulfhydryl content was not measured at pHs 2.5 to 4 while the greatest measure was obtained for samples at pH 5.5. Surface hydrophobicity was not detected from pHs 2.5 to 5.0; thereafter the content remained consistent through pH 11. Storage modulus, indicating the elastic element of myosin gels, was minimally affected at pH 2, indicating myosin was chemically denatured before the temperature sweep treatment. However, at pH 10, the thermal exposure of myosin, as evidenced by dynamic thermograms with deeper valleys at 40 to 60 °C, was noted, indicating myosin was not damaged by adjustment to pH 10 and therefore was still able to undergo thermal gelation. [ABSTRACT FROM AUTHOR]

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MI-IC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC20 has been associated with conformational changes in the 51 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ. [ABSTRACT FROM AUTHOR]

To elucidate the effects of seasonal temperature acclimatization on thermal gelation of grass carp myosin, myosins from fish in different seasons were prepared and investigated for the changes in dynamic viscoelastic parameters including storage modulus (G′), loss modulus (G′′) and damping factor (tan δ) upon heating. Myosins from fish in spring and summer had a temperature region of 38–44°C for the first marked increase of G′ higher than that of myosins from fish in autumn and winter (28–33°C). The measurement temperature-dependent changes in dynamic viscoelastic parameters such as G′′ and tan δ were also different among the four myosins. While gel formation was observed with the spring and summer myosins, apparently in two steps, three steps were found in the autumn myosin. Furthermore, the winter myosin exhibited more than three steps for gel formation. These differences in rheological properties among the four myosins were considered to be attributed to the differences in thermodynamic and structural properties of these myosins previously reported. [ABSTRACT FROM AUTHOR]

Enzymatic and structural properties of white croaker fast skeletal muscle myosin were determined and compared with those of walleye pollack counterpart. Ca2+-ATPase activity of white croaker myosin was decreased to approximately 70% of the original activity during 1 day of storage at 0°C and pH 7.0 in 0.5 M KCl and 0.1 mM dithiothreitol, whereas that of walleye pollack was decreased to approximately 20% under the same condition. The activation energy ( Ea) for inactivation of white croaker myosin calculated by the Arrhenius plot for inactivation rate constant (KD) was 1.2-fold higher than that of walleye pollack. While Ca2+-ATPase showed a similar KCl-dependency for the two species, the maximal activity was observed at pH 6.2 and 6.3 for white croaker and walleye pollack, respectively. Actin-activated myosin Mg2+-ATPase activity of white croaker was approximately half that of walleye pollack at 0.05 M KCl and pH 7.0, although the two myosins showed a similar affinity to F-actin with Km of 1.7 and 1.4, respectively. Limited proteolysis with α-chymotrypsin cleaved heat-denatured white croaker myosin mainly at heavy meromyosin/light meromyosin (HMM/LMM) junction, whereas walleye pollack myosin was cleaved at several sites in LMM as well as at the HMM/LMM junction. [ABSTRACT FROM AUTHOR]

BSTRACT Thermodynamic properties in differential scanning calorimetry (DSC) and changes in viscoelasticity upon heating of myosins from white croaker, Alaska pollock, and rabbit fast muscles were investigated in relation to their thermal gel formation abilities. Alaska pollock myosin unfolded in a wide temperature range of 19 to 69°C as revealed by DSC, whereas rabbit myosin unfolded in very narrow range of 32 to 56°C. Thermal unfolding of white croaker myosin occurred in an intermediate temperature range of 30 to 60°C. Viscoelastic properties determined as storage modulus, G′, and loss modulus, G″, reflected differences observed in DSC for the 3 myosins. [ABSTRACT FROM AUTHOR]

Based on the high level of extractability of myosin subunits (light chains), even after prolonged heat treatment of muscle, a new method to evaluate the dark muscle content in the fish meat and products of mackerel is proposed. Tissue-specific rabbit antisera with myosin light chains (A1 from ordinary muscle and D1 from dark muscle) from mackerel Scomber japonicus were obtained. Mackerel meat paste (surimi) was dissolved in 8 M urea containing 1% SDS, and diffused on agar plates containing antiserum against A1 or D1 by single radial immunodiffusion (SRID). The results obtained showed that the area of halos formed in the plates was quite proportional to the content of dark muscle. [ABSTRACT FROM AUTHOR]

Investigates age-related alterations in distribution of myosin structural states during muscle contraction. Model for the molecular mechanisms of force generation; Single-fiber contractile properties; Specificity of spin labeling; Deficits of semimembranosus fibers from aged animals relative to fibers from young adult animals.

