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Sample records for actin myosin heavy

  1. Actin and Myosin in Pea Tendrils 1

    PubMed Central

    Ma, Yong-Ze; Yen, Lung-Fei

    1989-01-01

    We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K+-EDTA and Ca2+ and is inhibited by Mg2+. At low ionic strength, the Mg2+-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril. Images Figure 1 Figure 3 Figure 4 PMID:16666586

  2. Upregulation of alpha-skeletal muscle actin and myosin heavy polypeptide gene products in degenerating rotator cuff muscles.

    PubMed

    Fuchs, Bruno; Zumstein, Matthias; Regenfelder, Felix; Steinmann, Patrick; Fuchs, Thomas; Husmann, Knut; Hellermann, Jens; Jost, Bernhard; Hodler, Jürg; Born, Walter; Gerber, C

    2008-07-01

    Impaired function of shoulder muscles, resulting from rotator cuff tears, is associated with abnormal deposition of fat in muscle tissue, but corresponding cellular and molecular mechanisms, likely reflected by altered gene expression profiles, are largely unknown. Here, an analysis of muscle gene expression was carried out by semiquantitative RT-PCR in total RNA extracts of supraspinatus biopsies collected from 60 patients prior to shoulder surgery. A significant increase of alpha-skeletal muscle actin (p = 0.0115) and of myosin heavy polypeptide 1 (p = 0.0147) gene transcripts was observed in parallel with progressive fat deposition in the muscle, assessed on parasagittal T1-weighted turbo-spin-echo magnetic resonance images according to Goutallier. Upregulation of alpha-skeletal muscle actin and of myosin heavy polypeptide-1 has been reported to be associated with increased muscle tissue metabolism and oxidative stress. The findings of the present study, therefore, challenge the hypothesis that increased fat deposition in rotator cuff muscle after injury reflects muscle degeneration.

  3. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  4. The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

    PubMed

    Ducret, Axel; Nguyen, Mai; Breckenridge, David G; Shore, Gordon C

    2003-01-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.

  5. Structure of the Rigor Actin-Tropomyosin-Myosin Complex

    PubMed Central

    Behrmann, Elmar; Müller, Mirco; Penczek, Pawel A.; Mannherz, Hans Georg; Manstein, Dietmar J.; Raunser, Stefan

    2014-01-01

    The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin. PMID:22817895

  6. Myosin lever arm directs collective motion on cellular actin network.

    PubMed

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

  7. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

    PubMed

    Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

    2010-08-06

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

  8. Regulation of myosin II activity by actin architecture

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Stam, Samantha; McCall, Patrick; Munro, Edwin; Gardel, Margaret

    2015-03-01

    Networks of actin filaments containing myosin II motors generate forces and motions that promote biological processes such as cell division, motility, and cargo transport. In cells, actin filaments are arranged in various structures from disordered meshworks to tight bundles. Clusters of myosin II motors, known as myosin filaments, crosslink and generate force on neighboring actin filaments. We hypothesized that the local actin architecture controls the magnitude and duration of force generated by myosin II motors. We used fluorescence imaging to directly measure the mobility of myosin II filaments on actin networks and bundles with varying actin filament polarity, orientation, spacing, and length. On unipolar bundles, myosin exhibits fast, unidirectional motion consistent with their unloaded gliding speed. On mixed polarity bundles, myosin speed is reduced by one order of magnitude and marked by direction switching and trapping. Increasing filament spacing and bundle flexibility reduces the duration of trapping and enhances the mobility of motors. Simulations indicate that stable trapping is a signature of large generated forces while increased mobility indicates force release. Our data underscore that the efficiency of force generation by myosin motors in an actin network depends sensitively on its architecture and suggests actin crosslinking proteins are tuned to optimize actomyosin contractility.

  9. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-08

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  10. Magnesium Modulates Actin Binding and ADP Release in Myosin Motors*

    PubMed Central

    Swenson, Anja M.; Trivedi, Darshan V.; Rauscher, Anna A.; Wang, Yuan; Takagi, Yasuharu; Palmer, Bradley M.; Málnási-Csizmadia, András; Debold, Edward P.; Yengo, Christopher M.

    2014-01-01

    We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg2+-dependent manner (0.3–9.0 mm free Mg2+) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg2+ in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg2+ in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg2+ coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg2+ concentrations, demonstrating that the ADP release rate constant is slowed by Mg2+ in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg2+ reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg2+ inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg2+-dependent alterations in actin binding. Overall, our results suggest that Mg2+ reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins. PMID:25006251

  11. How Actin Initiates the Motor Activity of Myosin

    PubMed Central

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H. Lee; Houdusse, Anne

    2015-01-01

    SUMMARY Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential to use the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin, and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement, and may represent a strategy common to many molecular machines. PMID:25936506

  12. How actin initiates the motor activity of Myosin.

    PubMed

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H Lee; Houdusse, Anne

    2015-05-26

    Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

  13. Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

    PubMed Central

    Brawley, Crista M.; Rock, Ronald S.

    2009-01-01

    Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments. PMID:19478066

  14. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  15. Self-organization of actin networks by a monomeric myosin

    PubMed Central

    Saczko-Brack, Dario; Warchol, Ewa; Rogez, Benoit; Kröss, Markus; Heissler, Sarah M.; Sellers, James R.; Batters, Christopher; Veigel, Claudia

    2016-01-01

    The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase–activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration. PMID:27956608

  16. The effects of phosphorylation and dephosphorylation of brain myosin on its actin-activated Mg2+-ATPase and contractile activities.

    PubMed

    Matsumura, S; Takashima, T; Ohmori, H; Kumon, A

    1988-02-01

    Purified bovine brain myosin contained approximately 1 and 3 mol of protein-bound phosphate/mol myosin in the light chains and heavy chains, respectively. Large portions of this light chain- and heavy chain-bound phosphate (about 0.8 and 2.4 mol, respectively) were removed by incubation with a brain phosphoprotein phosphatase and potato acid phosphatase, respectively. Upon phosphorylation of the dephosphorylated brain myosin with myosin light chain kinase and casein kinase II, about 1.6 and 3.0 mol of phosphate was incorporated into the light chains and heavy chains, respectively, while much lower levels of phosphate were incorporated into the non-dephosphorylated brain myosin under the same conditions. The actin-activated Mg2+-ATPase activity of brain myosin rephosphorylated with myosin light chain kinase was about twice as high as that of dephosphorylated brain myosin (about 30 and 15 nmol phosphate/mg/min, respectively). On the other hand, whereas the rephosphorylated brain myosin superprecipitated rapidly with F-actin, the rate of superprecipitation of the dephosphorylated brain myosin was extremely low. Under appropriate conditions, a loose network of tiny superprecipitates, which formed initially throughout the solution, contracted to form eventually a large and dense particle. These results indicate that phosphorylation of the light chains of brain myosin is a prerequisite for the contraction of brain actomyosin. The role of phosphorylation of the heavy chains by casein kinase II remains to be elucidated.

  17. Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

    PubMed

    Wong, J Z; Woodcock-Mitchell, J; Mitchell, J; Rippetoe, P; White, S; Absher, M; Baldor, L; Evans, J; McHugh, K M; Low, R B

    1998-05-01

    In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

  18. Actin and myosin isoforms in aneural and malformed chick hearts.

    PubMed

    Kirby, M L; Shimizu, N; Gagnon, J; Toyofuku, T; Kennedy, J; Conrad, D C; Zak, R

    1990-09-01

    Although it is generally accepted that actin and myosin isoforms adapt to their functional requirements, the sequence of expression of these proteins in hearts developing abnormally is unknown. In the chick embryo it is possible to change various aspects of heart development without direct manipulation of the cardiovascular system, by removing various regions of the neural crest from early embryos. The neural crest provides both neural (sympathetic and parasympathetic) and ectomesenchymal components to the heart, and selective removal of various areas results in embryos with sympathetically aneural hearts, or persistent truncus arteriosus with or without parasympathetic denervation. Myosin isoform expression was studied in each of these types of hearts using an array of myosin antibodies specific for atrium, ventricle or the conduction system. Myosin expression in experimental hearts was found to follow the normal pattern of development using these antibodies. Actin expression was studied using cDNA probes for the 3' untranslated region of actin mRNA of the alpha-skeletal, alpha-cardiac and beta-actin isoforms. Using slot-blot hybridization analysis, the pattern of actin expression in atrium and ventricle was followed throughout the period of incubation in normal hearts. The pattern of actin expression was found to be abnormal in hearts which were sympathetically aneural and those which had persistent truncus arteriosus combined with parasympathetic denervation. ATPase activity was increased only in atria of hearts with persistent truncus arteriosus. It appears from these experiments that actin isoform expression is influenced in the chick heart by autonomic innervation.

  19. Myosin Vs organize actin cables in fission yeast

    PubMed Central

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G.

    2012-01-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  20. Reversal of cell polarity and actin-myosin cytoskeleton reorganization under mechanical and chemical stimulation.

    PubMed

    Dalous, Jérémie; Burghardt, Emmanuel; Müller-Taubenberger, Annette; Bruckert, Franz; Gerisch, Günther; Bretschneider, Till

    2008-02-01

    To study reorganization of the actin system in cells that invert their polarity, we stimulated Dictyostelium cells by mechanical forces from alternating directions. The cells oriented in a fluid flow by establishing a protruding front directed against the flow and a retracting tail. Labels for polymerized actin and filamentous myosin-II marked front and tail. At 2.1 Pa, actin first disassembled at the previous front before it began to polymerize at the newly induced front. In contrast, myosin-II slowly disappeared from the previous tail and continuously redistributed to the new tail. Front specification was myosin-II independent and accumulation of polymerized actin was even more focused in mutants lacking myosin-II heavy chains. We conclude that under mechanical stimulation, the inversion of cell polarity is initiated by a global internal signal that turns down actin polymerization in the entire cell. It is thought to be elicited at the most strongly stimulated site of the cell, the incipient front region, and to be counterbalanced by a slowly generated, short-range signal that locally activates actin polymerization at the front. Similar pattern of front and tail interconversion were observed in cells reorienting in strong gradients of the chemoattractant cyclic AMP.

  1. A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects

    PubMed Central

    Augière, Céline; Mégy, Simon; El Malti, Rajae; Boland, Anne; El Zein, Loubna; Verrier, Bernard; Mégarbané, André; Deleuze, Jean-François; Bouvagnet, Patrice

    2015-01-01

    Background A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5. Methods and Results A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface. Conclusions Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin. PMID:26061005

  2. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    PubMed

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  3. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

  4. Maleimidobenzoyl-G-actin: Structural properties and interaction with skeletal myosin subfragment-1

    SciTech Connect

    Bettache, N.; Bertrand, R.; Kassab, R. )

    1990-09-25

    The authors have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt-and myosin subfragment 1 (S-1)--induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain. The far-ultraviolet CD spectrum and {alpha}-helix content of the MBS-actin were identical with those displayed by native G-actin. {sup 45}Ca{sup 2+} measurements showed the same content of tightly bound Ca{sup 2+} in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl+5 mM MgCl{sub 2} led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6mg/mL, at 25{degree}C, pH 8.0. The MBS-F-actin formed activated the Mg{sup 2+}-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was to be at all affected by its specific covalent conjugation to S-1. This finding led them to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions.

  5. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  6. The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.

    PubMed

    Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P

    2013-03-05

    Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length β-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.

  7. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

  8. Actin- and myosin-like filaments in rat brain pericytes.

    PubMed

    Le Beux, Y J; Willemot, J

    1978-04-01

    Heavy meromyosin (HMM) labeling was used to identify the nature of the filaments which form bundles in the cytoplasm of the pericytes in brain tissue. Rat brain tissue pieces were incubated in glycerol solutions at 4 degrees and then transferred into buffer (pH 7.0), (1) without HMM, (2) with HMM, (3) with HMM + 5 mM ATP, and (4) with HMM + 2.5 mM Na+ pyrophosphate. In pericytes from untreated tissue, smooth-surfaced microfilaments, averaging 6 nm in diameter, appear to branch and anastomose and to anchor on the plasma membrane. After exposure to HMM, the number and the density of the microfilaments are strikingly increased. These tightly-packed microfilaments are now heavily coated with exogeneous HMM thus increasing in width to 18-20 mm. They intertwine in closely-woven networks. After incubation in HMM solutions containing ATP or Na+ phosphate, they are no longer coated with thick sidearms. It can thus be concluded that these microfilaments are of actin-like nature. In addition, after incubation in ATP, they are intermingled with, and converge onto the surfaces of, thick, tapered filaments, which we have tentatively identified as of myosin-like nature. Thus, it appears that certain of the major elements necessary for contraction are present in brain pericytes.

  9. Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes.

    PubMed Central

    Labbé, J P; Méjean, C; Benyamin, Y; Roustan, C

    1990-01-01

    Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction. Images Fig. 1. Fig. 4. PMID:2146951

  10. The association of myosin IB with actin waves in dictyostelium requires both the plasma membrane-binding site and actin-binding region in the myosin tail.

    PubMed

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A; Korn, Edward D

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.

  11. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    PubMed Central

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  12. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    SciTech Connect

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K.

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  13. Maximum limit to the number of myosin II motors participating in processive sliding of actin

    PubMed Central

    Rastogi, Khushboo; Puliyakodan, Mohammed Shabeel; Pandey, Vikas; Nath, Sunil; Elangovan, Ravikrishnan

    2016-01-01

    In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads. PMID:27554800

  14. Multiple Myosins Are Required to Coordinate Actin Assembly with Coat Compression during Compensatory Endocytosis

    PubMed Central

    Bement, William M.

    2007-01-01

    Actin is involved in endocytosis in organisms ranging from yeast to mammals. In activated Xenopus eggs, exocytosing cortical granules (CGs) are surrounded by actin “coats,” which compress the exocytosing compartments, resulting in compensatory endocytosis. Here, we examined the roles of two myosins in actin coat compression. Myosin-2 is recruited to exocytosing CGs late in coat compression. Inhibition of myosin-2 slows coat compression without affecting actin assembly. This differs from phenotype induced by inhibition of actin assembly, where exocytosing CGs are trapped at the plasma membrane (PM) completely. Thus, coat compression is likely driven in part by actin assembly itself, but it requires myosin-2 for efficient completion. In contrast to myosin-2, the long-tailed myosin-1e is recruited to exocytosing CGs immediately after egg activation. Perturbation of myosin-1e results in partial actin coat assembly and induces CG collapse into the PM. Intriguingly, simultaneous inhibition of actin assembly and myosin-1e prevents CG collapse. Together, the results show that myosin-1e and myosin-2 are part of an intricate machinery that coordinates coat compression at exocytosing CGs. PMID:17699600

  15. Regulation of the actin-activated MgATPase activity of Acanthamoeba myosin II by phosphorylation of serine 639 in motor domain loop 2.

    PubMed

    Liu, Xiong; Lee, Duck-Yeon; Cai, Shutao; Yu, Shuhua; Shu, Shi; Levine, Rodney L; Korn, Edward D

    2013-01-02

    It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

  16. The myosin X motor is optimized for movement on actin bundles

    PubMed Central

    Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana; Blanc, Florian; Zhou, Kaifeng; Lin, Tianming; Liu, Xiaoyan; Hissier, Pascale; Samazan, Frédéric; Amigues, Béatrice; Yang, Eric D.; Park, Hyokeun; Pylypenko, Olena; Cecchini, Marco; Sindelar, Charles V.; Sweeney, H. Lee; Houdusse, Anne

    2016-01-01

    Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles. PMID:27580874

  17. Intra-axonal myosin and actin in nerve regeneration.

    PubMed

    McQuarrie, Irvine G; Lund, Linda M

    2009-10-01

    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin.

  18. Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm-Tn chain.

    PubMed

    Mijailovich, Srboljub M; Kayser-Herold, Oliver; Li, Xiaochuan; Griffiths, Hugh; Geeves, Michael A

    2012-12-01

    The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k(-I). The resulting equilibrium constant K(B) ≡ 1/K(I) varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.

  19. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.

  20. Microscopic model of the actin-myosin interaction in muscular contractions

    NASA Astrophysics Data System (ADS)

    Gaveau, B.; Moreau, M.; Schuman, B.

    2004-01-01

    We define and study a detailed many body model for the muscular contraction taking into account the various myosin heads. The state of the system is defined by the position of the actin and by an internal coordinate of rotation for each myosin head. We write a system of Fokker-Planck equations and calculate the average for the position, the number of attached myosin heads, and the total force exerted on the actin. We also study the correlation between these quantities, in particular between the number of attached myosin heads and the force on the actin.

  1. Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

    SciTech Connect

    Matsuo, Tatsuhito; Arata, Toshiaki; Oda, Toshiro; Nakajima, Kenji; Ohira-Kawamura, Seiko; Kikuchi, Tatsuya; Fujiwara, Satoru

    2015-04-10

    Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than for S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1. - Highlights: • We studied the internal dynamics of F-actin and myosin S1 by neutron scattering. • The correlation times of the atomic motions were smaller for F-actin than for S1. • The fraction of the immobile atoms was also smaller for F-actin than for S1. • Our results suggest that mobility of atoms in F-actin is higher than that in S1. • We propose that high flexibility of F-actin facilitates the binding of myosin.

  2. Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation

    PubMed Central

    1995-01-01

    The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and

  3. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-04

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning.

  4. Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport

    PubMed Central

    Evans, Richard D.; Robinson, Christopher; Briggs, Deborah A.; Tooth, David J.; Ramalho, Jose S.; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V.; Hume, Alistair N.

    2014-01-01

    Summary In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11]. PMID:25065759

  5. Actin-myosin network is required for proper assembly of influenza virus particles

    SciTech Connect

    Kumakura, Michiko; Kawaguchi, Atsushi Nagata, Kyosuke

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  6. Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

    PubMed

    Tomatis, Vanesa M; Papadopulos, Andreas; Malintan, Nancy T; Martin, Sally; Wallis, Tristan; Gormal, Rachel S; Kendrick-Jones, John; Buss, Folma; Meunier, Frédéric A

    2013-02-04

    Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.

  7. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    SciTech Connect

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  8. Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions.

    PubMed

    Merritt, Raymond C; Manor, Uri; Salles, Felipe T; Grati, M'hamed; Dose, Andrea C; Unrath, William C; Quintero, Omar A; Yengo, Christopher M; Kachar, Bechara

    2012-02-21

    Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

  9. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    PubMed

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A Michael; Månsson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  10. The movement of actin-myosin biomolecular linear motor under AC electric fields: an experimental study.

    PubMed

    Lee, Yongkuk; Famouri, Parviz

    2013-03-15

    The role of actin-myosin as a biomolecular linear motor is considered a transport system at nanoscale because of their size, efficiency and functionality. To utilize the ability to transport, it is essential to control the random movement of actin filaments (F-actin) on myosin coated substrate. In the presence of an alternating current (AC) electric field, the direction of F-actin movement is regulated by electro-orientation torque and, as a result, its movement is perpendicularly toward the electrode edges. Our data confirm such aligned movement is proportional to the strength of applied electric field. Interestingly, the aligned movement is found frequency-dependent and the electrothermal effect is observed by means of the velocity measurement of aligned F-actin movement. The findings in this study may provide constructive information for manipulating actin-myosin nanotransport system to build functional nanodevices in future work.

  11. F-actin and myosin II accelerate catecholamine release from chromaffin granules

    PubMed Central

    Berberian, Khajak; Torres, Alexis J; Fang, Qinghua; Kisler, Kassandra

    2009-01-01

    The roles of non-muscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with Blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using Cytochalasin-D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces. PMID:19158310

  12. And the dead shall rise: Actin and myosin return to the spindle

    PubMed Central

    Sandquist, Joshua C.; Kita, Angela M.; Bement, William M.

    2011-01-01

    The spindle directs chromosome partitioning in eukaryotes and, for the last three decades, has been considered primarily a structure based on microtubules, microtubule motors, and other microtubule binding proteins. However, a surprisingly large body of both old and new studies suggests roles for actin filaments (F-actin) and myosins (F-actin-based motor proteins) in spindle assembly and function. Here we review these data, and conclude that in several cases the evidence for F-actin and myosins participation in spindle function is very strong, and in the situations where it is less strong, there is nevertheless enough evidence to warrant further investigation. PMID:21920311

  13. Myosin-Va binds to and mechanochemically couples microtubules to actin filaments.

    PubMed

    Cao, Tracy T; Chang, Wakam; Masters, Sarah E; Mooseker, Mark S

    2004-01-01

    Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

  14. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

    PubMed

    Hong, Feng; Brizendine, Richard K; Carter, Michael S; Alcala, Diego B; Brown, Avery E; Chattin, Amy M; Haldeman, Brian D; Walsh, Michael P; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

    2015-10-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.

  15. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

    PubMed

    Beach, Jordan R; Bruun, Kyle S; Shao, Lin; Li, Dong; Swider, Zac; Remmert, Kirsten; Zhang, Yingfan; Conti, Mary A; Adelstein, Robert S; Rusan, Nasser M; Betzig, Eric; Hammer, John A

    2017-02-01

    The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

  16. Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1982-01-01

    The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D.L. Mykles and D.M. Skinner, 1981, J. Ultrastruct. Res. 75, 314 to 325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca/sup 2 +/ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of crysteine proteinases, leupeptin, and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.