A cDNA library constructed from the dorsal fast skeletal muscle of white croaker Pen-nahia argentata was screened for myosin heavy chain using antibody raised against carp fast skele-tal myosin. A full-length cDNA was further cloned by reverse transcription--PCR and 5-RACE using first-strand cDNA as a template together with appropriate sets of primers. The entire cDNA consisted of 5986 nucleotides (nt) with 64 nt 5-untranslated and 129 nt 3-untranslated regions. This full-length cDNA had an open reading frame encoding a polypeptide of 1930 amino acid residues. Amino acid alignment with myosins from various vertebrates revealed some striking differences between fish and mammalian sequences which could be due to their position in the vertebrate evolutionary process. Hydrophilicity analysis revealed two different features of the myosin molecule: S1 heavy chain showed a mixed profile of hydrophobicity and hydrophilicity, whereas rod had a profile of only hydrophilicity. Comparison of amino acid sequences in the rod region between white croaker and walleye pollack showed a markedly high identity of 92%. White croaker myosin rod had a charac-teristic seven-residue (heptad) repeat (a, b, c, d, e, f, g)n, where positions a and d were normally occupied by hydrophobic residues, and positions b, c and f by oppositely charged residues, which may lead to interhelical electrostatic attractions stabilizing the coiled-coil of a -helices. [ABSTRACT FROM AUTHOR]

BackgroundProtein synthesis in skeletal muscle is reduced following starvation and restored by feeding. The mediators and mechanisms are incompletely understood. The aim of this study was to evaluate whether prolongation of undernutrition induced changes in muscle gene expression at the level of mRNA and protein. Materials and methodsThe changes in myosin heavy-chain 2X mRNA in adult partially starved (50% of ad libitum standard rodent chow intake for 4 or 7 days) C57BL mice or subsequently refed mice were studied. Ad libitum-fed mice were used as controls. Protein synthesis, total RNA and myosin heavy-chain 2X mRNA concentrations were determined. Plasma concentrations of amino acids were measured by high-performance liquid chromatography. ResultsPartial starvation of 4 and 7 days reduced body weight by 15.6 ± 1% and 17.1 ± 2.1% (P ≤ 0.05) vs. ad libitum fed controls. Protein synthesis was reduced by 32 ± 9% and protein content by 20 ± 4% (P ≤ 0.05) following 7 days of partial starvation. Plasma amino acid concentrations were increased (6297 ± 853) in refed animals vs. ad libitum-fed controls (3057 ± 141, P ≤ 0.05). Total RNA concentration (μg RNA μg-1 DNA) in skeletal muscle was unchanged. Myosin heavy-chain 2X mRNA concentration did not change following 4 days of partial starvation but increased by 24 ± 5% (P ≤ 0.05) following 7 days of partial starvation, hence suggesting that expression of myosin mRNA was nutritionally altered. ConclusionPostprandial stimulation of protein synthesis following starvation may thus be a combination of increased mRNA availability and increased translation. This effect may be activated by peak concentrations of amino acids in plasma following feeding. [ABSTRACT FROM AUTHOR]

SUMMARY:The amino acid sequences of myosin rod containing subfragment-2 (S2) and light meromyosin (LMM) were determined by cDNA cloning for walleye pollack fast skeletal myosin heavy chain. While S2 and LMM were composed of 442 and 656 amino acid residues, a total of 1937 amino acid residues accounted for the whole myosin heavy chain molecule with previously determined sequence for the subfragment-1 heavy chain region of this fish. Both regions for S2 and LMM showed a seven-residue repeat pattern characteristic to fibrous proteins with a coiled-coil structure of two α-helices, displaying a, b, c, d, e, f, and g where positions a and d were frequently occupied by hydrophobic amino acids and c and g often contained charged residues. The occurrence of a 28-residue unit with repetitive sequence was also strongly suggested, when one and three skip residues were adopted into S2 and LMM, respectively. Thus, walleye pollack S2 and LMM consisted of 17 and 24 zones with a 28-residue repeat rearrangement. There were several amino acid substitutions which might account for a low thermal stability of walleye pollack myosin heavy chain in comparison with the sequences of higher vertebrate counterparts. However, it seemed difficult to interpret such low stability only from the comparison in the 28-residue repeat arrangement at the primary structure. [ABSTRACT FROM AUTHOR]