  17. Criticalities in crosslinked actin networks due to myosin activity

    NASA Astrophysics Data System (ADS)

    Sheinman, Michael

    2013-03-01

    Many essential processes in cells and tissues, like motility and morphogenesis, are orchestrated by molecular motors applying internal, active stresses on crosslinked networks of actin filaments. Using scaling analysis, mean-field calculation, numerical modelling and in vitro experiments of such active networks we predict and observe different mechanical regimes exhibiting interesting critical behaviours with non-trivial power-law dependencies. Firstly, we find that the presence of active stresses can dramatically increase the stiffness of a floppy network, as was observed in reconstituted intracellular F-actin networks with myosin motors and extracellular gels with contractile cells. Uniform internal stress results in an anomalous, critical mechanical regime only in the vicinity of the rigidity percolation points of the network. However, taking into account heterogeneity of motors, we demonstrate that the motors, stiffening any floppy network, induce large non-affine fluctuations, giving rise to a critical mechanical regime. Secondly, upon increasing motor concentration, the resulting large internal stress is able to significantly enhance unbinding of the network's crosslinks and, therefore, disconnect the initially well-connected network to isolated clusters. However, during this process, when the network approaches marginal connectivity the internal stresses are expected to drop drastically such that the connectivity stabilizes. This general argument and detailed numerical simulations show that motors should drive a well connected network to a close vicinity of a critical point of marginal connectivity. Experiments clearly confirm this conclusion and demonstrate robust critical connectivity of initially well-connected networks, ruptured by the motor activity for a wide range of parameters. M. Sheinman, C.P. Broedersz and F.C. MacKintosh, Phys. Rev. Lett, in press. J. Alvarado, M. Sheinman, A. Sharma, F.C. MacKintosh and G. Koenderink, in preparation.

  18. Effects of filament rigidity in myosin II-induced actin network contractility and dynamics

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Gardel, Margaret

    2014-03-01

    Cells change shape, deforming to move and divide. The dynamic protein scaffold that shapes the cell is the cortex, a disordered, thin network of actin filaments. Random, local stresses generated by myosin II in the network create cellular-scale deformations. Myosin induced buckling and severing of actin filaments has been shown to underlie the contractility of two-dimensional disordered actin networks. This non-linear elastic response of actin filaments is thought to be an essential symmetry breaking mechanism to produce robust contractility in disordered actomyosin networks. To test this idea, we explore the effects of an actin bundling protein fascin, a crosslinker which induces polarity specific bundling of actin filaments, to create a network of F-actin bundles. We investigate myosin-induced stresses in a network of randomly oriented actin filaments, confined to a thin sheet at a supported lipid bilayer surface through a crowding agent. We find fascin-bundled filaments are less prone to filament buckling and show increased filament sliding, causing the myosin activity to induce network reorganization rather than contraction. Thus, changes in the filament bending rigidity in motor-filament systems can drive the system between distinct states with unique dynamic and mechanical signatures.

  19. Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

    PubMed Central

    Whitten, Andrew E.; Jeffries, Cy M.; Harris, Samantha P.; Trewhella, Jill

    2008-01-01

    Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin. PMID:19011110

  20. Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.

    PubMed

    Oztug Durer, Zeynep A; Kamal, J K Amisha; Benchaar, Sabrina; Chance, Mark R; Reisler, Emil

    2011-11-25

    Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function.

  1. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    SciTech Connect

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2010-10-22

    Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  2. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

    PubMed Central

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. DOI: http://dx.doi.org/10.7554/eLife.17523.001 PMID:27623148

  3. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes.

    PubMed

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas; Houdusse, Anne; Kerkhoff, Eugen

    2016-09-13

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.

  4. Myosin VI deafness mutation prevents the initiation of processive runs on actin.

    PubMed

    Pylypenko, Olena; Song, Lin; Shima, Ai; Yang, Zhaohui; Houdusse, Anne M; Sweeney, H Lee

    2015-03-17

    Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.

  5. Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

    PubMed Central

    Roman, Horia Nicolae; Zitouni, Nedjma B.; Kachmar, Linda; Ijpma, Gijs; Hilbert, Lennart; Matusovskiy, Oleg; Benedetti, Andrea; Sobieszek, Apolinary; Lauzon, Anne-Marie

    2013-01-01

    Background Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. Methods To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Results Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). Conclusions This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. General Significance This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved. PMID:23747303

  6. ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

    PubMed Central

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

    2014-01-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

  7. ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

    PubMed

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

    2014-04-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

  8. Electric field modulation of the motility of actin filaments on myosin-functionalised surfaces

    NASA Astrophysics Data System (ADS)

    Ramsey, L. C.; Aveyard, J.; van Zalinge, H.; Persson, M.; Mânsson, A.; Nicolau, D. V.

    2013-02-01

    We investigated the difference in electrically guided acto-myosin motility on two surfaces. Rabbit skeletal muscle heavy meromyosin (HMM) was absorbed onto surfaces coated with Nitrocellulose (NC) and Poly(butyl methacrylate) (PBMA). A modified in vitro motility assay with sealed chambers for the insertion of electrodes allowed an electrical field to be applied across the flow cell. On all surfaces a small increase in velocity and general guidance of the actin filaments towards the positive electrode is seen at field strengths in the range of ~3000 - 4000Vm-1. A large increase in velocity was observed at ~5000Vm-1 and a significant change in the velocity of the actin filaments present in field strengths higher than this. NC supported the highest percentage of motile filaments and at a field of 8000Vm-1 reached ~66%. PBMA however supported the least percentage of motile filaments and irregular motility was observed even at higher fields where guidance was expected to be strong. The change in velocity in the range of fields tested varied significantly on the surfaces with NC displaying a 46% increase from 0 to 8000Vm-1 whereas on PBMA this value was just 37%.

  9. Humoral immune response against contractile proteins (actin and myosin) during cardiovascular disease.

    PubMed

    De Scheerder, I K; De Buyzere, M; Delanghe, J; Maas, A; Clement, D L; Wieme, R

    1991-08-01

    Sensitive and highly specific ELISA assays were developed to determine humoral immune response against actin and myosin in 122 patients suffering from various cardiovascular diseases: acute viral myocarditis (n = 10, MYO), acute myocardial infarction (n = 28, AMI), valve surgery (n = 35, VALVE), coronary bypass surgery (n = 35, CABG), and peripheral vascular surgery (n = 14, VASC). Anti-actin and anti-myosin antibodies were determined on admission and serially during a period of 90 days. Anti-actin and anti-myosin immune response (IgG, IgM) was expressed comparing absorbance of the patients' serum with a reference serum. In the different patient groups significantly (P less than 0.01) higher anti-actin and anti-myosin antibody concentrations were found on admission compared with age-matched control groups. During follow-up, all patient groups except the vascular surgery group showed a significant immune response against actin and myosin, with an immune response ratio (peak/admission) for AMA IgG and IgM respectively of 2.12 and 2.40 in the VALVE group, 1.30 and 1.99 in the CABG group, 1.42 and 1.48 in the AMI group and 1.66 and 1.25 in the MYO group; and for AAA IgG and IgM respectively of 1.57 and 3.00 in the VALVE group, 1.54 and 1.64 in the CABG group, 1.25 and 1.07 in the AMI group, and 1.42 and 1.42 in the MYO group. A significant correlation between pre-cardiac injury and peak post-cardiac injury anti-myosin and anti-actin autoantibody levels could be demonstrated suggesting that pre-injury sensitization to these antigens plays an important role in evoking post-cardiac injury immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Conformational selection during weak binding at the actin and myosin interface.

    PubMed Central

    Xu, J; Root, D D

    2000-01-01

    The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF(4), ADP-BeF(x), and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin. PMID:10969011

  11. Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom

    PubMed Central

    1975-01-01

    The control systems regulating muscle contraction in approximately 100 organisms have been categorized. Both myosin control and actin control operate simultaneously in the majority of invertebrates tested. These include insects, chelicerates, most crustaceans, annelids, priapulids, nematodes, and some sipunculids. Single myosin control is present in the muscles of molluscs, brachiopods, echinoderms, echiuroids, and nemertine worms. Single actin control was found in the fast muscles of decapods, in mysidacea, in a single sipunculid species, and in vertebrate striated muscles. Classification is based on functional tests that include measurements of the calcium dependence of the actomyosin ATPase activity in the presence and the absence of purified rabbit actin and myosin. In addition, isolated thin filaments and myosins were also analyzed. Molluscs lack actin control since troponin is not present in sufficient quantities. Even though the functional tests indicate the complete lack of myosin control in vertebrate striated muscle, it is difficult to exclude unambiguously the in vivo existence of this regulation. Both control systems have been found in animals from phyla which evolved early. We cannot ascribe any simple correlation between ATPase activity, muscle structure, and regulatory mechanisms. PMID:125778

  12. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    PubMed Central

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

  13. Direct observation of motion of single F-actin filaments in the presence of myosin

    NASA Astrophysics Data System (ADS)

    Yanagida, Toshio; Nakase, Michiyuki; Nishiyama, Katsumi; Oosawa, Fumio

    1984-01-01

    Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems1-4, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell5. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.

  14. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    PubMed

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles.

  15. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase.

  16. Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

    PubMed Central

    Kiehart, D P; Lutz, M S; Chan, D; Ketchum, A S; Laymon, R A; Nguyen, B; Goldstein, L S

    1989-01-01

    In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis. Images PMID:2498088

  17. Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

    PubMed Central

    Noguchi, Tatsuhiko; Frank, Deborah J.; Isaji, Mamiko

    2009-01-01

    Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo. PMID:19005209

  18. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

    PubMed Central

    Arden, Susan D.; Tumbarello, David A.; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-01-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

  19. Selective localization of myosin-I proteins in macropinosomes and actin waves.

    PubMed

    Brzeska, Hanna; Koech, Hilary; Pridham, Kevin J; Korn, Edward D; Titus, Margaret A

    2016-02-01

    Class I myosins are widely expressed with roles in endocytosis and cell migration in a variety of cell types. Dictyostelium express multiple myosin Is, including three short-tailed (Myo1A, Myo1E, Myo1F) and three long-tailed (Myo1B, Myo1C, Myo1D). Here we report the molecular basis of the specific localizations of short-tailed Myo1A, Myo1E, and Myo1F compared to our previously determined localization of long-tailed Myo1B. Myo1A and Myo1B have common and unique localizations consistent with the various features of their tail region; specifically the BH sites in their tails are required for their association with the plasma membrane and heads are sufficient for relocalization to the front of polarized cells. Myo1A does not localize to actin waves and macropinocytic protrusions, in agreement with the absence of a tail region which is required for these localizations of Myo1B. However, in spite of the overall similarity of their domain structures, the cellular distributions of Myo1E and Myo1F are quite different from Myo1A. Myo1E and Myo1F, but not Myo1A, are associated with macropinocytic cups and actin waves. The localizations of Myo1E and Myo1F in macropinocytic structures and actin waves differ from the localization of Myo1B. Myo1B colocalizes with F-actin in the actin waves and at the tips of mature macropinocytic cups whereas Myo1E and Myo1F are in the interior of actin waves and along the entire surface of macropinocytic cups. Our results point to different mechanisms of targeting of short- and long-tailed myosin Is, and are consistent with these myosins having both shared and divergent cellular functions.

  20. [Conformational changes of actin induced by strong or weak myosin subfragment-1 binding].

    PubMed

    Dedova, I V; Avrova, S V; Vikhoreva, N N; Vikhorev, R G; Hazlett, T L; Van der Meer, W; Dos Remedios, C G; Borovikov, Iu S

    2004-01-01

    Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.

  1. Effect of nucleotides and actin on the intramolecular cross-linking of myosin subfragment-1.

    PubMed

    Blotnick, E; Muhlrad, A

    1994-06-07

    The heavy chain of myosin subfragment-1 (S1) is cleaved by limited trypsinolysis into three fragments, 27, 50, and 20 kDa--aligned in this order from the N-terminus. The tertiary structure of the molecule is essentially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-induced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of cross-linked products was monitored by SDS-PAGE, using the fluorescent probes 9-anthronitrile and N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifically label the 27- and 20-kDa fragments, respectively. The reaction with the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. Of these, the most intensive was the formation of the 50-kDa/20-kDa products, which appeared as a doublet on the SDS-PAGE with all the cross-linkers. This indicates that the interface between the two fragments is rather extended. The presence of MgATP or MgADP promoted the formation of the 20-kDa/50-kDa cross-linked products, especially with the lower electrophoretic mobility band, when EEDQ was used as a cross-linker. With PDG as a cross-linker, MgATP also affected the cross-link formation between the 20-kDa fragment and the light chains whereas it had no influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers was quite extensive, and it was manifested in the reduction of cross-link formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induced by actin, which extend to larger

  2. Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

    PubMed Central

    Léguillette, Renaud; Zitouni, Nedjma B.; Govindaraju, Karuthapillai; Fong, Laura M.; Lauzon, Anne-Marie

    2008-01-01

    Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (νmax) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of νmax versus [MgADP] was significantly greater for tonic (−0.51 ± 0.04) than phasic muscle myosin (−0.15 ± 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 ± 0.022 pN for phasic muscle and 0.084 ± 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state. PMID:18614813

  3. Molecular mechanisms underlying skeletal muscle weakness in human cancer: reduced myosin-actin cross-bridge formation and kinetics.

    PubMed

    Toth, Michael J; Miller, Mark S; Callahan, Damien M; Sweeny, Andrew P; Nunez, Ivette; Grunberg, Steven M; Der-Torossian, Hirak; Couch, Marion E; Dittus, Kim

    2013-04-01

    Many patients with cancer experience physical disability following diagnosis, although little is known about the mechanisms underlying these functional deficits. To characterize skeletal muscle adaptations to cancer in humans, we evaluated skeletal muscle structure and contractile function at the molecular, cellular, whole-muscle, and whole-body level in 11 patients with cancer (5 cachectic, 6 noncachectic) and 6 controls without disease. Patients with cancer showed a 25% reduction in knee extensor isometric torque after adjustment for muscle mass (P < 0.05), which was strongly related to diminished power output during a walking endurance test (r = 0.889; P < 0.01). At the cellular level, single fiber isometric tension was reduced in myosin heavy chain (MHC) IIA fibers (P = 0.05) in patients with cancer, which was explained by a reduction (P < 0.05) in the number of strongly bound cross-bridges. In MHC I fibers, myosin-actin cross-bridge kinetics were reduced in patients, as evidenced by an increase in myosin attachment time (P < 0.01); and reductions in another kinetic parameter, myosin rate of force production, predicted reduced knee extensor isometric torque (r = 0.689; P < 0.05). Patients with cancer also exhibited reduced mitochondrial density (-50%; P < 0.001), which was related to increased myosin attachment time in MHC I fibers (r = -0.754; P < 0.01). Finally, no group differences in myofilament protein content or ultrastructure were noted that explained the observed functional alterations. Collectively, our results suggest reductions in myofilament protein function as a potential molecular mechanism contributing to muscle weakness and physical disability in human cancer.

  4. Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

    PubMed

    Salles, Felipe T; Merritt, Raymond C; Manor, Uri; Dougherty, Gerard W; Sousa, Aurea D; Moore, Judy E; Yengo, Christopher M; Dosé, Andréa C; Kachar, Bechara

    2009-04-01

    Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

  5. Regulation of muscle force in the absence of actin-myosin-based cross-bridge interaction.

    PubMed

    Leonard, T R; Herzog, W

    2010-07-01

    For the past half century, the sliding filament-based cross-bridge theory has been the cornerstone of our understanding of how muscles contract. According to this theory, active force can only occur if there is overlap between the contractile filaments, actin and myosin. Otherwise, forces are thought to be caused by passive structural elements and are assumed to vary solely because of the length of the muscle. We observed increases in muscle force by a factor of 3 to 4 above the purely passive forces for activated and stretched myofibrils in the absence of actin-myosin overlap. We show that this dramatic increase in force is crucially dependent on the presence of the structural protein titin, cannot be explained with calcium activation, and is regulated by actin-myosin-based cross-bridge forces before stretching. We conclude from these observations that titin is a strong regulator of muscle force and propose that this regulation is based on cross-bridge force-dependent titin-actin interactions. These results suggest a mechanism for stability of sarcomeres on the "inherently unstable" descending limb of the force-length relationship, and they further provide an explanation for the protection of muscles against stretch-induced muscle injuries.

  6. Myosin motor function: the ins and outs of actin-based membrane protrusions

    PubMed Central

    Nambiar, Rajalakshmi; McConnell, Russell E.

    2011-01-01

    Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion. PMID:20107861

  7. Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay.

    PubMed

    Debold, Edward P; Turner, Matthew A; Stout, Jordan C; Walcott, Sam

    2011-06-01

    Elevated levels of inorganic phosphate (P(i)) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of P(i) can also affect muscle velocity, but the molecular basis underlying these effects remains unclear. We directly examined the effect of P(i) (30 mM) on skeletal muscle myosin's ability to translocate actin (V(actin)) in an in vitro motility assay. Manipulation of the pH enabled us to probe rebinding of P(i) to myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of P(i) significantly increased V(actin) at both pH 6.8 and 6.5, causing a doubling of V(actin) at pH 6.5. To probe the mechanisms underlying this increase in speed, we repeated these experiments while varying the ATP concentration. At pH 7.4, the effects of P(i) were highly ATP dependent, with P(i) slowing V(actin) at low ATP (<500 μM), but with a minor increase at 2 mM ATP. The P(i)-induced slowing of V(actin), evident at low ATP (pH 7.4), was minimized at pH 6.8 and completely reversed at pH 6.5. These data were accurately fit with a simple detachment-limited kinetic model of motility that incorporated a P(i)-induced prolongation of the rigor state, which accounted for the slowing of V(actin) at low ATP, and a P(i)-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in V(actin) at high ATP. These findings suggest that P(i) differentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.

  8. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

    PubMed Central

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  9. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis.

    PubMed

    Stossel, T P; Hartwig, J H

    1976-03-01

    Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.

  10. Myosin IIA is critical for organelle distribution and F-actin organization in megakaryocytes and platelets.

    PubMed

    Pertuy, Fabien; Eckly, Anita; Weber, Josiane; Proamer, Fabienne; Rinckel, Jean-Yves; Lanza, François; Gachet, Christian; Léon, Catherine

    2014-02-20

    During proplatelet formation, a relatively homogeneous content of organelles is transported from the megakaryocyte (MK) to the nascent platelets along microtubule tracks. We found that platelets from Myh9(-/-) mice and a MYH9-RD patient were heterogeneous in their organelle content (granules and mitochondria). In addition, Myh9(-/-) MKs have an abnormal cytoplasmic clustering of organelles, suggesting that the platelet defect originates in the MKs. Myosin is not involved in the latest stage of organelle traffic along microtubular tracks in the proplatelet shafts as shown by confocal observations of proplatelet buds. By contrast, it is required for the earlier distribution of organelles within the large MK preplatelet fragments shed into the sinusoid circulation before terminal proplatelet remodeling. We show here that F-actin is abnormally clustered in the cytoplasm of Myh9(-/-) MKs and actin polymerization is impaired in platelets. Myosin IIA is required for normal granule motility and positioning within MKs, mechanisms that may be dependent on organelle traveling and tethering onto F-actin cytoskeleton tracks. Altogether, our results indicate that the distribution of organelles within platelets critically depends on a homogeneous organelle distribution within MKs and preplatelet fragments, which requires myosin IIA.

  11. A mechanical model for the motility of actin filaments on myosin

    NASA Astrophysics Data System (ADS)

    Nicolau, Dan V., Jr.; Fulga, Florin; Nicolau, Dan V.

    2004-03-01

    The interaction of actin filaments with myosin is crucial to cell motility, muscular contraction, cell division and other processes. The in vitro motility assay involves the motion of actin filaments on a substrate coated with myosin, and is used extensively to investigate the dynamics of the actomyosin system. Following on from previous work, we propose a new mechanical model of actin motility on myosin, wherein a filament is modeled as a chain of beads connected by harmonic springs. This imposes a limitation on the "stretching" of the filament. The rotation of one bead with respect to its neighbours is also constrained in similar way. We implemented this model and used Monte Carlo simulations to determine whether it can predict the directionality of filament motion. The principal advantages of this model over our previous one are that we have removed the empirically correct but artificial assumption that the filament moves like a "worm" i.e. the head determines the direction of movement and the rest of the filament "follows" the head as well as the inclusion of dependencies on experimental rate constants (and so also on e.g. ATP concentration) via the cross-bridge cycle.

  12. Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

    PubMed

    Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2004-05-11

    Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

  13. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  14. Cytoplasmic myosin from Drosophila melanogaster

    PubMed Central

    1986-01-01

    Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized. PMID

  15. Continued Expression of Neonatal Myosin Heavy Chain in Adult Dystrophic Skeletal Muscle

    NASA Astrophysics Data System (ADS)

    Bandman, Everett

    1985-02-01

    The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.

  16. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Warshaw, David M.

    2016-01-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite’s intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein–tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  17. Convergence and extension at gastrulation require a myosin IIB dependent cortical actin network

    PubMed Central

    Skoglund, Paul; Rolo, Ana; Chen, Xuejun; Gumbiner, Barry M.; Keller, Ray

    2009-01-01

    Summary Force-producing convergence (narrowing) and extension (lengthening) of tissues by active intercalation of cells along the axis of convergence play a major role in axial morphogenesis during development of both vertebrate and invertebrate embryos, and failure of these processes in human embryos leads to embryonic defects including spina bifida and anencephaly. Here we use Xenopus laevis, a system in which the polarized cell motility that drives this active cell intercalation has been related to development of forces that close the blastopore and elongate the body axis, to examine the role of myosin IIB in convergence and extension. We find that myosin IIB is localized in the cortex of intercalating cells, and that morpholino knockdown of this myosin isoform shows that it is essential for maintenance of a stereotypical, cortical actin cytoskeleton that we visualize with time-lapse fluorescent confocal microscopy. We show that this actin network consists of foci or nodes connected by cables and is polarized relative to the embryonic axis, preferentially cyclically shortening and lengthening parallel to the axis of cell polarization, elongation, and intercalation, and also parallel to the axis of convergence forces during gastrulation. MHC-B-depletion results in disruption of this polarized cytoskeleton, loss of the polarized protrusive activity characteristic of intercalating cells, eventual loss of cell-cell and cell matrix adhesion, and dose-dependent failure of blastopore closure, arguably because of failure to develop convergence forces parallel to the myosin IIB-dependent dynamics of the actin cytoskeleton. These findings bridge the gap between a molecular-scale motor protein and tissue-scale embryonic morphogenesis. PMID:18550716

  18. Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin.

    PubMed

    Liu, Haiyang; Li, Jianchao; Raval, Manmeet H; Yao, Ningning; Deng, Xiaoying; Lu, Qing; Nie, Si; Feng, Wei; Wan, Jun; Yengo, Christopher M; Liu, Wei; Zhang, Mingjie

    2016-01-19

    Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1.

  19. Apical domain polarization localizes actin-myosin activity to drive ratchet-like apical constriction.

    PubMed

    Mason, Frank M; Tworoger, Michael; Martin, Adam C

    2013-08-01

    Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cells exhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twist is required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cell relaxation. We find that during constriction, Rho-associated kinase (Rok) is polarized to the middle of the apical domain (medioapical cortex), separate from adherens junctions. Rok recruits or stabilizes medioapical myosin II (Myo-II), which contracts dynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherin localization and form stable connections between the medioapical contractile network and adherens junctions. Twist is not required for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity of Rok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

  20. Myosin di-phosphorylation and peripheral actin bundle formation as initial events during endothelial barrier disruption.

    PubMed

    Hirano, Mayumi; Hirano, Katsuya

    2016-02-11

    The phosphorylation of the 20-kD myosin light chain (MLC) and actin filament formation play a key role in endothelial barrier disruption. MLC is either mono- or di-phosphorylated (pMLC and ppMLC) at T18 or S19. The present study investigated whether there are any distinct roles of pMLC and ppMLC in barrier disruption induced by thrombin. Thrombin induced a modest bi-phasic increase in pMLC and a robust mono-phasic increase in ppMLC. pMLC localized in the perinuclear cytoplasm during the initial phase, while ppMLC localized in the cell periphery, where actin bundles were formed. Later, the actin bundles were rearranged into stress fibers, where pMLC co-localized. Rho-kinase inhibitors inhibited thrombin-induced barrier disruption and peripheral localization of ppMLC and actin bundles. The double, but not single, mutation of phosphorylation sites abolished the formation of peripheral actin bundles and the barrier disruption, indicating that mono-phosphorylation of MLC at either T18 or S19 is functionally sufficient for barrier disruption. Namely, the peripheral localization, but not the degree of phosphorylation, is suggested to be essential for the functional effect of ppMLC. These results suggest that MLC phosphorylation and actin bundle formation in cell periphery are initial events during barrier disruption.

  1. Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network.

    PubMed

    Almeida, Claudia G; Yamada, Ayako; Tenza, Danièle; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

    2011-06-12

    The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.

  2. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    PubMed

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system.

  3. Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments.

    PubMed

    Okubo, Hiroyuki; Iwai, Masanori; Iwai, Sosuke; Chaen, Shigeru

    2010-05-28

    Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.

  4. Actin-myosin network influences morphological response of neuronal cells to altered osmolarity.

    PubMed

    Bober, Brian G; Love, James M; Horton, Steven M; Sitnova, Mariya; Shahamatdar, Sina; Kannan, Ajay; Shah, Sameer B

    2015-04-01

    Acute osmotic fluctuations in the brain occur during a number of clinical conditions and can result in a variety of adverse neurological symptoms. Osmotic perturbation can cause changes in the volumes of intra- and extracellular fluid and, due to the rigidity of the skull, can alter intracranial pressure thus making it difficult to analyze purely osmotic effects in vivo. The present study aims to determine the effects of changes in osmolarity on SH-SY5Y human neuroblastoma cells in vitro, and the role of the actin-myosin network in regulating this response. Cells were exposed to hyper- or hypoosmotic media and morphological and cytoskeletal responses were recorded. Hyperosmotic shock resulted in a drop in cell body volume and planar area, a persisting shape deformation, and increases in cellular translocation. Hypoosmotic shock did not significantly alter planar area, but caused a transient increase in cell body volume and an increase in cellular translocation via the development of small protrusions rich in actin. Disruption of the actin-myosin network with latrunculin and blebbistatin resulted in changes to volume and shape regulation, and a decrease in cellular translocation. In both osmotic perturbations, no apparent disruptions to cytoskeletal integrity were observed by light microscopy. Overall, because osmotically induced changes persisted even after volume regulation occurred, it is possible that osmotic stress may play a larger role in neurological dysfunction than currently believed.

  5. Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton

    PubMed Central

    1996-01-01

    The organization of the actin cytoskeleton plays a critical role in cell physiology in motile and nonmotile organisms. Nonetheless, the function of the actin based motor molecules, members of the myosin superfamily, is not well understood. Deletion of MYO3, a yeast gene encoding a "classic" myosin I, has no detectable phenotype. We used a synthetic lethality screen to uncover genes whose functions might overlap with those of MYO3 and identified a second yeast myosin 1 gene, MYO5. MYO5 shows 86 and 62% identity to MYO3 across the motor and non- motor regions. Both genes contain an amino terminal motor domain, a neck region containing two IQ motifs, and a tail domain consisting of a positively charged region, a proline-rich region containing sequences implicated in ATP-insensitive actin binding, and an SH3 domain. Although myo5 deletion mutants have no detectable phenotype, yeast strains deleted for both MYO3 and MYO5 have severe defects in growth and actin cytoskeletal organization. Double deletion mutants also display phenotypes associated with actin disorganization including accumulation of intracellular membranes and vesicles, cell rounding, random bud site selection, sensitivity to high osmotic strength, and low pH as well as defects in chitin and cell wall deposition, invertase secretion, and fluid phase endocytosis. Indirect immunofluorescence studies using epitope-tagged Myo5p indicate that Myo5p is localized at actin patches. These results indicate that MYO3 and MYO5 encode classical myosin I proteins with overlapping functions and suggest a role for Myo3p and Myo5p in organization of the actin cytoskeleton of Saccharomyces cerevisiae. PMID:8682864

  6. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    PubMed

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning

    2009-08-01

    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style.

  7. The Role of Caldesmon and its Phosphorylation by ERK on the Binding Force of Unphosphorylated Myosin to Actin

    PubMed Central

    Roman, Horia Nicolae; Zitouni, Nedjma B.; Kachmar, Linda; Benedetti, Andrea; Sobieszek, Apolinary; Lauzon, Anne-Marie

    2014-01-01

    Background Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low ATP consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin. Methods To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb) in the absence or presence of caldesmon, ERK-phosphorylated CaD, or CaD plus tropomyosin. Results Funb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06 ± 0.01 pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment. Conclusions CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels. General Significance This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state. PMID:25108062

  8. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

    PubMed

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

  9. Ginsenoside Rg1 Protects against Oxidative Stress-induced Neuronal Apoptosis through Myosin IIA-actin Related Cytoskeletal Reorganization

    PubMed Central

    Wang, Yan; Liu, Qian; Xu, Yingqiong; Zhang, Yuanyuan; Lv, Yanni; Tan, Yisha; Jiang, Nan; Cao, Guosheng; Ma, Xiaonan; Wang, Jingrong; Cao, Zhengyu; Yu, Boyang; Kou, Junping

    2016-01-01

    Oxidative stress-induced cytoskeletal dysfunction of neurons has been implicated as a crucial cause of cell apoptosis or death in the central nervous system (CNS) diseases, such as neurodegenerative and psychiatric diseases. The application of neuroprotectants rescuing the neurons from cytoskeletal damage and apoptosis can be a potential treatment for these CNS diseases. Ginsenoside Rg1 (Rg1), one of the major active components of ginseng, has been reported possessing notable neuroprotective activities. However, there is rare report about its effect on cytoskeleton and its undergoing mechanism. The current study is to reveal the regulatory effects of Rg1 on cytoskeletal and morphological lesion in oxidative stress-induced neuronal apoptosis. The results demonstrated that pre-treatment with Rg1 (0.1-10 μM) attenuated hydrogen peroxide (H2O2)-induced neuronal apoptosis and oxidative stress through reducing the intracellular reactive oxygen species (ROS) production and methane dicarboxylic aldehyde (MDA) level. The Rg1 treatment also abolished H2O2-induced morphological changes, including cell rounding, membrane blebbing, neurite retraction and nuclei condensation, which were generated by myosin IIA-actin interaction. These effects were mediated via the down-regulation of caspase-3, ROCK1 (Rho-associated kinase1) activation and myosin light chain (MLC, Ser-19) phosphorylation. Furthermore, inhibiting myosin II activity with blebbistatin partly blocked the neuroprotective effects of Rg1. The computer-aided homology modelling revealed that Rg1 preferentially positioned in the actin binding cleft of myosin IIA and might block the binding of myosin IIA to actin filaments. Accordingly, the neuroprotective mechanism of Rg1 is related to the activity that inhibits myosin IIA-actin interaction and the caspase-3/ROCK1/MLC signaling pathway. These findings put some insights into the unique neuroprotective properties of Rg1 associated with the regulation of myosin IIA-actin

  10. HDAC3-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity*

    PubMed Central

    Samant, Sadhana A.; Courson, David S.; Sundaresan, Nagalingam R.; Pillai, Vinodkumar B.; Tan, Minjia; Zhao, Yingming; Shroff, Sanjeev G.; Rock, Ronald S.; Gupta, Mahesh P.

    2011-01-01

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:21177250

  11. HDAC3-dependent reversible lysine acetylation of cardiac myosin heavy chain isoforms modulates their enzymatic and motor activity.

    PubMed

    Samant, Sadhana A; Courson, David S; Sundaresan, Nagalingam R; Pillai, Vinodkumar B; Tan, Minjia; Zhao, Yingming; Shroff, Sanjeev G; Rock, Ronald S; Gupta, Mahesh P

    2011-02-18

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

  12. Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles.

    PubMed

    Ali, M Yusuf; Vilfan, Andrej; Trybus, Kathleen M; Warshaw, David M

    2016-11-15

    Myosin Va (myoVa) is a processive, actin-based molecular motor essential for intracellular cargo transport. When a cargo is transported by an ensemble of myoVa motors, each motor faces significant physical barriers and directional challenges created by the complex actin cytoskeleton, a network of actin filaments and actin bundles. The principles that govern the interaction of multiple motors attached to the same cargo are still poorly understood. To understand the mechanical interactions between multiple motors, we developed a simple in vitro model in which two individual myoVa motors labeled with different-colored Qdots are linked via a third Qdot that acts as a cargo. The velocity of this two-motor complex was reduced by 27% as compared to a single motor, whereas run length was increased by only 37%, much less than expected from multimotor transport models. Therefore, at low ATP, which allowed us to identify individual motor steps, we investigated the intermotor dynamics within the two-motor complex. The randomness of stepping leads to a buildup of tension in the linkage between motors-which in turn slows down the leading motor-and increases the frequency of backward steps and the detachment rate. We establish a direct relationship between the velocity reduction and the distribution of intermotor distances. The analysis of run lengths and dwell times for the two-motor complex, which has only one motor engaged with the actin track, reveals that half of the runs are terminated by almost simultaneous detachment of both motors. This finding challenges the assumptions of conventional multimotor models based on consecutive motor detachment. Similar, but even more drastic, results were observed with two-motor complexes on actin bundles, which showed a run length that was even shorter than that of a single motor.

  13. A single-headed fission yeast myosin V transports actin in a tropomyosin-dependent manner

    PubMed Central

    Tang, Qing; Krementsova, Elena B.; Bookwalter, Carol S.; Lord, Matthew

    2016-01-01

    Myo51, a class V myosin in fission yeast, localizes to and assists in the assembly of the contractile ring, a conserved eukaryotic actomyosin structure that facilitates cytokinesis. Rng8 and Rng9 are binding partners that dictate the cellular localization and function of Myo51. Myo51 was expressed in insect cells in the presence or absence of Rng8/9. Surprisingly, electron microscopy of negatively stained images and hydrodynamic measurements showed that Myo51 is single headed, unlike most class V myosins. When Myo51–Rng8/9 was bound to actin-tropomyosin, two attachment sites were observed: the typical ATP-dependent motor domain attachment and a novel ATP-independent binding of the tail mediated by Rng8/9. A modified motility assay showed that this additional binding site anchors Myo51–Rng8/9 so that it can cross-link and slide actin-tropomyosin filaments relative to one another, functions that may explain the role of this motor in contractile ring assembly. PMID:27432898

  14. The Intensity Of The 2.7nm Reflection As A Constraint For Models Of Myosin Docking To Actin

    SciTech Connect

    Reconditi, Massimo; Irving, Tom C.

    2009-03-16

    Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data (Mendelson and Morris, 1997 PNAS 94:8533; Holmes et al. 2003 Nature 425:423). We have used data from small angle X-ray fiber diffraction from living muscle to check the predictions of these models. Whole sartorius muscles from Rana pipiens were mounted in a chamber containing Ringer's solution at 10 C and at rest length at the BioCAT beamline (18 ID, Advanced Photon Source, Argonne, IL-U.S.A.). The muscles were activated by electrical stimulation and the force was recorded with a muscle lever system type 300B (Aurora Scientific). X-ray patterns were collected with 1s total exposures at rest and during isometric contraction out to 0.5 nm{sup -1} in reciprocal space, as the higher angle reflections are expected to be more sensitive to the arrangement of myosin heads on actin. We observed that during isometric contraction the meridional reflection originating from the 2.73nm repeat of the actin monomers along the actin filament increases its intensity by a factor 2.1 {+-} 0.2 relative to rest. Among the models tested, Holmes et al. fits the data when the actin filament is decorated with 30-40% the total available myosin heads, a fraction similar to that estimated with fast single fiber mechanics by Piazzesi et al. (2007, Cell 131:784). However, when the mismatch between the periodicities of actin and myosin filaments is taken into account, none of the models can reproduce the fiber diffraction data. We suggest that the fiber diffraction data should be used as a further constraint on new high resolution models for the docking of the myosin heads on actin.

  15. Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

    PubMed Central

    Lu, Hailong; Fagnant, Patricia M.; Bookwalter, Carol S.; Joel, Peteranne; Trybus, Kathleen M.

    2015-01-01

    Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD. PMID:26153420

  16. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    PubMed

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

  17. Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model.

    PubMed

    Walcott, Sam; Docken, Steffen; Harris, Samantha P

    2015-01-06

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.

  18. Effects of Cardiac Myosin Binding Protein-C on Actin Motility Are Explained with a Drag-Activation-Competition Model

    PubMed Central

    Walcott, Sam; Docken, Steffen; Harris, Samantha P.

    2015-01-01

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin. PMID:25564844

  19. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

    PubMed Central

    1995-01-01

    The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion. PMID:7490299

  20. Porcine myosin-VI: characterization of a new mammalian unconventional myosin

    PubMed Central

    1994-01-01

    We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain. PMID:7929586

  1. Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin

    PubMed Central

    Liu, Haiyang; Li, Jianchao; Raval, Manmeet H; Yao, Ningning; Deng, Xiaoying; Lu, Qing; Nie, Si; Feng, Wei; Wan, Jun; Yengo, Christopher M; Liu, Wei; Zhang, Mingjie

    2016-01-01

    Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1. DOI: http://dx.doi.org/10.7554/eLife.12856.001 PMID:26785147

  2. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  3. Adaptations in myosin heavy chain profile in chronically unloaded muscles

    NASA Technical Reports Server (NTRS)

    Talmadge, R. J.; Roy, R. R.; Bodine-Fowler, S. C.; Pierotti, D. J.; Edgerton, V. R.

    1995-01-01

    In this review, myosin heavy chain (MHC) adaptations in response to several models of decreased neuromuscular activity (i.e. electrical activation and loading of a muscle) are evaluated. In each of these "reduced-activity" models it is important to: a) quantify the changes in electrical activation of the muscle as a result of the intervention; b) quantify the forces generated by the muscle; and c) determine whether the neuromuscular junction remains normal. Most of the models, including spaceflight, hindlimb suspension, spinal cord isolation, spinal cord transection, denervation, and limb immobilization in a shortened position, result in increases in the percentage of fast MHCs (or fast MHC mRNA) in normally slow rat muscles. It also can be inferred from histochemical data that increases in fast MHCs occur with TTX application and bed rest. The only "reduced-activity" model to consistently increase slow muscle myosin mRNA, and slow fibers is limb immobilization in a stretched position; however, this model results in at least a temporary increase in tension. It appears that the most common feature of these models that might induce MHC adaptations is the modification in loading rather than a change in the neuromuscular activity.

  4. Compliance Accelerates Relaxation in Muscle by Allowing Myosin Heads to Move Relative to Actin.

    PubMed

    Campbell, Kenneth S

    2016-02-02

    The mechanisms that limit the speed at which striated muscles relax are poorly understood. This work presents, to our knowledge, novel simulations that show that the time course of relaxation is accelerated by interfilamentary movement resulting from series compliance; force drops faster when myosin heads move relative to actin during relaxation. This insight was obtained by using cross-bridge distribution techniques to simulate the mechanical behavior of half-sarcomeres that were connected in series with springs of varying stiffness. (The springs mimic the combined effects of half-sarcomere heterogeneity and muscle's series elastic component.) Half-sarcomeres that shortened by >∼10 nm when they were activated subsequently relaxed with a biphasic profile; force initially declined slowly and approximately linearly before collapsing with a fast exponential time course. Stretches imposed during the linear phase quickened relaxation, while shortening movements prolonged the time course. These predictions are consistent with data from experiments performed by many other groups using single muscle fibers and isolated myofibrils. When half-sarcomeres were linked to stiff springs (so that they did not shorten appreciably during the simulations), force relaxed with a slow exponential time course and did not show biphasic behavior. Together, these results suggest that fast relaxation of striated muscle is an emergent property that reflects multiscale interactions within the muscle architecture. The nonlinear behavior during relaxation reflects perturbations to the dynamic coupling of regulated binding sites and cycling myosin heads that are induced by interfilamentary movement.

  5. Compliance Accelerates Relaxation in Muscle by Allowing Myosin Heads to Move Relative to Actin

    PubMed Central

    Campbell, Kenneth S.

    2016-01-01

    The mechanisms that limit the speed at which striated muscles relax are poorly understood. This work presents, to our knowledge, novel simulations that show that the time course of relaxation is accelerated by interfilamentary movement resulting from series compliance; force drops faster when myosin heads move relative to actin during relaxation. This insight was obtained by using cross-bridge distribution techniques to simulate the mechanical behavior of half-sarcomeres that were connected in series with springs of varying stiffness. (The springs mimic the combined effects of half-sarcomere heterogeneity and muscle’s series elastic component.) Half-sarcomeres that shortened by >∼10 nm when they were activated subsequently relaxed with a biphasic profile; force initially declined slowly and approximately linearly before collapsing with a fast exponential time course. Stretches imposed during the linear phase quickened relaxation, while shortening movements prolonged the time course. These predictions are consistent with data from experiments performed by many other groups using single muscle fibers and isolated myofibrils. When half-sarcomeres were linked to stiff springs (so that they did not shorten appreciably during the simulations), force relaxed with a slow exponential time course and did not show biphasic behavior. Together, these results suggest that fast relaxation of striated muscle is an emergent property that reflects multiscale interactions within the muscle architecture. The nonlinear behavior during relaxation reflects perturbations to the dynamic coupling of regulated binding sites and cycling myosin heads that are induced by interfilamentary movement. PMID:26840730

  6. Structural Characterization of the Binding of Myosin*ADP*Pi to Actin in Permeabilized Rabbit Psoas Muscle

    SciTech Connect

    Xu,S.; Gu, J.; Belknap, B.; White, H.; Yu, L.

    2006-01-01

    When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A{center_dot}M{center_dot}ADP and A{center_dot}M) and the weakly bound A{center_dot}M{center_dot}ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ('stereospecific' attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A{center_dot}M{center_dot}ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A{center_dot}M{center_dot}ADP{center_dot}P{sub i}, however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A{center_dot}M{center_dot}ADP{center_dot}P{sub i} state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M{center_dot}ATP, M{center_dot}ADP{center_dot}P{sub i} states and the weakly attached A{center_dot}M{center_dot}ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A{center_dot}M{center_dot}ADP{center_dot}P{sub i}. The series of experiments presented in this article were carried out under relaxing conditions at 25{sup o}C, where {approx}95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in

  7. Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum.

    PubMed Central

    Warrick, H M; De Lozanne, A; Leinwand, L A; Spudich, J A

    1986-01-01

    The 2116-amino acid myosin heavy chain sequence from Dictyostelium discoideum was determined from DNA sequence analysis of the cloned gene. The gene product can be divided into two distinct regions, a globular head region and a long alpha-helical, rod-like tail. In comparisons with nematode and mammalian muscle myosins, specific areas of the head region are highly conserved. These areas presumably reflect conserved functional and structural domains. Certain features that are present in the head region of nematode and mammalian muscle myosins, and that have been assumed to be important for myosin function, are missing in the Dictyostelium myosin sequence. The protein sequence of the Dictyostelium tail region is very poorly conserved with respect to the other myosins but displays the periodicities similar to those of muscle myosins. These periodicities are believed to play a role in filament formation. The 196-residue repeating unit that determines the 14.3-nm repeat seen in muscle thick filaments, the 28-residue charge repeating unit, and the 1,4 hydrophobic repeat previously described for the nematode myosin are all present in the Dictyostelium myosin rod sequence, suggesting that the filament structures of muscle and Dictyostelium myosins must be similar. PMID:3540939

  8. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    SciTech Connect

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  9. EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

    PubMed

    Prasath, Thiruketheeswaran; Greven, Hartmut; D'Haese, Jochen

    2012-06-01

    Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

  10. Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.