SUMMARY:Carboxyl groups of myosin rod were chemically modified by means of carbodiimide-mediated amidation using glycine ethyl ether as an amino donor. Mobility of the rod on urea-polyacrylamide gel electrophoresis was reduced on modification, which indicates a reduced negative charge of rod. The modification significantly promoted the filament formation of rod especially at alkaline pH. Modified rod required neither lowering pH (pH 6.0) nor Mg2+ addition for its filament formation. Amino groups of rod were also modified using the same principle of carbodiimide-mediated modification using sodium formate as a donor. This modification led to an opposite effect on the filament formation of rod. Modification rather inhibited the self-assembly of rod. Thus, it was concluded that surface negative charges of the rod play an important role in filament formation. Factors that decrease the negative charge of rod such as lowering pH, adding Mg2+, or blocking carboxyl groups led to the promotion of the filament formation similarly. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the mechanisms and molecules involved in the assembly of a macromolecular machine using the myosin-containing thick filament as a model. Structure of the thick filament backbone; Properties of myosin related to self-assembly; Regulation of the thick filament.

Heavy meromyosin subfragment-1 (S1) was prepared by α-chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca- and K (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 μmol P·min·mg, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K) of S1 from cold-acclimated carp was 32.1x10· s, compared to 13.2x10·s for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle. [ABSTRACT FROM AUTHOR]

Myosin in pollen tubes of Lilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin. [ABSTRACT FROM AUTHOR]

Background: Immunohistochemical studies of hearts from the lesser spotted dogfish, Scyliorhinus canicula (Chondrichthyes) revealed that the pan-myosin heavy chain (pan-MyHC) antibody MF20 homogeneously labels all the myocardium, while the pan-MyHC antibody A4.1025 labels the myocardium of the inflow (sinus venosus and atrium) but not the outflow (ventricle and conus arteriosus) cardiac segments, as opposed to other vertebrates. We hypothesized that the conventional pattern of cardiac MyHC isoform distribution present in most vertebrates, i.e. MYH6 in the inflow and MYH7 in the outflow segments, has evolved from a primitive pattern that persists in Chondrichthyes. In order to test this hypothesis, we conducted protein detection techniques to identify the MyHC isoforms expressed in adult dogfish cardiac segments and to assess the pan-MyHC antibodies reactivity against the cardiac segments of representative species from different vertebrate groups. Results: Western and slot blot results confirmed the specificity of MF20 and A4.1025 for MyHC in dogfish and their differential reactivity against distinct myocardial segments. HPLC-ESI-MS/MS and ESI-Quadrupole-Orbitrap revealed abundance of MYH6 and MYH2 in the inflow and of MYH7 and MYH7B in the outflow segments. Immunoprecipitation showed higher affinity of A4.1025 for MYH2 and MYH6 than for MYH7 and almost no affinity for MYH7B. Immunohistochemistry showed that A4.1025 signals are restricted to the inflow myocardial segments of elasmobranchs, homogeneous in all myocardial segments of teleosts and acipenseriforms, and low in the ventricle of polypteriforms. Conclusions: The cardiac inflow and outflow segments of the dogfish show predominance of fast- and slow-twitch MyHC isoforms respectively, what can be considered a synapomorphy of gnathostomes. The myocardium of the dogfish contains two isomyosins (MYH2 and MYH7B) not expressed in the adult heart of other vertebrates. We propose that these isomyosins lost their function in cardiac contraction during the evolution of gnathostomes, the later acquiring a regulatory role in myogenesis through its intronic miRNA. Loss of MYH2 and MYH7B expression in the heart possibly occurred before the origin of Osteichthyes, being the latter reacquired in polypteriforms. We raise the hypothesis that the slow tonic MYH7B facilitates the peristaltic contraction of the conus arteriosus of fish with a primitive cardiac anatomical design and of the vertebrate embryo. [ABSTRACT FROM AUTHOR]