    PubMed

    Raval, Manmeet H; Quintero, Omar A; Weck, Meredith L; Unrath, William C; Gallagher, James W; Cui, Runjia; Kachar, Bechara; Tyska, Matthew J; Yengo, Christopher M

    2016-10-21

    Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

  11. Quantitative Proteomics Reveals Myosin and Actin as Promising Saliva Biomarkers for Distinguishing Pre-Malignant and Malignant Oral Lesions

    PubMed Central

    Onsongo, Getiria; Stone, Matthew D.; Chen, Xiao-Bing; Kooren, Joel A.; Refsland, Eric W.; Griffin, Robert J.; Ondrey, Frank G.; Wu, Baolin; Le, Chap T.; Rhodus, Nelson L.; Carlis, John V.; Griffin, Timothy J.

    2010-01-01

    Background Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need. Methodology/Principal Findings To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells. Conclusions/Significance Salivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early. PMID:20567502

  12. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    PubMed

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  13. Kinetic properties and small-molecule inhibition of human myosin-6

    PubMed Central

    Heissler, Sarah M.; Selvadurai, Jayashankar; Bond, Lisa M.; Fedorov, Roman; Kendrick-Jones, John; Buss, Folma; Manstein, Dietmar J.

    2012-01-01

    Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion. PMID:22884421

  14. Myosin heavy chain expression in respiratory muscles of the rat.

    PubMed

    LaFramboise, W A; Watchko, J F; Brozanski, B S; Daood, M J; Guthrie, R D

    1992-03-01

    Myosin heavy chain (MHC) isoforms of hind limb adult rat muscles and muscles with a range of respiratory activities were analyzed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis technique that allowed electrophoretic separation of the three fast and one slow MHC isoform found in typical rat muscle. Costal and crural diaphragm muscle samples expressed a mixture of MHC beta/slow, MHC2A, and MHC2X but little MHC2B. In contrast, MHC2B was the dominant MHC isoform in the genioglossus, intercostal, and three abdominal muscles, all of which exhibited minimal expression of MHC beta/slow. The amount of MHC2X (relative to total MHC composition) was similar in the diaphragm, genioglossus, and transversus abdominis muscles, while considerably less was detected in the rectus abdominis and external oblique muscles. These results indicate that MHC2X is broadly and variably distributed among respiratory muscles. Furthermore, these data suggest that a large portion of 2X fibers (containing MHC2X), which cannot be detected by standard histochemical analysis, may be present in the genioglossus and transversus abdominis muscles as has been demonstrated for the diaphragm muscle. We speculate that an association exists between the level of MHC2X expression and frequency of respiratory recruitment.

  15. Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-D-aspartate Receptor Trafficking during Synaptic Plasticity.

    PubMed

    Bu, Yunfei; Wang, Ning; Wang, Shaoli; Sheng, Tao; Tian, Tian; Chen, Linlin; Pan, Weiwei; Zhu, Minsheng; Luo, Jianhong; Lu, Wei

    2015-10-16

    N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity.

  16. Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes

    PubMed Central

    Kollmar, Martin; Hatje, Klas

    2014-01-01

    Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times

  17. The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

    PubMed

    Henson, John H; Ditzler, Casey E; Germain, Aphnie; Irwin, Patrick M; Vogt, Eric T; Yang, Shucheng; Wu, Xufeng; Shuster, Charles B

    2017-03-01

    Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

  18. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity*

    PubMed Central

    Samant, Sadhana A.; Pillai, Vinodkumar B.; Sundaresan, Nagalingam R.; Shroff, Sanjeev G.; Gupta, Mahesh P.

    2015-01-01

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:25911107

  19. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

    PubMed

    Samant, Sadhana A; Pillai, Vinodkumar B; Sundaresan, Nagalingam R; Shroff, Sanjeev G; Gupta, Mahesh P

    2015-06-19

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

  20. Monomeric Acanthamoeba myosins I support movement in vitro.

    PubMed

    Albanesi, J P; Fujisaki, H; Hammer, J A; Korn, E D; Jones, R; Sheetz, M P

    1985-07-25

    Acanthamoeba myosins IA and IB were found to have molecular weights of 159,000 and 150,000 and Stokes radii of 6.2 and 5.9 nm, respectively. Both enzymes have frictional ratios of 1.7. Myosin IA consists of 22% alpha-helix, 32% beta-structure, and 46% unordered structure, while myosin IB is 16% alpha-helix, 46% beta-structure, and 38% unordered. Both myosins remain monomolecular under conditions in which other myosins form filaments. Beads coated with myosin IA or IB move unidirectionally on actin cables of Nitella. Movement requires ATP and phosphorylation of the myosin I heavy chain which is also required for actin-activated Mg2+-ATPase activity. Movement is inhibited by myosin I antiserum that inhibits actin-activated ATPase activity. These studies establish that these nonfilamentous, monomolecular myosins with single heavy chains of 130,000 and 125,000 daltons (IA and IB, respectively) can support actin-dependent movement analogous to that supported by filamentous myosins.

  1. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    PubMed

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  2. Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers.

    PubMed

    Losinno, Antonella D; Morales, Alfonsina; Fernández, Dario; Lopez, Luis A

    2012-05-01

    In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.

  3. Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR.

    PubMed

    Klein, Jennifer C; Burr, Adam R; Svensson, Bengt; Kennedy, Daniel J; Allingham, John; Titus, Margaret A; Rayment, Ivan; Thomas, David D

    2008-09-02

    We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.

  4. Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform

    PubMed Central

    1990-01-01

    Motile activities such as chemotaxis and phagocytosis, which occur in Dictyostelium cells lacking myosin II, may be dependent upon myosin I. To begin to explore this possibility, we have engineered a disruption of the Dictyostelium myosin I heavy chain (DMIHC) gene described recently (Jung, G., C. L. Saxe III, A. R. Kimmel, and J. A. Hammer III. 1989. Proc. Natl. Acad. Sci. USA. 86:6186-6190). The double-crossover, gene disruption event that occurred resulted in replacement of the middle approximate one-third of the gene with the neomycin resistance marker. The resulting cells are devoid of both the 3.6-kb DMIHC gene transcript and the 124-kD DMIHC polypeptide. DMIHC- cells are capable of chemotactic streaming and aggregation, but these processes are delayed. Furthermore, the rate of phagocytosis by DMIHC- cells is reduced, as assessed by growth rate on lawns of heat-killed bacteria and on the initial rate of uptake of FITC-labeled bacteria. Therefore, this Dictyostelium myosin I isoform appears to play a role in supporting chemotaxis and phagocytosis, but it is clearly not required for these processes to occur. Using a portion of the DMIHC gene as a probe, we have cloned three additional Dictyostelium small myosin heavy chain genes. Comparison of these four genes with three genes described recently by Titus et al. (Titus, M. A., H. M. Warrick, and J. A. Spudich. 1989. Cell Reg. 1:55-63) indicates that there are at least five small myosin heavy chain genes in Dictyostelium. The probability that there is considerable overlap of function between these small myosin isoforms indicates that multiple gene disruptions within a single cell may be necessary to generate a more striking myosin I- phenotype. PMID:2141028

  5. A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex

    PubMed Central

    Evangelista, Marie; Klebl, Bert M.; Tong, Amy H.Y.; Webb, Bradley A.; Leeuw, Thomas; Leberer, Ekkehard; Whiteway, Malcolm; Thomas, David Y.; Boone, Charles

    2000-01-01

    Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly. PMID:10648568

  6. Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain.

    PubMed Central

    Grinnell, F; Ho, C H

    1995-01-01

    We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626021

  7. Is sarcomere lattice geometry optimal? Analysis of several potential virtual polygon cross-sectional patterns for actin and myosin myofilaments in muscle.

    PubMed

    Kepner, Gordon R

    2014-09-01

    The hexagonal arrangement of actin filaments in skeletal muscle is not the fundamental geometrical or functioning myofilament unit. This analysis of several possible sarcomere lattice geometries for the arrangement of the actin and myosin filaments identifies several geometrical constraints that can be compared for their effect on muscle sarcomere functioning and efficiency. Three distinct virtual polygons, with myosins at their vertices and that tessellate the plane, are compared for both centered actin and perimeter actin arrangements. The analysis evaluates the optimal ratio of myosin to actin filaments, the packing density, and the effect on new myofilament formation in muscle hypertrophy for the various lattice geometries. The results support the view that no single measure of geometrical effectiveness can evaluate definitively the efficiency of any particular arrangement of the myofilaments. The analysis provides quantitative measures of several parameters that, taken overall, support the effectiveness of the myofilament arrangement in Nature. It provides a new definition of the fundamental myofilament unit (FMU). It is possible to calculate the number of actin and myosin myofilaments that need to be added to each polygon arrangement of the myofilaments to create a new FMU for that specific geometry. This leads to useful conclusions about the biochemical efficiency involved in where such units arise in the course of muscle hypertrophy. It supports the idea that the evolutionary endpoint for optimizing muscle's force-generating function can be better understood via the concepts of a FMU and the polygon arrangement of the sarcomere lattice geometry.

  8. Ligand-induced changes in the location of actin, myosin, 95K (alpha- actinin), and 120K protein in amebae of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier

  9. Re-evaluating the roles of myosin 18Aα and F-actin in determining Golgi morphology.

    PubMed

    Bruun, Kyle; Beach, Jordan R; Heissler, Sarah M; Remmert, Kirsten; Sellers, James R; Hammer, John A

    2017-03-22

    The peri-centrosomal localization and morphology of the Golgi apparatus depends largely on the microtubule cytoskeleton and the microtubule motor protein dynein. Recent studies proposed that myosin 18Aα (M18Aα) also contributes to Golgi morphology by binding the Golgi protein GOLPH3 and walking along adjacent actin filaments to stretch the Golgi into its classic ribbon structure. Biochemical analyses have shown, however, that M18A is not an actin-activated ATPase and lacks motor activity. Our goal, therefore, was to define the precise molecular mechanism by which M18Aα determines Golgi morphology. We show that purified M18Aα remains inactive in the presence of GOLPH3, arguing against the Golgi-specific activation of the myosin. Using M18A-specific antibodies and expression of GFP-tagged M18Aα, we find no evidence that it localizes to the Golgi. Moreover, several cell lines with reduced or eliminated M18Aα expression exhibited normal Golgi morphology. Interestingly, actin filament disassembly resulted in a marked reduction in lateral stretching of the Golgi in both control and M18Aα-deficient cells. Importantly, this reduction was accompanied by an expansion of the Golgi in the vertical direction, vertical movement of the centrosome, and increases in the height of both the nucleus and the cell. Collectively, our data indicate that M18Aα does not localize to the Golgi or play a significant role in determining its morphology, and suggest that global F-actin disassembly alters Golgi morphology indirectly by altering cell shape. This article is protected by copyright. All rights reserved.

  10. Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions.

    PubMed Central

    Kirshenbaum, K.; Papp, S.; Highsmith, S.

    1993-01-01

    Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact. Compared to pPDM-S1, the fluorescence intensity of pPDM-S1 *MgADP is increased 50%(compared to 8 and 31% increases, respectively, for MgADP and MgATP binding to S1). Compared to acto-pPDM-S1, the absolute value of the product of the net effective electric charge at the actin binding interface of acto-pPDM-S1 *MgADP increases 7.3 esu2 (compared to a 0.9 esu2 decrease and an 11.0 esu2

  11. Myosin IIB and F-actin control apical vacuolar morphology and histamine-induced trafficking of H-K-ATPase-containing tubulovesicles in gastric parietal cells.

    PubMed

    Natarajan, Paramasivam; Crothers, James M; Rosen, Jared E; Nakada, Stephanie L; Rakholia, Milap; Okamoto, Curtis T; Forte, John G; Machen, Terry E

    2014-04-15

    Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle.

  12. Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin.

    PubMed

    Adhikari, Arjun S; Kooiker, Kristina B; Sarkar, Saswata S; Liu, Chao; Bernstein, Daniel; Spudich, James A; Ruppel, Kathleen M

    2016-12-13

    Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

  13. A novel mammalian myosin I from rat with an SH3 domain localizes to Con A-inducible, F-actin-rich structures at cell-cell contacts

    PubMed Central

    1995-01-01

    In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head domain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca(2+)-binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and alpha-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact. PMID:7730414

  14. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  15. Random walk of processive, quantum dot-labeled myosin Va molecules within the actin cortex of COS-7 cells.

    PubMed

    Nelson, Shane R; Ali, M Yusuf; Trybus, Kathleen M; Warshaw, David M

    2009-07-22

    Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (actin network.

  16. Random Walk of Processive, Quantum Dot-Labeled Myosin Va Molecules within the Actin Cortex of COS-7 Cells

    PubMed Central

    Nelson, Shane R.; Ali, M. Yusuf; Trybus, Kathleen M.; Warshaw, David M.

    2009-01-01

    Abstract Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (≤0.5 s) of ATP-sensitive linear motion. The mean velocity of these trajectories was 604 nm/s and independent of the number of myoVa molecules attached to the Qdot. With high time (16.6 ms) and spatial (15 nm) resolution imaging, Qdot-labeled myoVa moved with sequential 75 nm steps per head, at a rate of 16 s−1, similarly to myoVa in vitro. Monte Carlo modeling suggests that the random nature of the trajectories represents processive myoVa motors undergoing a random walk through the dense and randomly oriented cortical actin network. PMID:19619465

  17. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  18. Movement and force produced by a single myosin head

    NASA Astrophysics Data System (ADS)

    Molloy, J. E.; Burns, J. E.; Kendrick-Jones, J.; Tregear, R. T.; White, D. C. S.

    1995-11-01

    MUSCLE contraction is driven by the cyclical interaction of myosin with actin, coupled to the breakdown of ATP. Studies of the interaction of filamentous myosin1 and of a double-headed proteolytic fragment, heavy meromyosin (HMM)2,3, with actin have demonstrated discrete mechanical events, arising from stochastic interaction of single myosin molecules with actin. Here we show, using an optical-tweezers transducer2,4, that a single myosin subfragment-1 (S1), which is a single myosin head, can act as an independent generator of force and movement. Our analysis accounts for the broad distribution of displacement amplitudes observed, and indicates that the underlying movement (working stroke) produced by a single acto-Sl interaction is ˜4 nm, considerably shorter than previous estimates1-3,5 but consistent with structural data6. We measure the average force generated by S1 or HMM to be at least 1.7 pN under isometric conditions.

  19. Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed.

    PubMed

    Shaffer, Justin F; Kier, William M

    2012-01-15

    The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds.

  20. The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

    PubMed

    Collings, David A; Harper, John D I; Vaughn, Kevin C

    2003-12-01

    We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

  1. Masseter Myosin Heavy Chain Composition Varies With Mandibular Asymmetry

    PubMed Central

    Raoul, Gwénaël; Rowlerson, Anthea; Sciote, James; Codaccioni, Emmanuel; Stevens, Laurence; Maurage, Claude-Alain; Duhamel, Alain; Ferri, Joël

    2014-01-01

    Human jaw dysmorphologies are frequent and often affect young patients, resulting in malocclusion of teeth and inappropriate jaw relationships. Treatment is performed by means of orthodontics with orthognathic surgery as required. Mandibular asymmetry is one of the most frequent dysmorphologies, but in many cases, the specific cause is unknown. In healthy patients who were undergoing orthognathic surgery for correction of malocclusion, we tested the hypothesis that masseter muscle phenotype composition, which determines contractile properties, was different between sides in patients with mandibular asymmetry but not in those without mandibular asymmetry. After cephalometric analysis, 50 patients from whom we obtained samples of both right and left masseter muscles were separated into 2 groups: with or without mandibular lateral deviation. Samples were immunostained with myosin-isoform–specific antibodies to identify 4 skeletal muscle fiber types, and their fiber areas and proportions were measured. Two-tailed Wilcoxon test for paired samples was used to compare the 4 fiber-type compositions by means of percent occupancy and mean fiber area on both sides. Patients with mandibular asymmetry were associated with a significant increase of type II fiber occupancy (P = 0.0035) on the same side as the deviation. This finding that masseter muscle phenotype is significantly linked to mandibular asymmetry is of relevance to physiotherapeutic and surgical managements of jaw discrepancies and merits further investigation in the light of its possible role in the etiology of this condition. PMID:21586952

  2. The Hill model for binding myosin S1 to regulated actin is not equivalent to the McKillop-Geeves model.

    PubMed

    Mijailovich, Srboljub M; Li, Xiaochuan; Griffiths, R Hugh; Geeves, Michael A

    2012-03-16

    The Hill two-state cooperativity model and the McKillop-Geeves (McK-G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338-2349]. Here, we compared the Hill model and the McK-G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK-G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only K(B) and K(T) show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK-G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use.

  3. Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail

    PubMed Central

    1987-01-01

    We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity. PMID:3040773

  4. Myosin flares and actin leptomeres as myofibril assembly/disassembly intermediates in sonic muscle fibers.

    PubMed

    Nahirney, Patrick C; Fischman, Donald A; Wang, Kuan

    2006-04-01

    The sonic muscle of type 1 male midshipman fish produces loud and enduring mating calls. Each sonic muscle fiber contains a tubular contractile apparatus with radially arranged myofibrillar plates encased in a desmin-rich cytoskeleton that is anchored to broad Z bands (approximately 1.2 micro m wide). Immunomicroscopy has revealed patches of myosin-rich "flares" emanating from the contractile tubes into the peripheral sarcoplasm along the length of the fibers. These flares contain swirls of thick filaments devoid of associated thin filaments. In other regions of the sarcoplasm at the inner surface of the sarcolemma and near Z bands, abundant ladder-like leptomeres occur with rungs every 160 nm. Leptomeres consist of dense arrays of filaments (approximately 4 nm) with a structure that resembles myofibrillar Z band structure. We propose that flares and leptomeres are distinct filamentous arrays representing site-specific processing of myofibrillar components during the assembly and disassembly of the sarcomere. Recent reports that myosin assembles into filamentous aggregates before incorporating into the A band in the skeletal muscles of vertebrates and Caenorhabditis elegans suggest that sonic fibers utilize a similar pathway. Thus, sonic muscle fibers, with their tubular design and abundant sarcoplasmic space, may provide an attractive muscle model to identify myofibrillar intermediates by structural and molecular techniques.

  5. Fission Yeast Myosin-I, Myo1p, Stimulates Actin Assembly by Arp2/3 Complex and Shares Functions with Wasp

    PubMed Central

    Lee, Wei-Lih; Bezanilla, Magdalena; Pollard, Thomas D.

    2000-01-01

    Fission yeast myo1+ encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1+ is not lethal, Δmyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1+ is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Δmyo1 defects. However, it fails to rescue the Δmyo1 Δwsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity. PMID:11076964

  6. Correlation between the distribution of smooth muscle or non muscle myosins and alpha-smooth muscle actin in normal and pathological soft tissues.

    PubMed

    Benzonana, G; Skalli, O; Gabbiani, G

    1988-01-01

    The distribution of smooth muscle (SM) and non muscle myosins was compared with that of alpha-SM actin in various normal and pathological tissues and in cultured cells by means of indirect immunofluorescence using a monoclonal antibody specific for alpha-SM actin [anti-alpha sm-1, Skalli et al., 1986b] and two polyclonal antibodies raised against bovine aortic myosin (ABAM) and human platelet myosin (AHPM), respectively. In normal tissues ABAM stained vascular and parenchymal smooth muscle cells (SMC), myoepithelial cells and myoid cells of the testis in a pattern similar to that reported by other authors with antisera raised against non vascular SM myosin. Cells stained with ABAM were always positive for anti-alpha sm-1. In human and experimental atheromatous plaques, most cells were positive for AHPM; a variable proportion was also stained for ABAM plus anti-alpha sm-1. Myofibroblasts from rat granulation tissue, Dupuytren's nodule and stroma from breast carcinoma were constantly positive for AHPM and negative for ABAM; however, myofibroblasts from Dupuytren's nodule and breast carcinoma were anti-alpha sm-1 positive. Early primary cultures of rat aortic SMC were positive for ABAM and anti-alpha sm-1 and became negative for ABAM and positive for AHPM after a few days in culture. They remained positive for AHPM and anti-alpha sm-1 after passages; the staining of AHPM and anti-alpha sm-1 appeared to be colocalized along the same stress fibers. These results may be relevant for the understanding of SMC function and adaptation, and show that in non malignant SMC proliferation, alpha-SM actin represents a more general marker of SM origin than SM myosin.

  7. An invertebrate smooth muscle with striated muscle myosin filaments

    PubMed Central

    Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio; Márquez, Gustavo; Méndez, Franklin; Padrón, Raúl; Craig, Roger

    2015-01-01

    Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components. PMID:26443857

  8. An invertebrate smooth muscle with striated muscle myosin filaments.

    PubMed

    Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio; Márquez, Gustavo; Méndez, Franklin; Padrón, Raúl; Craig, Roger

    2015-10-20

    Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components.

  9. Characteristics of light chains of Chara myosin revealed by immunological investigation

    PubMed Central

    KAKEI, Toshihito; SUMIYOSHI, Hiroki; HIGASHI-FUJIME, Sugie

    2012-01-01

    Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca++-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively. PMID:22687741

  10. Nucleocapsid of Tomato spotted wilt tospovirus forms mobile particles that traffic on an actin/endoplasmic reticulum network driven by myosin XI-K.

    PubMed

    Feng, Zhike; Chen, Xiaojiao; Bao, Yiqun; Dong, Jiahong; Zhang, Zhongkai; Tao, Xiaorong

    2013-12-01

    A number of viral proteins from plant viruses, other than movement proteins, have been shown to traffic intracellularly along actin filaments and to be involved in viral infection. However, there has been no report that a viral capsid protein may traffic within a cell by utilizing the actin/endoplasmic reticulum (ER) network. We used Tomato spotted wilt tospovirus (TSWV) as a model virus to study the cell biological properties of a nucleocapsid (N) protein. We found that TSWV N protein was capable of forming highly motile cytoplasmic inclusions that moved along the ER and actin network. The disruption of actin filaments by latrunculin B, an actin-depolymerizing agent, almost stopped the intracellular movement of N inclusions, whereas treatment with a microtubule-depolymerizing reagent, oryzalin, did not. The over-expression of a myosin XI-K tail, functioning in a dominant-negative manner, completely halted the movement of N inclusions. Latrunculin B treatment strongly inhibited the formation of TSWV local lesions in Nicotiana tabacum cv Samsun NN and delayed systemic infection in N. benthamiana. Collectively, our findings provide the first evidence that the capsid protein of a plant virus has the novel property of intracellular trafficking. The findings add capsid protein as a new class of viral protein that traffics on the actin/ER system.

  11. Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha actinin, or actin

    PubMed Central

    1986-01-01

    Striational autoantibodies (StrAb), which react with elements of skeletal muscle cross-striations, occur frequently in patients with thymoma associated with myasthenia gravis (MG). Dissociated thymic lymphocytes from 22 of 72 MG patients secreted StrAb when cultured with PWM. A high yield of EBV-transformed B cell lines was established from thymus, thymoma, and peripheral blood of seven patients with MG, but clones secreting StrAb arose only from the three patients who had StrAb in their sera. The monoclonal StrAb bound to A bands or I bands in skeletal muscle of human, rat, and frog. One bound to mitochondria in addition to myofibrillar I bands. None bound to nuclei, smooth muscle, or gastric mucosal cells. In immunoblot analyses and ELISAs the monoclonal StrAb bound to muscle and nonmuscle isotypes of myosin, alpha actinin, and/or actin. All bound to contractile proteins common to thymus and muscle, and one selectively immunostained epithelial cells of the thymic medulla. From these antigenic specificities we suggest that StrAb might arise as an immune response directed against the cytoskeletal anchoring proteins associated with nicotinic acetylcholine receptors in thymic epithelial cells undergoing neoplastic transformation to thymoma. PMID:3020150

  12. Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue-specific muscle myosin heavy chain isoforms

    PubMed Central

    Shaffer, Justin F.; Kier, William M.

    2015-01-01

    The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities. PMID:26997860

  13. Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue-specific muscle myosin heavy chain isoforms.

    PubMed

    Shaffer, Justin F; Kier, William M

    2016-03-01

    The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities.

  14. UNC-45/CRO1/She4p (UCS) Protein Forms Elongated Dimer and Joins Two Myosin Heads Near Their Actin Binding Region

    SciTech Connect

    H Shi; G Blobel

    2011-12-31

    UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 {angstrom} resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-{angstrom}-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p's C-terminal region as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 {angstrom} thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments.

  15. Mammalian class I myosin, Myo1b, is monomeric and cross-links actin filaments as determined by hydrodynamic studies and electron microscopy.

    PubMed

    Stafford, Walter F; Walker, Matt L; Trinick, John A; Coluccio, Lynne M

    2005-01-01

    The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 x 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.

  16. The effects of various nucleotides on the structure of actin-attached myosin subfragment-1 studied by quick-freeze deep-etch electron microscopy.

    PubMed

    Katayama, E

    1989-11-01

    Stereo electron microscopy of negatively stained images showed that myosin heads in acto-subfragment-1 (S1) covalently cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide were predominantly short and round when ATP was added, in contrast to their uniform tilted appearance in the rigor state. As an attempt to exclude molecules which were actually dissociated but still tethered to actin by artificial cross-links, quick-freeze deep-etch electron microscopy was coupled with the mica flake method to observe uncross-linked native acto-S1 in the presence of ATP. To maintain the low affinity S1 associated to actin in the presence of ATP, a high concentration of acto-S1 was applied to mica flakes whose absorption had been chemically modified. The image of acto-S1 with added ATP agreed well with the expected time-course of reversible dissociation and reassociation, confirming the applicability of this approach to examination of the structural changes of acto-S1. S1 molecules attached to F-actin under rigor conditions or in the presence of ADP were elongated, with the long axis tilted to F-actin. Actin-attached S1 became short and round upon addition of ATP or ADP-inorganic vanadate. Adenyl-5'-yl imidodiphosphate and inorganic pyrophosphate each partially dissociated S1 from actin, as expected.

  17. RNA-protein interactions of the 3' untranslated regions of myosin heavy chain transcripts.

    PubMed

    Kiri, Arpna; Goldspink, Geoffrey

    2002-01-01

    The RNA-protein interactions of the myosin heavy chain (MyHC) 3' untranslated regions (3'UTRs) were investigated using gel mobility shift assays. Marine skeletal myosin heavy chain mRNAs were amplified using reverse transcription coupled with the polymerase chain reaction (RT-PCR). Four cloned MyHC sequences were identified as slow type 1, fast 2a, fast 2b and fast 2x. The 3'UTRs of the four MyHC mRNAs were shown to interact with muscle protein in a tissue-specific manner as illustrated by gel retardation assays with protein extracts from various tissues. Competition assays indicate that this interaction is specific to the MyHC 3'UTR sequence. UV cross-linking suggests that several small proteins bind to the 3'UTR's. Peptide sequencing identified aldolase A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as MyHC 3'UTR RNA-binding proteins. The implications of these interactions and post-transcriptional regulation of the MyHC genes are discussed.

  18. Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding.

    PubMed

    Risal, Dipesh; Gourinath, S; Himmel, Daniel M; Szent-Györgyi, Andrew G; Cohen, Carolyn

    2004-06-15

    Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-A x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm =45 degrees to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called "front door." In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-A nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.

  19. Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator.

    PubMed

    Fabian, Lacramioara; Xia, Xuequin; Venkitaramani, Deepa V; Johansen, Kristen M; Johansen, Jørgen; Andrew, Deborah J; Forer, Arthur

    2007-07-01

    Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein.

  20. A millennial myosin census.

    PubMed

    Berg, J S; Powell, B C; Cheney, R E

    2001-04-01

    The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms.

  1. Myosin subunit interactions. Properties of the 19,000-dalton light chain-deficient myosin.

    PubMed

    Pastra-Landis, S C; Lowey, S

    1986-11-05

    The 19,000-dalton light chain (LC2) can be completely and reversibly removed from chicken pectoralis myosin in 1 mM EDTA and 5 mM ATP using immunoaffinity chromatography at 37 degrees C. Earlier methods have led to only partial removal of LC2 or have caused limited degradation of the heavy chain. Electron microscopy of LC2-deficient myosin showed it to have a marked tendency to aggregate into oligomers through the "neck" region of the myosin head. Myosin reverted to the monomeric form when it was reconstituted with light chains. LC2-deficient myosin retained full K+ (EDTA) or Ca2+-ATPase activity, and the actin-activated Mg2+-ATPase was similar to that of the native molecule. Alkali light chain exchange at 37 degrees C, which has been demonstrated in subfragment 1 prepared with chymotrypsin, does not occur with intact myosin molecules or with papain subfragment 1, both of which contain LC2. However, a temperature-dependent exchange of alkali light chains was observed in myosin lacking LC2. The interaction of the alkali light chain with the heavy chain thus appears to be influenced by the presence of LC2, which may have an important stabilizing effect on the myosin molecule.

  2. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries.

    PubMed

    Hubbell, Margaret C; Semotiuk, Andrew J; Thorpe, Richard B; Adeoye, Olayemi O; Butler, Stacy M; Williams, James M; Khorram, Omid; Pearce, William J

    2012-11-15

    Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A(165) similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility.

  3. Lampreys have a single gene cluster for the fast skeletal myosin heavy chain gene family.

    PubMed

    Ikeda, Daisuke; Ono, Yosuke; Hirano, Shigeki; Kan-no, Nobuhiro; Watabe, Shugo

    2013-01-01

    Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5'-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.

  4. Myosin IIA Heavy Chain Phosphorylation Mediates Adhesion Maturation and Protrusion in Three Dimensions.

    PubMed

    Rai, Vandana; Thomas, Dustin G; Beach, Jordan R; Egelhoff, Thomas T

    2017-02-24

    Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.

  5. Myosin heavy chain composition in the rat diaphragm - Effect of age and exercise training

    NASA Technical Reports Server (NTRS)

    Gosselin, Luc E.; Betlach, Michael; Vailas, Arthur C.; Greaser, Marion L.; Thomas, D. P.

    1992-01-01

    The effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Treadmill running at 75 percent maximal oxygen consumption resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23mo) trained animals (P less than 0.05). It was found that the ratio of fast to slow MHC was significantly higher (P less than 0.005) in the crural compared with costal diaphragm region in both age groups. A significant age-related increase in persentage of slow MHC was observed in both diaphragm regions. The relative proportion of slow MHC in either costal or crural region was not changed by exercise training.

  6. Developmental transitions in the myosin heavy chain phenotype of human respiratory muscle.

    PubMed

    Lloyd, J S; Brozanski, B S; Daood, M; Watchko, J F

    1996-01-01

    We studied the expression of myosin heavy chain (MHC) isoforms in the costal diaphragm (DIA) and the genioglossus (GG) muscles from 16 to 42 weeks gestation in the human using Western blotting techniques. Embryonic/neonatal MHC (MHCemb/neo) was the predominant isoform expressed in the DIA and GG at 16-24 weeks gestation. Subsequently, MHCemb/neo expression declined and the expression of MHCslow and MHC2A increased. At term, the DIA MHC phenotype was a composite of MHCemb/neo (15% of the total MHC complement), MHCslow (32%), MHC2A (47%), and MHC2B (6%); whereas, the GG was largely comprised of MHC2A (74%). We conclude that human DIA and GG demonstrate temporally dependent changes in MHC expression during gestation- and muscle-specific MHC phenotypes as they approach term.

  7. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  8. Non-Invasive Assessment of Skeletal Muscle Myosin Heavy Chain Expression in Trained and Untrained Men.

    PubMed

    Fry, Andrew C; Housh, Terry J; Cramer, Joel B; Weir, Joseph P; Beck, Travis W; Schilling, Brian K; Miller, Jonathan D; Nicoll, Justin X

    2016-09-20

    Numerous conditions and types of physical activity (e.g., exercise, aging, muscle-related diseases) can influence muscle fiber types and the proteins expressed. To date, muscle fibers can only be characterized by actually obtaining a tissue sample using the invasive muscle biopsy procedure. Mechanomyography (MMG) is the assessment of the vibration properties of contracting skeletal muscle, and has been proposed as a possible non-invasive method for muscle fiber analysis. Therefore, the purpose of this project was to examine the feasibility of using MMG and muscle performance measures to non-invasively assess muscle fiber characteristics. Fifteen men (5 endurance-trained [End], 5 weight-trained [WT], and 5 sedentary [Sed]) provided muscle samples from their vastus lateralis muscle. These samples were analyzed for relative myosin heavy chain protein expression, which is highly correlated with % muscle fiber type areas. Additionally, each subject performed several muscle performance tests, and MMG of the quadriceps was assessed during a knee extension exercise. Multiple regression was used to develop prediction equations for determining relative muscle content of myosin heavy chain (MHC) types I, IIa, and IIx. A combination of MMG and knee extension performance variables estimated types I, IIa, and IIx MHC with approximately 80% accuracy. Although preliminary, these data suggest that muscle performance tests in addition to MMG assessments during a simple muscle performance task (knee extension) can be used to estimate muscle fiber type composition in a healthy male population. Such methods could ultimately be used to non-invasively monitor muscle health and fitness.

  9. Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

    PubMed Central

    Catera, Rosa; Hatzi, Katerina; Yan, Xiao-Jie; Zhang, Lu; Wang, Xiao Bo; Fales, Henry M.; Allen, Steven L.; Kolitz, Jonathan E.; Rai, Kanti R.; Chiorazzi, Nicholas

    2008-01-01

    Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells. PMID:18812466

  10. Dilated Cardiomyopathy Mutation (R134W) in Mouse Cardiac Troponin T Induces Greater Contractile Deficits against α-Myosin Heavy Chain than against β-Myosin Heavy Chain.

    PubMed

    Gollapudi, Sampath K; Chandra, Murali

    2016-01-01

    Many studies have demonstrated that depressed myofilament Ca(2+) sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant-such as myofilament Ca(2+) sensitivity-is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT), which is known to modulate crossbridge (XB) recruitment dynamics in a myosin heavy chain (MHC)-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W) of the human DCM mutation (R131W), we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC) and transgenic mice (β-MHC). TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca(2+) sensitivity to a greater extent in α-MHC (0.14 pCa units) than in β-MHC fiber bundles (0.08 pCa units). Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.

  11. Dilated Cardiomyopathy Mutation (R134W) in Mouse Cardiac Troponin T Induces Greater Contractile Deficits against α-Myosin Heavy Chain than against β-Myosin Heavy Chain

    PubMed Central

    Gollapudi, Sampath K.; Chandra, Murali

    2016-01-01

    Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant—such as myofilament Ca2+ sensitivity—is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT), which is known to modulate crossbridge (XB) recruitment dynamics in a myosin heavy chain (MHC)-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W) of the human DCM mutation (R131W), we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC) and transgenic mice (β-MHC). TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units) than in β-MHC fiber bundles (0.08 pCa units). Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background. PMID:27757084

  12. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  13. Structural studies of rigor bovine myofibrils using fluorescence microscopy. II. Influence of sarcomere length on the binding of myosin subfragment-1, alpha-actinin and G-actin to rigor myofibrils.

    PubMed

    Swartz, D R; Greaser, M L; Marsh, B B

    1993-01-01

    Sarcomere length influences the textural quality of meat, yet the molecular basis for the mechanism of post-mortem shortening and the toughness associated with shortened muscle remain obscure. Bovine and rabbit myofibrillar structure was investigated over a range of sarcomere lengths (SL), using the high affinity of the myosin head for actin and of alpha-actinin for the Z-line. Myofibrils were incubated with fluorescent conjugates of myosin subfragment-1 (S1), alpha-actinin and actin. When S1 and alpha-actinin were added together, S1 bound in the I-band and A-band but not at the Z-line, while alpha-actinin bound at the Z-line. The pattern of S1 binding was highly influenced by SL, and could be explained using a model with the ratio of myofibrillar actin to myosin heads in the overlap region of 2:1 and thin filament penetration into opposing half sarcomeres. Double staining with S1 and actin demonstrated that, once the tip of the thin filament reached the bare zone, few intrinsic myosin heads were available for fluorescent actin. The patterns observed for both S1 and actin staining suggest that myofibrillar rigor bonds form even at very short SL. These observations lead to the hypothesis that the toughness associated with short sarcomeres is due to thick-filament interactions and not to the density of rigor bonds in the myofibril. Regulation of S1 binding was investigated by incubating myofibrils with low levels of fluorescent S1 in the presence and absence of calcium; S1 binding was in the overlap region when calcium was absent, but in the I-band when it was present. These results suggest that the thin filament can be activated for muscle shortening by the binding of myosin heads, a mechanism that may contribute to post-mortem muscle shortening.

  14. Protein Kinase A-Mediated Phosphorylation of cMyBP-C Increases Proximity of Myosin Heads to Actin in Resting Myocardium

    SciTech Connect

    Colson, Brett A; Bekyarova, Tanya; Locher, Matthew R; Fitzsimons, Daniel P; Irving, Thomas C; Moss, Richard L

    2008-09-16

    Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I{sub 11}/I{sub 10}, in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C{sup -/-}) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I{sub 11}/I{sub 10} (0.22{+-}0.03 versus 0.33{+-}0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I{sub 11}/I{sub 10} between untreated and PKA-treated cMyBP-C{sup -/-} myocardium (0.40{+-}0.06 versus 0.42{+-}0.05). Although lattice spacing did not change after treatment in wild-type (45.68{+-}0.84 nm versus 45.64{+-}0.64 nm), treatment of cMyBP-C{sup -/-} myocardium increased lattice spacing (46.80{+-}0.92 nm versus 49.61{+-}0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

  15. Sequence of the 20-kilodalton heavy chain peptide from the carboxyl-terminus of bovine cardiac myosin subfragment-1.

    PubMed Central

    Flink, I L; Morkin, E

    1984-01-01

    An almost complete amino acid sequence of the carboxyl-terminal 20-kD tryptic heavy chain peptide from bovine cardiac myosin Subfragment-1 (S-1) has been determined by automated sequential degradation of the undigested peptide and subfragments derived by chemical and enzymatic digestion. The fragment contains 169 residues, including two reactive cysteinyl residues which are located nine residues apart. At six positions in the sequence, two amino acid residues were present and two different versions of a chymotryptic peptide were isolated in approximately 53 and 24% yields, suggesting that there are two cardiac myosin beta-type heavy chains in this species. Analysis of the secondary structure of the 20-kD peptide predicts that there are two distinct regions within the fragment. The first region (residues 1-121) contains 12% alpha-helix, 25% beta-sheet, 40% beta-bends, and 19% coil; the second region (residues 122-169) may form an extended alpha-helix. Comparison of the bovine sequence with the deduced amino acid sequence of a recombinant plasmid containing DNA sequences coding for the beta-heavy chain of rabbit cardiac myosin (pMHC beta 174) reveals approximately 86% homology. Images PMID:6746911

  16. Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa, and actin.

    PubMed

    Fukuda, Mitsunori; Kuroda, Taruho S

    2002-11-08

    Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2 domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A.Slac2-a.myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432-12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B both in vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400-590) and Slac2-c (amino acids 670-856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery.

  17. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

    PubMed

    Morin, Nicole A; Oakes, Patrick W; Hyun, Young-Min; Lee, Dooyoung; Chin, Y Eugene; Chin, Eugene Y; King, Michael R; Springer, Timothy A; Shimaoka, Motomu; Tang, Jay X; Reichner, Jonathan S; Kim, Minsoo

    2008-01-21

    Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

  18. Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats.

    PubMed

    Cao, Ruihua; Huang, Fei; Wang, Peihuan; Chen, Chen; Zhu, Guoxiong; Chen, Lei; Wu, Gaoyi

    2015-05-01

    To investigate the changes in myosin heavy chain (MyHC) isoforms of rat masseter muscle fibres caused by chronic sleep deprivation and a possible link with the pathogenesis of disorders of the temporomandibular joint (TMJ). A total of 180 male rats were randomly divided into three groups (n=60 in each): cage controls, large platform controls, and chronic sleep deprivation group. Each group was further divided into three subgroups with different observation periods (7, 14, and 21 days). We investigated he expression of MyHC isoforms in masseter muscle fibres by real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunohistochemical staining. In rats with chronic sleep deprivation there was increased MyHC-I expression in layers of both shallow and deep muscles at 7 and 21 days compared with the control groups, whereas sleep deprivation was associated with significantly decreased MyHC-II expression. At 21 days, there were no differences in MyHC-I or MyHC-II expression between the groups and there were no differences between the two control groups at any time point. These findings suggest that chronic sleep deprivation alters the expression of MyHC isoforms, which may contribute to the pathogenesis of disorders of the TMJ.

  19. Myosin heavy chain is stabilized by BCL-2 interacting cell death suppressor (BIS) in skeletal muscle

    PubMed Central

    Hong, Jin; Park, Jun-Sub; Lee, Hyun; Jeong, Jaemin; Hyeon Yun, Hye; Yun Kim, Hye; Ko, Young-Gyu; Lee, Jeong-Hwa

    2016-01-01

    BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle. PMID:27034027

  20. Segmental distribution of myosin heavy chain isoforms within single muscle fibers.

    PubMed

    Zhang, Ming; Gould, Maree

    2017-02-18

    Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the 3 EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I&cenveo_unknown_entity_wingdings_F0F3;IIA&cenveo_unknown_entity_wingdings_F0F3;IIX&cenveo_unknown_entity_wingdings_F0F3;IIB, however in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be due to the myonuclear domain. This article is protected by copyright. All rights reserved.

  1. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  2. Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

    NASA Technical Reports Server (NTRS)

    di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

    2000-01-01

    The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

  3. Developmental Modulation of a beta myosin heavy chain promoter-driven transgene.

    PubMed

    Knotts, S; Sánchez, A; Rindt, H; Robbins, J

    1996-06-01

    The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

  4. In vivo regulation of the mouse beta myosin heavy chain gene.

    PubMed

    Knotts, S; Rindt, H; Neumann, J; Robbins, J

    1994-12-09

    The interactions of trans-acting factors with their respective cis-acting elements in the 5' upstream region of the beta myosin heavy chain gene (MyHC) regulate its tissue- and developmental stage-specific expression. The role of three conserved elements, an MCAT or TEF-1 binding site, a C-rich region, and a beta e3 region, in muscle-specific gene expression was analyzed in vivo. Each cis-acting site was ablated in the context of the beta MyHC promoter, fused to the chloramphenicol acetyltransferase reporter gene, and used to generate transgenic mice. In contrast to results obtained in vitro, the data demonstrate that mutating any one of these cis-acting elements does not affect the level or tissue specificity of transgene expression. Sequences upstream of -600 can functionally substitute for any one of these regulatory cassettes and are important both for high levels of expression as well as for controlled muscle specificity. Mutation of any two of the cis-acting elements also does not affect transgene expression. However, simultaneous mutation of the three sites significantly reduces expression, indicating that these conserved sequences do play an important role and that combinatorial interactions underlie the beta MyHC's regulation.

  5. In vivo analysis of the murine beta-myosin heavy chain gene promoter.

    PubMed

    Rindt, H; Gulick, J; Knotts, S; Neumann, J; Robbins, J

    1993-03-05

    The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.

  6. Transcriptional regulation of the Drosophila melanogaster muscle myosin heavy-chain gene

    PubMed Central

    Hess, Norbert K.; Singer, Phillip A.; Trinh, Kien; Nikkhoy, Massoud; Bernstein, Sanford I.

    2007-01-01

    We show that a 2.6 kb fragment of the muscle myosin heavy-chain gene (Mhc) of Drosophila melanogaster (containing 458 base pairs of upstream sequence, the first exon, the first intron and the beginning of the second exon) drives expression in all muscles. Comparison of the minimal promoter to Mhc genes of ten Drosophila species identified putative regulatory elements in the upstream region and in the first intron. The first intron is required for expression in four small cells of the tergal depressor of the trochanter (jump) muscle and in the indirect flight muscle. The 3′ end of this intron is important for Mhc transcription in embryonic body wall muscle and contains AT–rich elements that are protected from DNase I digestion by nuclear proteins of Drosophila embryos. Sequences responsible for expression in embryonic, adult body wall and adult head muscles are present both within and outside the intron. Elements important for expression in leg muscles and in the large cells of the jump muscle flank the intron. We conclude that multiple transcriptional regulatory elements are responsible for Mhc expression in specific sets of Drosophila muscles. PMID:17194628

  7. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state

    NASA Technical Reports Server (NTRS)

    Adams, G. R.; McCue, S. A.; Zeng, M.; Baldwin, K. M.

    1999-01-01

    During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

  8. Effects of inactivity on myosin heavy chain composition and size of rat soleus fibers

    NASA Technical Reports Server (NTRS)

    Grossman, E. J.; Roy, R. R.; Talmadge, R. J.; Zhong, H.; Edgerton, V. R.

    1998-01-01

    Myosin heavy chain (MHC) and fiber size properties of the adult rat soleus were determined after 4-60 days of complete inactivity, i.e., lumbar spinal cord isolation. Soleus atrophy was rapid and progressive, i.e., 25% and 64% decrease in weight and 33% and 75% decrease in fiber size after 4 and 60 days of inactivity, respectively. Changes in MHC occurred at a slower rate than the atrophic response. After 15 days there was de novo expression of type IIx MHC (approximately 10%). By 60 days, type IIx MHC accounted for 33% of the total MHC content, and 7% of the fibers contained only type IIx MHC. The relative amount of type I MHC was reduced from 93% in control to 49% after 60 days of inactivity. Therefore, the effects of 60 days of inactivity suggest that during this time period at least 75% of fiber size and approximately 40% of type I MHC composition of the adult rat soleus can be attributed to activation-related events.

  9. Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Adams, Gregory R.; Baldwin, Kenneth M.

    1995-01-01

    This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid (creatine depletion juvenile (CDJ) group). Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens (normal diet adult and creatine depletion adult (CDA) groups). After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC ditribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC tranitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of the animals to be used.

  10. Myosin heavy chain composition of tiger (Panthera tigris) and cheetah (Acinonyx jubatus) hindlimb muscles.

    PubMed

    Hyatt, Jon-Philippe K; Roy, Roland R; Rugg, Stuart; Talmadge, Robert J

    2010-01-01

    Felids have a wide range of locomotor activity patterns and maximal running speeds, including the very fast cheetah (Acinonyx jubatas), the roaming tiger (Panthera tigris), and the relatively sedentary domestic cat (Felis catus). As previous studies have suggested a relationship between the amount and type of activity and the myosin heavy chain (MHC) isoform composition of a muscle, we assessed the MHC isoform composition of selected hindlimb muscles from these three felid species with differing activity regimens. Using gel electrophoresis, western blotting, histochemistry, and immunohistochemistry with MHC isoform-specific antibodies, we compared the MHC composition in the tibialis anterior, medial gastrocnemius (MG), plantaris (Plt), and soleus muscles of the tiger, cheetah, and domestic cat. The soleus muscle was absent in the cheetah. At least one slow (type I) and three fast (types IIa, IIx, and IIb) MHC isoforms were present in the muscles of each felid. The tiger had a high combined percentage of the characteristically slower isoforms (MHCs I and IIa) in the MG (62%) and the Plt (86%), whereas these percentages were relatively low in the MG (44%) and Plt (55%) of the cheetah. In general, the MHC isoform characteristics of the hindlimb muscles matched the daily activity patterns of these felids: the tiger has daily demands for covering long distances, whereas the cheetah has requirements for speed and power.

  11. An electrophoretic study of myosin heavy chain expression in skeletal muscles of the toad Bufo marinus.

    PubMed

    Nguyen, L T; Stephenson, G M

    1999-10-01

    In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight < 1 g) to late adulthood (body weight 200-450 g). The developmental changes in the MHC isoform composition of the toad IF muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.

  12. Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide.

    PubMed Central

    Tamura, Y; Suzuki, N; Mihashi, K

    1993-01-01

    The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber. PMID:8298019

  13. Differential susceptibility on myosin heavy chain isoform following eccentric-induced muscle damage.

    PubMed

    Choi, Seung Jun

    2014-12-01

    Based on myosin heavy chain (MHC) isoform, human skeletal muscle fibers can be categorized into three fiber types, type I, IIa, IIx fibers, and each fiber type has different characteristics. Typical characteristics are difference in force production, shortening velocity, and fatigue resistance. When the muscle is contract and stretched by a force that is greater than the force generated by the muscle, contraction-induced muscle damage frequently occurs. Several experimental models involving both human and animal have considered the susceptibility of different muscle fiber type and part of muscles to eccentric induced muscle damage. General consensus is a greater susceptibility of fast-twitch fiber or type II fiber to damage following eccentric contractions. However, the results from these previous efforts were not enough to conclude the susceptibility between each individual fiber types after eccentric contraction. Therefore, the purpose of this review is to explore detail limitation and interpretation of previous studies, and review the recent study that eliminated the prior limitations, such as strain magnitude, lengthening velocity, fiber type heterogeneity, and muscle architecture issue.

  14. Limited Expression of Slow Tonic Myosin Heavy Chain in Human Cranial Muscles

    PubMed Central

    Sokoloff, Alan J.; Li, Haiyan; Burkholder, Thomas J.

    2013-01-01

    Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular and developmental muscle with antibodies (Abs) ALD-58 and S46 considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform. PMID:17486578

  15. Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles

    PubMed Central

    Xu, Jin; Gao, Jie; Li, Junling; Xue, Liangyi; Clark, Karl J.; Ekker, Stephen C.; Du, Shao Jun

    2014-01-01

    Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles. PMID:22361506

  16. Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles.

    PubMed

    Xu, Jin; Gao, Jie; Li, Junling; Xue, Liangyi; Clark, Karl J; Ekker, Stephen C; Du, Shao Jun

    2012-02-01

    Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.

  17. Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

    PubMed Central

    Bershitsky, S Y; Tsaturyan, A K

    1995-01-01

    To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature. PMID:8519956

  18. Biochemical and Immunocytochemical Characterization of Two Types of Myosins in Cultured Tobacco Bright Yellow-2 Cells1

    PubMed Central

    Yokota, Etsuo; Yukawa, Chiharu; Muto, Shoshi; Sonobe, Seiji; Shimmen, Teruo

    1999-01-01

    We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 μm/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 μm/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 μm/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca2+ at concentrations higher than 10−6 m. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants. PMID:10517844

  19. CLIC5 Stabilizes Membrane-Actin Filament Linkages at the Base of Hair Cell Stereocilia in a Molecular Complex with Radixin, Taperin, and Myosin VI

    PubMed Central

    Salles, Felipe T.; Andrade, Leonardo R.; Tanda, Soichi; Grati, M’hamed; Plona, Kathleen L.; Gagnon, Leona H.; Johnson, Kenneth R.; Kachar, Bechara; Berryman, Mark A.

    2015-01-01

    Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. PMID:24285636

  20. Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats

    PubMed Central

    Yousefzadeh, Nasibeh; Jeddi, Sajad; Alipour, Mohammad Reza

    2016-01-01

    Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart. PMID:27411095

  1. Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

    PubMed Central

    Štrbenc, Malan; Smerdu, Vika; Pogačnik, Azra; Fazarinc, Gregor

    2006-01-01

    To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (α-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4–6 weeks of age, which coincides with the increased physical activity of puppies. PMID:16879596

  2. Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Herrick, R. E.; Adams, G. R.; Baldwin, K. M.

    1993-01-01

    This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

  3. Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure

    PubMed Central

    Neary, Joseph M.; Lund, Gretchen K.; Holt, Timothy N.; Garry, Franklyn B.; Mohun, Timothy J.; Breckenridge, Ross A.

    2014-01-01

    Abstract Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension. PMID:25621163

  4. Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

    PubMed Central

    Bottinelli, R; Canepari, M; Pellegrino, M A; Reggiani, C

    1996-01-01

    1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force-velocity curves of sixty-seven fibres were obtained by load-clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross-sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack-test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I-IIA and IIA-IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty-one fibres in the range 12-22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol-1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12-17 degrees C range, but less dependent between 17 and 22 degrees C. Images Figure 1 Figure 3 Figure 6 PMID:8887767

  5. Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

    PubMed Central

    Kim, Dong Kwan; Zhu, Jianliang; Kozyak, Benjamin W; Burkman, James M; Rubinstein, Neal A; Lankford, Edward B; Stedman, Hansell H; Nguyen, Taitan; Levine, Sanford; Shrager, Joseph B

    2003-01-01

    Background Several physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema. Methods We sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro. Results In addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 ± 2.7 ms versus 53.9 ± 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema. Conclusion This is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans. PMID:12617755

  6. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  7. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    SciTech Connect

    Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X.

    2010-04-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  8. Myosin Heavy Chain Expression Can Vary over the Length of Jaw and Leg Muscles

    PubMed Central

    Korfage, J.A.M.; Kwee, K.E.; Everts, V.; Langenbach, G.E.J.

    2016-01-01

    Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the same over its length. We analyzed this in detail in three muscle types: the temporalis (closer) and digastricus (opener; both first brachial arch), and the medial gastrocnemius (somite). Sections of the muscles were incubated with monoclonal antibodies against various MyHC isoforms, and the distribution of these isoforms within individual fibers was followed over a distance of approximately 1 mm. The staining intensity of a fiber was measured and compared with the other fibers in the section. In the temporalis, digastricus, and gastrocnemius, 46, 11, and 15%, respectively, of their MyHC-I fibers showed a variation in the staining intensity over the length of their fibers, as well as 47, 87, and 22%, respectively, of their MyHC-IIA fibers. Most variable fibers were found amongst those with an overall relative intermediate staining intensity, which are presumably hybrid fibers. We conclude that different parts of a muscle fiber can have different fiber type compositions and, thus, contractile properties. Some muscle parts might reach their maximum contraction peak sooner or later than a muscle part a few microns further away. Next to stimulation by the nerve and systemic influences, local influences might also have an impact on the MyHC expression of the fiber. PMID:26950765

  9. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    PubMed

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P < 0.01; mean ± standard error of the mean (SEM), Student's unpaired t-test], as well as in the tongue muscle (65.7 versus 73.8 per cent; P < 0.05). Similarly, the content of MyHC-2x isoforms in mdx tongue muscle was lower than in the controls (25.9 versus 30.8 per cent; P < 0.05). The observed down-regulation of the MyHC-2x and MyHC-2b mRNA in the masticatory muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  10. Identification of myosin in a flowering plant, Egeria densa.

    PubMed

    Ohsuka, K; Inoue, A

    1979-02-01

    A myosin-like protein was extracted and partially purified from a flowering plant, Egeria densa. It had no p-nitrophenyl phosphatase activity, but exhibited EDTA(K+)-ATPase [EC 3.6.1.3] activity at high ionic strength. Its molecular weight as estimated by gel filtration was 4-5 X 10(5). The presence of a heavy chain (MW = about 1.8 X 10(5)) was indicated by SDS-gel electrophoresis. Egeria myosin aggregated in an environment of low ionic strength and formed bipolar filaments. It bound with skeletal muscle F-actin with a periodicity of 40 nm.

  11. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  12. Temperature and ionic strength dependence of the subunit interactions in vertebrate skeletal myosin. A comparison of the interaction between the alkali light and heavy chains of mammalian and avian myosin.

    PubMed

    Zaager, S; Burke, M

    1988-09-25

    The stability of the interaction of A1 in myosin and subfragment 1 isolated from fast-twitch mammalian and avian muscles with respect to temperature and ionic strength has been examined. This was done by determining the extent of exchange of the endogenous free A1 light chain into these proteins from the two species. Whereas the extent of exchange at 37 degrees C into mammalian S1, occurring after 60 min, is about 80% of the theoretically expected amount at physiological ionic conditions, the level of exchange observed with the avian S1 is significantly lower. However, close to the theoretical limit is observed for the avian S1 when exchange is done at 43 degrees C which is close to average avian body temperature. A similar dependence with temperature is observed in the case of exchanges into avian myosin. In the case of mammalian myosin, 50% of the theoretical exchange is observed at 37 degrees C under physiological ionic strength, whereas the level of exchange observed under these conditions with the avian protein is much lower in agreement with recent observations (Waller, G. S., and Lowey, S. (1985) J. Biol. Chem. 260, 14368-14373; Pastra-Landis, S. C., and Lowey, S. (1986) J. Biol. Chem. 261, 14811-14816). If, however, the exchanges are done at 43 degrees C in physiological ionic strength, significant extents of exchange can be observed in avian myosin. These results suggest that at physiological ionic and temperature conditions relevant for the source of myosin and S1 being investigated, the alkali light chains are in dynamic equilibrium between free and heavy chain associated states. Therefore, the failure to observe alkali light chain exchange in avian myosin at 37 degrees C appears to be related to the higher temperature stability of its interaction with the heavy chain.

  13. Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization.

    PubMed

    Vassilopoulos, Stéphane; Gentil, Christel; Lainé, Jeanne; Buclez, Pierre-Olivier; Franck, Agathe; Ferry, Arnaud; Précigout, Guillaume; Roth, Robyn; Heuser, John E; Brodsky, Frances M; Garcia, Luis; Bonne, Gisèle; Voit, Thomas; Piétri-Rouxel, France; Bitoun, Marc

    2014-05-12

    The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM-sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.

  14. The heavy chain has its day

    PubMed Central

    Dulyaninova, Natalya G; Bresnick, Anne R

    2013-01-01

    Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain. PMID:24002531

  15. Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

    PubMed

    Bee, G; Solomon, M B; Czerwinski, S M; Long, C; Pursel, V G

    1999-08-01

    Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (P<.05). There were no correlations between FOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important

  16. Increased myocardial short-range forces in a rodent model of diabetes reflect elevated content of β myosin heavy chain.

    PubMed

    Chung, Charles S; Mitov, Mihail I; Callahan, Leigh Ann; Campbell, Kenneth S

    2014-06-15

    Diastolic dysfunction is a clinically significant problem for patients with diabetes and often reflects increased ventricular stiffness. Attached cross-bridges contribute to myocardial stiffness and produce short-range forces, but it is not yet known whether these forces are altered in diabetes. In this study, we tested the hypothesis that cross-bridge-based short-range forces are increased in the streptozotocin (STZ) induced rat model of type 1 diabetes. Chemically permeabilized myocardial preparations were obtained from 12week old rats that had been injected with STZ or vehicle 4weeks earlier, and activated in solutions with pCa (=-log10[Ca(2+)]) values ranging from 9.0 to 4.5. The short-range forces elicited by controlled length changes were ∼67% greater in the samples from the diabetic rats than in the control preparations. This change was mostly due to an increased elastic limit (the length change at the peak short-range force) as opposed to increased passive muscle stiffness. The STZ-induced increase in short-ranges forces is thus unlikely to reflect changes to titin and/or collagen filaments. Gel electrophoresis showed that STZ increased the relative expression of β myosin heavy chain. This molecular mechanism can explain the increased short-ranges forces observed in the diabetic tissue if β myosin molecules remain bound between the filaments for longer durations than α molecules during imposed movements. These results suggest that interventions that decrease myosin attachment times may be useful treatments for diastolic dysfunction associated with diabetes.

  17. Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

    PubMed

    Sari-Hassoun, Meryem; Clement, Marie-Jeanne; Hamdi, Imane; Bollot, Guillaume; Bauvais, Cyril; Joshi, Vandana; Toma, Flavio; Burgo, Andrea; Cailleret, Michel; Rosales-Hernández, Martha Cecilia; Macias Pérez, Martha Edith; Chabane-Sari, Daoudi; Curmi, Patrick A

    2016-02-15

    Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.

  18. Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site

    SciTech Connect

    Garabedian, T.E.; Yount, R.G. )

    1990-12-25

    The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with ({sup 3}H)UDP. ({sup 3}H) UDP was stably trapped at the active site by addition of vanadate (Vi) and Co{sup 2+}. The extraordinary stability of the myosin.Co2+.(3H)UDP.Vi complex (t1/2 greater than 5 days at 0{degrees}C) allowed it to be purified free of extraneous ({sup 3}H)UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped ({sup 3}H)UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results using ({sup 3}H)UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a zero-length cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by ({sup 3}H)UTP photolabeling in Acanthamoeba myosin II.

  19. Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

    PubMed

    Dausse, E; Komajda, M; Fetler, L; Dubourg, O; Dufour, C; Carrier, L; Wisnewsky, C; Bercovici, J; Hengstenberg, C; al-Mahdawi, S

    1993-12-01

    Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

  20. High fat/low carbohydrate diet attenuates left ventricular hypertrophy and prevents myosin heavy chain isoform switching induced by chronic hypertenstion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A switch in the expression of myosin heavy chain isoform (MHC) alpha to beta is observed with left ventricular hypertrophy (LVH) and heart failure. This switch is associated with a defect in myocardial energy production and contractile dysfunction. Similar MHC isoform profile is observed in the fe...

  1. [Protective effect of tiacalix[4]arene-tetrasulphonate on heavy metal inhibition of myometrium myosin subfragment-1 ATP-hydrolase activity].

    PubMed

    Labyntseva, R D; Bevza, O V; Bevza, A A; Liul'ko, A O; Kharchenko, S H; Kal'chenko, V I; Kosterin, S O

    2014-01-01

    Heavy metals have a negative effect on the contractility of uterine smooth muscles (myometrium), these effects can lead to various pathologies of a women reproductive system. To overcome these effects the methods for correcting the myometrium contractile activity are to be developed. Catalyzed by myosin ATPase ATP hydrolysis is the most important reaction in the molecular mechanism of myometrium contraction. We have found an inhibitory effect of 0.03-0.3 mM Ni2+, Pb2+ and Cd2+ on enzymatic hydrolysis of ATP by myosin subfragment-1 obtained from swine uterine smooth muscles. We have demonstrated that 100 μM thiacalix[4]arene-tetrasulphonate (C-798) recovered to the control level of ATPase activity of myosin subfragment-1 in the presence of heavy metal cations. One of the most probable mechanisms of C-798 corrective activity is based on its ability to chelate heavy metals, thus cations Pb, Cd and Ni can be removed from the incubation medium. Computer simulation has demonstrated that the protective effect of C-798 may also be the result of weakening the interaction of heavy metal ions with amino acid residues of the myosin molecule near the active site of ATP hydrolase. The obtained results can be used for further research aimed at assessing the prospects of thiacalix[4]arene-tetrasulfonate as pharmacological compounds.

  2. Calcium inhibition as an intracellular signal for actin–myosin interaction

    PubMed Central

    KOHAMA, Kazuhiro

    2016-01-01

    Intracellular signaling pathways include both the activation and the inhibition of biological processes. The activation of Ca2+ regulation of actin-myosin interactions was examined first, whereas it took 20 years for the author to clarify the inhibitory mode by using Physarum polycephalum, a lower eukaryote. This review describes the investigation of the inhibitory mode since 1980. The inhibitory effect of Ca2+ on myosin was detected chemically by ATPase assays and mechanically by in vitro motility assays. The Ca2+-binding ability of Physarum myosin is as high as that of scallop myosin. Ca2+ inhibits Physarum myosin, whereas it activates scallop myosin. We cloned cDNA of the myosin heavy chain and light chains to express a hybrid of Physarum and scallop myosin, and found that the Ca-binding light chain (CaLc), which belongs to an alkali light chain class, plays a major role in Ca inhibition. The role of CaLc was confirmed by mutating its EF-hand, Ca-binding structure and expressing Physarum myosin as a recombinant protein. Thus, the data obtained by classical protein purification were confirmed by the results obtained with the modern recombinant techniques. However, there are some discrepancies that remain to be solved as described in Section XII. PMID:27941307

  3. Sexually Dimorphic Expression of a Laryngeal-Specific, Androgen-Regulated Myosin Heavy Chain Gene during Xenopus laevis Development

    PubMed Central

    Catz, Diana S.; Fischer, Leslie M.; Moschella, Maria C.; Tobias, Martha L.

    2012-01-01

    Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue. PMID:1426643

  4. Comparison of Characteristics of Myosin Heavy Chain-based Fiber and Meat Quality among Four Bovine Skeletal Muscles

    PubMed Central

    Kim, Gap-Don; Yang, Han-Sul; Jeong, Jin-Yeon

    2016-01-01

    Muscle fiber characteristics account for meat quality and muscle fibers are mainly classified into three or more types according to their contractile and metabolic properties. However, the majority of previous studies on bovine skeletal muscle are based on myosin ATPase activity. In the present study, the differences in the characteristics of muscle fibers classified by the expression of myosin heavy chain (MHC) among four bovine skeletal muscles such as longissimus thoracis (LT), psoas major (PM), semimembranosus (SM) and semitendinosus (ST) and their relationships to beef quality were investigated. MHCs 2x, 2a and slow were identified by LC-MS/MS and IIX, IIA and I fiber types were classified. PM, which had the smallest size and highest density of fibers regardless of type, showed the highest myoglobin content, CIE L*, a*, b* and sarcomere length (p<0.05), whereas ST with the highest composition of IIX, showed high shear force and low sarcomere length (p<0.05). The correlation coefficients between muscle fiber characteristics and meat quality showed that type IIX is closely related to poor beef quality and that a high density of small-sized fibers is related to redness and tenderness. Therefore, the differences in meat quality between muscles can be explained by the differences in muscle fiber characteristics, and especially, the muscles with good quality are composed of more small-sized fibers regardless of fiber type. PMID:28115894

  5. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  6. Molecular Basis of Dynamic Relocalization of Dictyostelium Myosin IB*

    PubMed Central

    Brzeska, Hanna; Guag, Jake; Preston, G. Michael; Titus, Margaret A.; Korn, Edward D.

    2012-01-01

    Class I myosins have a single heavy chain comprising an N-terminal motor domain with actin-activated ATPase activity and a C-terminal globular tail with a basic region that binds to acidic phospholipids. These myosins contribute to the formation of actin-rich protrusions such as pseudopodia, but regulation of the dynamic localization to these structures is not understood. Previously, we found that Acanthamoeba myosin IC binds to acidic phospholipids in vitro through a short sequence of basic and hydrophobic amino acids, BH site, based on the charge density of the phospholipids. The tail of Dictyostelium myosin IB (DMIB) also contains a BH site. We now report that the BH site is essential for DMIB binding to the plasma membrane and describe the molecular basis of the dynamic relocalization of DMIB in live cells. Endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The BH site is required for association of DMIB with the plasma membrane at all stages where it colocalizes with phosphoinositide bisphosphate/phosphoinositide trisphosphate (PIP2/PIP3). The charge-based specificity of the BH site allows for in vivo specificity of DMIB for PIP2/PIP3 similar to the PH domain-based specificity of other class I myosins. However, DMIB-head is required for relocalization of DMIB to the front of migrating cells. Motor activity is not essential, but the actin binding site in the head is important. Thus, dynamic relocalization of DMIB is determined principally by the local PIP2/PIP3 concentration in the plasma membrane and cytoplasmic F-actin. PMID:22367211

  7. Myosin light chains: Teaching old dogs new tricks

    PubMed Central

    Heissler, Sarah M; Sellers, James R

    2014-01-01

    The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

  8. Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness

    PubMed Central

    Wang, Yang; Melkani, Girish C.; Suggs, Jennifer A.; Melkani, Anju; Kronert, William A.; Cammarato, Anthony; Bernstein, Sanford I.

    2012-01-01

    Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients. PMID:22496423

  9. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

  10. Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis.

    PubMed

    Yumura, Shigehiko; Ueda, Masahiro; Sako, Yasushi; Kitanishi-Yumura, Toshiko; Yanagida, Toshio

    2008-12-01

    Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.

  11. Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis.

    PubMed Central

    Halsell, S R; Chu, B I; Kiehart, D P

    2000-01-01

    A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipper(Ebr) allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipper(Ebr), disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan. PMID:10880486

  12. Study of actin and its interactions with heavy meromyosin and the regulatory proteins by the pulse fluorimetry in polarized light of a fluorescent probe attached to an actin cysteine.

    PubMed

    Tawada, K; Wahl, P; Auchet, J C

    1978-08-01

    The decay of anisotropy of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine fluorescence attached to cysteine-373 of actin can be characterized by two correlation times theta1 and theta2. theta1 has a value of several nanoseconds and is thought to represent some local protein motion. theta2 is of the order of several hundreds of nanoseconds. Its value increases with actin concentration. It represents an average of the G and F actin correlation times. When actin interacts with heavy meromyosin, theta2 increases and becomes infinite at a molar ratio of one heavy meromyosin molecule per four actin protomers. It is concluded that a definite complex is then formed between F actin and heavy meromyosin. In the same time, G actin concentration becomes equal to zero. Finally, when F actin forms a complex with the regulatory proteins tropomyosin and troponin, the value of theta2 is greater in the absence than in the presence of Ca2+. This result indicates that micromolar concentrations of Ca2+ induces a conformation change of the complex of F actin with the regulatory proteins.

  13. Myosin is involved in postmitotic cell spreading

    PubMed Central

    1995-01-01

    We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II. PMID:7559774

  14. The role of myosin 1c and myosin 1b in surfactant exocytosis

    PubMed Central

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-01-01

    ABSTRACT Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression. PMID:26940917

  15. The role of myosin 1c and myosin 1b in surfactant exocytosis.

    PubMed

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-04-15

    Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression.

  16. Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

    PubMed

    Fong, Y; Rosenbaum, M; Tracey, K J; Raman, G; Hesse, D G; Matthews, D E; Leibel, R L; Gertner, J M; Fischman, D A; Lowry, S F

    1989-05-01

    A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.

  17. Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA-reductase inhibition on skeletal muscle functions.

    PubMed

    Trapani, Laura; Melli, Luca; Segatto, Marco; Trezza, Viviana; Campolongo, Patrizia; Jozwiak, Adam; Swiezewska, Ewa; Pucillo, Leopoldo Paolo; Moreno, Sandra; Fanelli, Francesca; Linari, Marco; Pallottini, Valentina

    2011-11-01

    The rate-limiting step of cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme reductase (HGMR), whose inhibitors, the statins, widely used in clinical practice to treat hypercholesterolemia, often cause myopathy, and rarely rhabdomyolysis. All studies to date are limited to the definition of statin-induced myotoxicity omitting to investigate whether and how HMGR inhibition influences muscle functions. To this end, 3-mo-old male rats (Rattus norvegicus) were treated for 3 wk with a daily intraperitoneal injection of simvastatin (1.5 mg/kg/d), and biochemical, morphological, mechanical, and functional analysis were performed on extensor digitorum longus (EDL) muscle. Our results show that EDL muscles from simvastatin-treated rats exhibited reduced HMGR activity; a 15% shift from the fastest myosin heavy-chain (MHC) isoform IIb to the slower IIa/x; and reduced power output and unloaded shortening velocity, by 41 and 23%, respectively, without any change in isometric force and endurance. Moreover, simvastatin-treated rats showed a decrease of maximum speed reached and the latency to fall off the rotaroad (∼-30%). These results indicate that the molecular mechanism of the impaired muscle function following statin treatment could be related to the plasticity of fast MHC isoform expression.

  18. Identification and quantification of myosin heavy chain isoforms in bovine and porcine longissimus muscles by LC-MS/MS analysis.

    PubMed

    Kim, Gap-Don; Jeong, Tae-Chul; Cho, Kye Man; Jeong, Jin-Yeon

    2017-03-01

    To identify and quantify the myosin heavy chain (MHC) isoforms in porcine and bovine longissimus thoracis (LT) muscles, LC-MS/MS and label-free quantification were conducted. 2a, 2x, 2b and slow isoforms were identified in porcine LT muscle, whereas bovine LT muscle contained 2a, 2x and slow isoforms. The highest peak intensity (44.32%) of MHC 2b was observed in porcine LT muscle, whereas the peak intensity of MHC 2a was the highest (71.15%) in bovine LT muscle (p<0.05). MHC-based fiber compositions for porcine and bovine LT were also the highest in types IIB (61.13%) and IIA (61.90%), respectively (p<0.05). Spectral count and peak intensity had positive correlations with MHC-based fiber composition (p<0.01), however the spectral count and peak intensity of total peptides were not acceptable for MHC quantification because of the limited reliability of the protein and common peptide, respectively. Therefore, the peak intensity of unique peptides would be the most acceptable trait for MHC quantification.

  19. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation

    PubMed Central

    Sabbir, Mohammad G.; Dillon, Rachelle; Mowat, Michael R. A.

    2016-01-01

    ABSTRACT The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

  20. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed Central

    Rindt, H; Knotts, S; Robbins, J

    1995-01-01

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7878016

  1. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed

    Rindt, H; Knotts, S; Robbins, J

    1995-02-28

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter.

  2. Transgenic analysis of the thyroid-responsive elements in the alpha-cardiac myosin heavy chain gene promoter.

    PubMed

    Subramaniam, A; Gulick, J; Neumann, J; Knotts, S; Robbins, J

    1993-02-25

    The role of two putative, cis-acting thyroid hormone-responsive elements, TRE1 and TRE2, located at -129 to -149 and -102 to -120, respectively, on the murine alpha-myosin heavy chain (MHC) gene, has been investigated in transgenic mice. These motifs are present in a 4.5-kilobase fragment lying upstream of the transcriptional start site of the mouse alpha-MHC gene: this fragment directs appropriate expression of a reporter gene in transgenic mice (Subramaniam, A., Jones, W. K., Gulick, J., Wert, S., Neumann, J., and Robbins, J. (1991) J. Biol. Chem. 266, 24613-24620). Here, we independently mutate the TRE1 and TRE2 elements by base substitution. The mice were analyzed for transgene expression in different muscle and non-muscle tissues including the atria and ventricles. Normal levels of transgene expression were observed in euthyroid mice carrying a mutation in TRE1. In contrast to these results, mice in which TRE2 was mutated showed reduced levels of CAT activity in both the atria and ventricles, suggesting a previously undefined role for this element in the constitutive up-regulation of the alpha-MHC gene. In hypothyroid mice carrying either of these mutations, the complete cessation of ventricular expression of the chloramphenicol acetyltransferase transcripts that takes place in the alpha-5.5 (wild type) animals did not occur.

  3. Fiber size, type, and myosin heavy chain content in rhesus hindlimb muscles after 2 weeks at 2 G

    NASA Technical Reports Server (NTRS)

    Tavakol, Morteza; Roy, Roland R.; Kim, Jung A.; Zhong, Hui; Hodgson, John A.; Hoban-Higgins, Tana M.; Fuller, Charles A.; Edgerton, V. Reggie

    2002-01-01

    BACKGROUND: Fiber atrophy and an increase in the percentage of fast fibers have been observed in Rhesus leg muscles after spaceflight. Hypothesis: Hypergravity will result in muscle fiber hypertrophy and an increase in the percentage of slow fibers. METHODS: Open muscle biopsies were obtained from Rhesus soleus, medial gastrocnemius (MG), and tibialis anterior (TA) muscles before and after 14 d of centrifugation (2 G) and in time-matched controls. Cage activity levels were measured by telemetry. RESULTS: Based on monoclonal antibody binding for myosin heavy chains (MHC), the fastest region of soleus contained a higher proportion of type I+II (27 vs. 13%) and had a tendency for a lower proportion of type I (38 vs. 61%, p = 0.10) fibers after than before centrifugation. There was a higher proportion of type I+II fibers in post- vs. pre-2 G (10 vs. 0.6%) MG biopsies. Fiber type distribution and MHC composition were unaffected in the TA. Overall, mean fiber sizes were unaffected by centrifugation. Average cage activity levels were 36% lower during than before 2 G. CONCLUSIONS: Our hypothesis was rejected. The changes in the proportion of fibers expressing type I MHC are the reverse of that expected with chronic loading of extensors and, paradoxically, are similar to changes observed with chronic unloading, such as occurs during spaceflight, in this primate model. The data are consistent with the observed decrease in total daily activity levels.

  4. bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion.

    PubMed

    Seward, D J; Haney, J C; Rudnicki, M A; Swoap, S J

    2001-02-01

    A strong correlative pattern between MyoD gene expression and myosin heavy chain IIB (MHC IIB) gene expression exists. To test whether this correlative relationship is causative, MHC gene expression in muscles from MyoD(-/-) mice was analyzed. The MHC IIB gene was not detectable in the MyoD(-/-) diaphragm, whereas the MHC IIB protein made up 10.0 +/- 1.7% of the MHC protein pool in the wild-type (WT) mouse diaphragm. Furthermore, the MHC IIA protein was not detectable in the MyoD(-/-) biceps brachii, and the MHC IIB protein was overexpressed in the masseter. To examine whether MyoD is required for the upregulation of the MHC IIB gene within slow muscle after disuse, MyoD(-/-) and WT hindlimb musculature was unweighted. MyoD(-/-) exhibited a diminished response in the upregulation of the MHC IIB mRNA within the soleus muscle as a result of the hindlimb unweighting. Collectively, these data suggest that MyoD plays a role in the MHC profile in a muscle-specific fashion.

  5. Synergistic ablation does not affect atrophy or altered myosin heavy chain expression in the non-weight bearing soleus muscle

    NASA Technical Reports Server (NTRS)

    Linderman, J. K.; Talmadge, R. J.; Gosselink, K. L.; Tri, P. N.; Roy, R. R.; Grindeland, R. E.

    1996-01-01

    The purpose of this study was to investigate whether the soleus muscle undergoes atrophy and alterations in myosin heavy chain (MHC) composition during non-weight bearing in the absence of synergists. Thirty-two female rats were randomly assigned to four groups: control (C), synergistic ablation (ABL) of the gastrocnemius and plantaris muscles to overload the soleus muscle, hindlimb suspension (HLS), or a combination of synergistic ablation and hindlimb suspension (HLS-ABL). After 28 days of hindlimb suspension, soleus atrophy was more pronounced in HLS (58%) than in HLS-ABL (43%) rats. Compared to C rats, non-weight bearing decreased mixed and myofibrillar protein contents and Type I MHC 49%, 45%, and 7%, respectively, in HLS animals. In addition, de novo expression of fast Type IIx and Type IIb MHC (5% and 2%, respectively) was observed in HLS animals. Similarly, when compared to C rats, mixed and myofibrillar protein contents and Type I MHC decreased 43%, 46%, and 4%, respectively, in HLS-ABL animals. Also, de novo expression of Type IIx (4%) and IIb (1%) MHC was observed. Collectively, these data indicate that the loss of muscle protein and Type I MHC, and the de novo expression of Type IIx and Type IIb MHC in the rat soleus occur independently of the presence of synergists during non-weight bearing. Furthermore, these results confirm the contention that soleus mass and MHC expression are highly sensitive to alterations in mechanical load.

  6. Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

    PubMed

    Eizema, Karin; van den Burg, Maarten; Kiri, Arpna; Dingboom, Elizabeth G; van Oudheusden, Hans; Goldspink, Geoffrey; Weijs, Wim A

    2003-09-01

    The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

  7. Ubiquitin Ligase, MuRF-1 regulates myosin heavy chain type IIa transcripts during muscle atrophy under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Kagawa, Sachiko

    Skeletal muscles are vulnerable to marked atrophy under microgravity conditions. We previously reported that gastrocnemius muscle atrophy by spaceflight was specifically sensitive to the ubiquitin-proteasome proteolytic pathway. We also screened more over 26,000 skeletal muscle genes in rats exposed to real weightlessness and found that the expression of Ubiquitin Ligase, Muscle specific Ring Finger-1 (MuRF-1) upregulated under microgravity. In the present study, we examined the role of MuRF-1 in microgravity-induced muscle atrophy. The amounts of MuRF-1 transcripts significantly increased in skeletal muscle after denervation, an in vivo model of microgravity-induced unloading. MuRF-1 deficient (MuRF-1-/-) mice significantly inhibited reduction of muscle weight for muscle atrophy, compared with wild type mice. Interestingly, MuRF-1-/- mice significantly inhibited upregulation of myosin heavy chain (MyHC) type IIa transcrips, while wild type mice significantly increased expression of MyHC type IIa transcripts in denervated skeletal muscle. Our present results suggest that MuRF-1 may play an important role in regulation of MyHC type IIa during muscle atrophy under microgravity conditions.

  8. The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

    PubMed Central

    Durst, Kristie L.; Lutterbach, Bart; Kummalue, Tanawan; Friedman, Alan D.; Hiebert, Scott W.

    2003-01-01

    Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor. PMID:12509458

  9. Structural abnormalities develop in the brain after ablation of the gene encoding nonmuscle myosin II-B heavy chain.

    PubMed

    Tullio, A N; Bridgman, P C; Tresser, N J; Chan, C C; Conti, M A; Adelstein, R S; Hara, Y

    2001-04-23

    Ablation of nonmuscle myosin heavy chain II-B (NMHC-B) in mice results in severe hydrocephalus with enlargement of the lateral and third ventricles. All B(-)/B(-) mice died either during embryonic development or on the day of birth (PO). Neurons cultured from superior cervical ganglia of B(-)/B(-) mice between embryonic day (E) 18 and P0 showed decreased rates of neurite outgrowth, and their growth cones had a distinctive narrow morphology compared with those from normal mice. Serial sections of E12.5, E13.5, and E15 mouse brains identified developmental defects in the ventricular neuroepithelium. On E12.5, disruption of the coherent ventricular surface and disordered cell migration of neuroepithelial and differentiated cells were seen at various points in the ventricular walls. These abnormalities resulted in the formation of rosettes in various regions of the brain and spinal cord. On E13.5 and E15, disruption of the ventricular surface and aberrant protrusions of neural cells into the ventricles became more prominent. By E18.5 and P0, the defects in cells lining the ventricular wall resulted in an obstructive hydrocephalus due to stenosis or occlusion of the third ventricle and cerebral aqueduct. These defects may be caused by abnormalities in the cell adhesive properties of neuroepithelial cells and suggest that NMHC-B is essential for both early and late developmental processes in the mammalian brain.

  10. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  11. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression.

    PubMed

    Miller, J B; Teal, S B; Stockdale, F E

    1989-08-05

    A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that

  12. The neck region of the myosin motor domain acts as a lever arm to generate movement.

    PubMed Central

    Uyeda, T Q; Abramson, P D; Spudich, J A

    1996-01-01

    The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8633089

  13. Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression.

    PubMed

    Clause, Kelly C; Tchao, Jason; Powell, Mary C; Liu, Li J; Huard, Johnny; Keller, Bradley B; Tobita, Kimimasa

    2012-01-01

    Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle.

  14. Severe atrophy of slow myofibers in aging muscle is concealed by myosin heavy chain co-expression.

    PubMed

    Purves-Smith, Fennigje M; Solbak, Nathan M; Rowan, Sharon L; Hepple, Russell T

    2012-12-01

    Although slow myofibers are considered less susceptible to atrophy with aging, slow fiber atrophy may have been underestimated previously. First, the marked atrophy of the aging rat soleus (Sol) muscle cannot be explained by the atrophy of only the fast fibers, due to their low abundance. Second, the increase in small fibers co-expressing both fast and slow myosin heavy chains (MHC) in the aging rat Sol is proportional to a decline in pure MHC slow fibers (Snow et al., 2005), suggesting that these MHC co-expressing fibers represent formerly pure slow fibers. Thus, we examined the size and proportion of MHC slow, MHC fast, and MHC fast-slow co-expressing fibers in the Sol and mixed region of the gastrocnemius (Gas) muscle in young adult (YA) and senescent (SEN) rats. Our results suggest that formerly pure MHC slow fibers are the source of MHC co-expressing fibers with aging in both muscle regions. Accounting for the atrophy of these fibers in calculating MHC slow fiber atrophy with aging revealed that MHC slow fibers atrophy on average by 40% in the Sol and by 38% in the mixed Gas, values which are similar to the 60% and 31% atrophy of pure MHC fast fibers in the Sol and mixed Gas, respectively. Probing for the atrophy-dependent ubiquitin ligase, MAFbx (atrogin 1), it was suggested that former slow fibers acquire atrophy potential via the up-regulation of MAFbx coincident with the co-expression of fast MHC. These results redefine the impact of aging on slow fiber atrophy, and emphasize the necessity of addressing the atrophy of fast and slow fibers in seeking treatments for aging muscle atrophy.

  15. Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues.

    PubMed

    Rinaldi, F; Terracciano, C; Pisani, V; Massa, R; Loro, E; Vergani, L; Di Girolamo, S; Angelini, C; Gourdon, G; Novelli, G; Botta, A

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a complex multisystemic disorder caused by an expansion of a CTG repeat located at the 3' untranslated region (UTR) of DMPK on chromosome 19q13.3. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms of DM1 including insulin resistance, muscle wasting and myotonia. In this paper we analyzed the expression of the MYH14 mRNA and protein in the muscle of DM1 patients (n=12) with different expansion lengths and normal subjects (n=7). The MYH14 gene is located on chromosome 19q13.3 and encodes for one of the heavy chains of the so called class II "nonmuscle" myosins (NMHCII). MYH14 has two alternative spliced isoforms: the inserted isoform (NMHCII-C1) which includes 8 amino acids located in the globular head of the protein, not encoded by the non inserted isoform (NMHCII-C0). Results showed a splicing unbalance of the MYH14 gene in DM1 muscle, with a prevalent expression of the NMHCII-C0 isoform more marked in DM1 patients harboring large CTG expansions. Minigene assay indicated that levels of the MBNL1 protein positively regulates the inclusion of the MYH14 exon 6. Quantitative analysis of the MYH14 expression revealed a significant reduction in the DM1 muscle samples, both at mRNA and protein level. No differences were found between DM1 and controls in the skeletal muscle localization of MYH14, obtained through immunofluorescence analysis. In line with the thesis of an "RNA gain of function" hypothesis described for the CTG mutation, we conclude that the alterations of the MYH14 gene may contribute to the DM1 molecular pathogenesis.

  16. Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle

    PubMed Central

    Andruchova, Olena; Stephenson, Gabriela M M; Andruchov, Oleg; Stephenson, D George; Galler, Stefan

    2006-01-01

    Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity. PMID:16644798

  17. Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle.

    PubMed

    Andruchova, Olena; Stephenson, Gabriela M M; Andruchov, Oleg; Stephenson, D George; Galler, Stefan

    2006-07-01

    Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.

  18. A continuum of myofibers in adult rabbit extraocular muscle: force, shortening velocity, and patterns of myosin heavy chain colocalization.

    PubMed

    McLoon, Linda K; Park, Han Na; Kim, Jong-Hee; Pedrosa-Domellöf, Fatima; Thompson, Ladora V

    2011-10-01

    Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.

  19. Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity.

    PubMed

    Kengyel, András; Bécsi, Bálint; Kónya, Zoltán; Sellers, James R; Erdődi, Ferenc; Nyitrai, Miklós

    2015-05-01

    The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

  20. An alternative domain near the nucleotide-binding site of Drosophila muscle myosin affects ATPase kinetics.

    PubMed

    Miller, Becky M; Zhang, Shuxing; Suggs, Jennifer A; Swank, Douglas M; Littlefield, Kimberly P; Knowles, Aileen F; Bernstein, Sanford I

    2005-10-14

    In Drosophila melanogaster expression of muscle myosin heavy chain isoforms occurs by alternative splicing of transcripts from a single gene. The exon 7 domain is one of four variable regions in the catalytic head and is located near the nucleotide-binding site. To ascribe a functional role to this domain, we created two chimeric myosin isoforms (indirect flight isoform-exon 7a and embryonic-exon 7d) that differ from the native indirect flight muscle and embryonic body-wall muscle isoforms only in the exon 7 region. Germline transformation and subsequent expression of the chimeric myosins in the indirect flight muscle of myosin-null Drosophila allowed us to purify the myosin for in vitro studies and to assess in vivo structure and function of transgenic muscles. Intriguingly, in vitro experiments show the exon 7 domain modulates myosin ATPase activity but has no effect on actin filament velocity, a novel result compared to similar studies with other Drosophila variable exons. Transgenic flies expressing the indirect flight isoform-exon 7a have normal indirect flight muscle structure, and flight and jump ability. However, expression of the embryonic-exon 7d chimeric isoform yields flightless flies that show improvements in both the structural stability of the indirect flight muscle and in locomotor abilities as compared to flies expressing the embryonic isoform. Overall, our results suggest the exon 7 domain participates in the regulation of the attachment of myosin to actin in order to fine-tune the physiological properties of Drosophila myosin isoforms.

  1. Alternative exon-encoding regions of Locusta migratoria muscle myosin modulate the pH dependence of ATPase activity.

    PubMed

    Li, J; Lu, Z; He, J; Chen, Q; Wang, X; Kang, L; Li, X-D

    2016-12-01

    Whereas the vertebrate muscle myosin heavy chains (MHCs) are encoded by a family of Mhc genes, most insects examined to date contain a single Mhc gene and produce all of the different MHC isoforms by alternative RNA splicing. Here, we found that the migratory locust, Locusta migratoria, has one Mhc gene, which contains 41 exons, including five alternative exclusive exons and one differently included penultimate exon, and potentially encodes 360 MHC isoforms. From the adult L. migratoria, we identified 14 MHC isoforms (including two identical isoforms): four from flight muscle (the thorax dorsal longitudinal muscle), three from jump muscle (the hind leg extensor tibiae muscle) and seven from the abdominal intersegmental muscle. We purified myosins from flight muscle and jump muscle and characterized their motor activities. At neutral pH, the flight and the jump muscle myosins displayed similar levels of in vitro actin-gliding activity, whereas the former had a slightly higher actin-activated ATPase activity than the latter. Interestingly, the pH dependences of the actin-activated ATPase activity of these two myosins are different. Because the dominant MHC isoforms in these two muscles are identical except for the two alternative exon-encoding regions, we propose that these two alternative regions modulate the pH dependence of L. migratoria muscle myosin.

  2. Purification of a protein phosphatase from Acanthamoeba that dephosphorylates and activates myosin II.

    PubMed

    McClure, J A; Korn, E D

    1983-12-10

    The actin-activated ATPase activity of myosin II from Acanthamoeba castellanii is inhibited by phosphorylation of 3 serine residues near the carboxyl end of the heavy chain of the molecule. We have purified a protein phosphatase from Acanthamoeba using myosin II as a substrate. This phosphatase has a molecular weight of 39,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point in urea of 5.2. The enzyme also is active against other phosphoserine protein substrates such as turkey gizzard smooth muscle myosin light chain, but not against a synthetic phosphotyrosine protein substrate. It does not hydrolyze ATP or p-nitrophenol phosphate. No effector has been found to increase substantially the activity of the enzyme as isolated, but it is inhibited by ATP, pyrophosphate, and NaF. This inhibition is reduced in the presence of MnCl2. The Mg2+-dependent actin-activated ATPase of myosin II is activated by dephosphorylation of phosphorylated myosin II by the phosphatase. Its broad substrate specificity, molecular weight, and response to protein phosphatase inhibitors suggest that the Acanthamoeba protein phosphatase is a type 2A phosphatase (Cohen, P. (1982) Nature (Lond.) 206, 613-620).

  3. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    PubMed

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  4. Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function.

    PubMed

    Kronert, William A; Dambacher, Corey M; Knowles, Aileen F; Swank, Douglas M; Bernstein, Sanford I

    2008-06-06

    The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.

  5. Responses of Myosin Heavy Chain Phenotypes and Gene Expressions in Neck Muscle to Micro- an Hyper-Gravity in Mice

    NASA Astrophysics Data System (ADS)

    Ohira, Tomotaka; Ohira, Takashi; Kawano, F.; Shibaguchi, T.; Okabe, H.; Ohno, Y.; Nakai, N.; Ochiai, T.; Goto, K.; Ohira, Y.

    2013-02-01

    Neck muscles are known to play important roles in the maintenance of head posture against gravity. However, it is not known how the properties of neck muscle are influenced by gravity. Therefore, the current study was performed to investigate the responses of neck muscle (rhomboideus capitis) in mice to inhibition of gravity and/or increase to 2-G for 3 months to test the hypothesis that the properties of neck muscles are regulated in response to the level of mechanical load applied by the gravitational load. Three male wild type C57BL/10J mice (8 weeks old) were launched by space shuttle Discovery (STS-128) and housed in Japanese Experimental Module “KIBO” on the International Space Station in mouse drawer system (MDS) project, which was organized by Italian Space Agency. Only 1 mouse returned to the Earth alive after 3 months by space shuttle Atlantis (STS-129). Neck muscles were sampled from both sides within 3 hours after landing. Cage and laboratory control experiments were also performed on the ground. Further, 3-month ground-based control experiments were performed with 6 groups, i.e. pre-experiment, 3-month hindlimb suspension, 2-G exposure by using animal centrifuge, and vivarium control (n=5 each group). Five mice were allowed to recover from hindlimb suspension (including 5 cage control) for 3 months in the cage. Neck muscles were sampled bilaterally before and after 3-month suspension and 2-G exposure, and at the end of 3-month ambulation recovery. Spaceflight-associated shift of myosin heavy chain phenotype from type I to II and atrophy of type I fibers were observed. In response to spaceflight, 17 genes were up-regulated and 13 genes were down-regulated vs. those in the laboratory control. Expression of 6 genes were up-regulated and that of 88 genes were down-regulated by 3-month exposure to 2-G vs. the age-matched cage control. In response to chronic hindlimb suspension, 4 and 20 genes were up- or down-regulated. Further, 98 genes responded

  6. Expression profiles of myostatin, myogenin, and Myosin heavy chain in skeletal muscles of two rabbit breeds differing in growth rate.

    PubMed

    Kuang, Liangde; Xie, Xiaohong; Zhang, Xiangyu; Lei, Min; Li, Congyan; Ren, Yongjun; Zheng, Jie; Guo, Zhiqiang; Zhang, Cuixia; Yang, Chao; Zheng, Yucai

    2014-01-01

    The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.

  7. Association between myosin heavy chain protein isoforms and intramuscular anabolic signaling following resistance exercise in trained men

    PubMed Central

    Gonzalez, Adam M.; Hoffman, Jay R.; Townsend, Jeremy R.; Jajtner, Adam R.; Wells, Adam J.; Beyer, Kyle S.; Willoughby, Darryn S.; Oliveira, Leonardo P.; Fukuda, David H.; Fragala, Maren S.; Stout, Jeffrey R.

    2015-01-01

    Abstract Resistance exercise stimulates an increase in muscle protein synthesis regulated by intracellular anabolic signaling molecules in a mammalian/mechanistic target of rapamycin (mTOR)‐dependent pathway. The purpose of this study was to investigate acute anabolic signaling responses in experienced, resistance‐trained men, and to examine the association between myosin heavy chain (MHC) isoform composition and the magnitude of anabolic signaling. Eight resistance‐trained men (24.9 ± 4.3 years; 91.2 ± 12.4 kg; 176.7 ± 8.0 cm; 13.3 ± 3.9 body fat %) performed a whole body, high‐volume resistance exercise protocol (REX) and a control protocol (CTL) in a balanced, randomized order. Participants were provided a standardized breakfast, recovery drink, and meal during each protocol. Fine needle muscle biopsies were completed at baseline (BL), 2 h (2H) and 6 h post‐exercise (6H). BL biopsies were analyzed for MHC isoform composition. Phosphorylation of proteins specific to the Akt/mTOR signaling pathway and MHC mRNA expression was quantified. Phosphorylation of p70S6k was significantly greater in REX compared to CTL at 2H (P = 0.04). MHC mRNA expression and other targets in the Akt/mTOR pathway were not significantly influenced by REX. The percentage of type IIX isoform was inversely correlated (P < 0.05) with type I and type IIA MHC mRNA expression (r = −0.69 to −0.93). Maximal strength was also observed to be inversely correlated (P < 0.05) with Type I and Type IIA MHC mRNA expression (r = −0.75 to −0.77) and p70S6k phosphorylation (r = −0.75). Results indicate that activation of p70S6k occurs within 2‐h following REX in experienced, resistance‐trained men. Further, results also suggest that highly trained, stronger individuals have an attenuated acute anabolic response. PMID:25626869

  8. C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

    PubMed

    Jóźwiak, Jolanta; Rzhepetskyy, Yuriy; Sobczak, Magdalena; Kocik, Elżbieta; Skórzewski, Radosław; Kłopocka, Wanda; Rędowicz, Maria Jolanta

    2011-02-01

    Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

  9. MicroRNA-23a reduces slow myosin heavy chain isoforms composition through myocyte enhancer factor 2C (MEF2C) and potentially influences meat quality.

    PubMed

    Shen, Linyuan; Chen, Lei; Zhang, Shunhua; Zhang, Yi; Wang, Jingyong; Zhu, Li

    2016-06-01

    MicroRNAs (miRNAs) are non-coding small RNAs that participate in the regulation of a variety of biological processes. Muscle fiber types were very important to meat quality traits, however, the molecular mechanism by which miRNAs regulate the muscle fiber type composition is not fully understood. The aim of this study was to investigate whether miRNA-23a can affect muscle fiber type composition. Luciferase reporter assays proved that miRNA-23a directly targets the 3' untranslated region (UTRs) of MEF2c. Overexpression of miRNA-23a significantly suppressed the expression of MEF2c both in mRNA and protein levels, thus caused down-regulation of the expression of some key downstream genes of MEF2c (PGC1-α, NRF1 and mtTFA). More interestingly, overexpression of miRNA-23a significantly restrained the myogenic differentiation and decreased the ratio of slow myosin heavy chain in myoblasts (p<0.05). Our findings hinted a novel role of miRNA-23a in the epigenetic regulation of meat quality via decreasing the ratio of slow myosin heavy chain isoforms.

  10. Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Peggion, Caterina; Massimino, Maria Lina; Biancotto, Giancarlo; Angeletti, Roberto; Reggiani, Carlo; Sorgato, Maria Catia; Bertoli, Alessandro; Stella, Roberto

    2017-03-01

    Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

  11. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

    ERIC Educational Resources Information Center

    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

  12. Myosin chaperones☆

    PubMed Central

    Hellerschmied, Doris; Clausen, Tim

    2014-01-01

    The folding and assembly of myosin motor proteins is essential for most movement processes at the cellular, but also at the organism level. Importantly, myosins, which represent a very diverse family of proteins, require the activity of general and specialized folding factors to develop their full motor function. The activities of the myosin-specific UCS (UNC-45/Cro1/She4) chaperones range from assisting acto-myosin dependent transport processes to scaffolding multi-subunit chaperone complexes, which are required to assemble myofilaments. Recent structure–function studies revealed the structural organization of TPR (tetratricopeptide repeat)-containing and TPR-less UCS chaperones. The observed structural differences seem to reflect the specialized and remarkably versatile working mechanisms of myosin-directed chaperones, as will be discussed in this review. PMID:24440450

  13. Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens

    PubMed Central

    Michael, Magdalene; Liang, Xuan; Gomez, Guillermo A.

    2017-01-01

    Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at various subcellular localizations (Ebrahim et al., 2013; Beach et al., 2014). At the zonula adherens (ZA) of epithelia, NMII minifilaments bind the circumferential actin bundles in a pseudo-sarcomeric manner (Ebrahim et al., 2013), a conformation required to maintain junctional tension and tissue integrity (Ratheesh et al., 2012). By expressing green fluorescent protein (GFP)-NMIIA heavy chain and immunolabel it using a NMIIA C-terminus specific antibody, we were able to visualize the NMII minifilaments bound to F-actin bundles in Caco-2 cells (Michael et al., 2016), as previously reported (Ebrahim et al., 2013; Beach et al., 2014). In addition, we designed an FIJI/MATLAB analysis module to quantify the size, distance and alignment of these minifilaments with respect to junctional F-actin at the ZA. Measurements of the dispersion of minifilaments angles were proven to be a useful parameter that closely correlated to the extent of contractility at junctions (Michael et al., 2016). PMID:28251171

  14. Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family

    PubMed Central

    1989-01-01

    The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625- 14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine. PMID:2687288

  15. Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins.

    PubMed

    Pasha, Shaik Naseer; Meenakshi, Iyer; Sowdhamini, Ramanathan

    2016-01-01

    Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes.

  16. Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins

    PubMed Central

    Pasha, Shaik Naseer; Meenakshi, Iyer; Sowdhamini, Ramanathan

    2016-01-01

    Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes. PMID:27597808

  17. MicroRNA deep sequencing in two adult stem cell populations identifies miR-501 as a novel regulator of myosin heavy chain during muscle regeneration

    PubMed Central

    Mizbani, Amir; Luca, Edlira; Rushing, Elisabeth J.

    2016-01-01

    MicroRNAs (miRNAs) are important regulators of skeletal muscle regeneration, but the underlying mechanisms are still incompletely understood. Here, comparative miRNA sequencing analysis of myogenic progenitor cells (MPs) and non-myogenic fibroblast-adipocyte progenitors (FAPs) during cardiotoxin (CTX)-induced muscle injury uncovered miR-501 as a novel muscle-specific miRNA. miR-501 is an intronic miRNA and its expression levels in MPs correlated with its host gene, chloride channel, voltage-sensitive 5 (Clcn5). Pharmacological inhibition of miR-501 dramatically blunted the induction of embryonic myosin heavy chain (MYH3) and, to a lesser extent, adult myosin isoforms during muscle regeneration, and promoted small-diameter neofibers. An unbiased target identification approach in primary myoblasts validated gigaxonin as a target of miR-501 that mimicked the effect of miR-501 inhibition on MYH3 expression. In the mdx mouse model, which models a pathological disease state, not only was miR-501 induced in regenerating skeletal muscle, but also its serum levels were increased, which correlated with the disease state of the animals. Our results suggest that miR-501 plays a key role in adult muscle regeneration and might serve as a novel serum biomarker for the activation of adult muscle stem cells. PMID:27707793

  18. Characterization of Amoeba proteus myosin VI immunoanalog.

    PubMed

    Dominik, Magdalena; Kłopocka, Wanda; Pomorski, Paweł; Kocik, Elzbieta; Redowicz, Maria Jolanta

    2005-07-01

    Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

  19. Actin crosslinkers: repairing the sense of touch.

    PubMed

    Sun, Sean X; Walcott, Sam

    2010-10-26

    Cells use actin bundles infused with myosin to exert contractile forces on the extracellular environment. This active tension is essential for cellular mechanosensation. Now, the role of actin crosslinkers in stabilizing and repairing the actin bundles is coming into clearer view.

  20. Load and Pi control flux through the branched kinetic cycle of myosin V.

    PubMed

    Kad, Neil M; Trybus, Kathleen M; Warshaw, David M

    2008-06-20

    Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (P(i)), both forward and backward steps become less load-dependent. From these data, we infer that P(i) release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of P(i). We propose a branched kinetic model to explain these data. Our model includes P(i) release prior to the most load-dependent step in the cycle, implying that P(i) release and load both act as checkpoints that control the flux through two parallel pathways.

  1. 3-Methylhistidine in actin and other muscle proteins

    PubMed Central

    Johnson, P.; Harris, C. I.; Perry, S. V.

    1967-01-01

    1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7·6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation. ImagesFig. 1.Fig. 2.Fig. 3. PMID:6056634

  2. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed

    Knotts, S; Rindt, H; Robbins, J

    1995-08-25

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.

  3. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed Central

    Knotts, S; Rindt, H; Robbins, J

    1995-01-01

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms. Images PMID:7667107

  4. Akirin2 promotes slow myosin heavy chain expression by CaN/NFATc1 signaling in porcine skeletal muscle satellite cells.

    PubMed

    Chen, Xiaoling; Luo, Yanliu; Huang, Zhiqing; Liu, Guangmang; Zhao, Hua

    2017-02-16

    The objective of this study was to evaluate the effect of Akirin2 on slow myosin heavy chain (slow MyHC, MyHC I) gene expression and its molecular mechanisms. In this study, we showed that the protein expression of Akirin2 in pig slow oxidative Psoas major muscle is higher than that in fast glycolytic tibialis anterior muscle, suggesting that Akirin2 may play a role in myofiber typing. Knockdown of Akirin2 decreased the MyHC I expression and the calcineurin (CaN) activity, and also decreased the expressions of NFATc1 and MCIP1.4. Conversely, overexpression of Akirin2 got the opposite results. Furthermore, inhibition of CaN or knockdown of NFATc1 attenuated Akirin2 overexpression-induced upregulation of MyHC I. Together, these results demonstrate that Akirin2 promotes MyHC I expression via CaN/NFATc1 signaling pathway in porcine skeletal muscle satellite cells.

  5. Cardiac beta-myosin heavy chain defects in two families with non-compaction cardiomyopathy: linking non-compaction to hypertrophic, restrictive, and dilated cardiomyopathies.

    PubMed

    Hoedemaekers, Yvonne M; Caliskan, Kadir; Majoor-Krakauer, Danielle; van de Laar, Ingrid; Michels, Michelle; Witsenburg, Maarten; ten Cate, Folkert J; Simoons, Maarten L; Dooijes, Dennis

    2007-11-01

    Cardiomyopathies are classified according to distinct morphological characteristics. They occur relatively frequent and are an important cause of mortality and morbidity. Isolated ventricular non-compaction or non-compaction cardiomyopathy (NCCM) is characterized by an excessively thickened endocardial layer with deep intertrabecular recesses, reminiscent of the myocardium during early embryogenesis. Aims Autosomal-dominant as well as X-linked inheritance for NCCM has been described and several loci have been associated with the disease. Nevertheless, a major genetic cause for familial NCCM remains to be identified. Methods and Results We describe, in two separate autosomal-dominant NCCM families, the identification of mutations in the sarcomeric cardiac beta-myosin heavy chain gene (MYH7), known to be associated with hypertrophic cardiomyopathy (HCM), restricted cardiomyopathy (RCM), and dilated cardiomyopathy (DCM). Conclusion These results confirm the genetic heterogeneity of NCCM and suggest that the molecular classification of cardiomyopathies includes an MYH7-associated spectrum of NCCM with HCM, RCM, and DCM.

  6. The Spatial Distribution of Actin and Mechanical Cycle of Myosin Are Different in Right and Left Ventricles of Healthy Mouse Hearts

    PubMed Central

    2014-01-01

    The contraction of the right ventricle (RV) expels blood into the pulmonary circulation, and the contraction of the left ventricle (LV) pumps blood into the systemic circulation through the aorta. The respective afterloads imposed on the LV and RV by aortic and pulmonary artery pressures create very different mechanical requirements for the two ventricles. Indeed, differences have been observed in the contractile performance between left and right ventricular myocytes in dilated cardiomyopathy, in congestive heart failure, and in energy usage and speed of contraction at light loads in healthy hearts. In spite of these functional differences, it is commonly believed that the right and left ventricular muscles are identical because there were no differences in stress development, twitch duration, work performance, or power among the RV and LV in dogs. This report shows that on a mesoscopic scale [when only a few molecules are studied (here three to six molecules of actin) in ex vivo ventricular myofibrils], the two ventricles in rigor differ in the degree of orientational disorder of actin within in filaments and during contraction in the kinetics of the cross-bridge cycle. PMID:25488019

  7. Postnatal development of epididymis and ductus deferens in the rat. A correlation between the ultrastructure of the epithelium and tubule wall, and the fluorescence-microscopic distribution of actin, myosin, fibronectin, and basement membrane.

    PubMed

    Francavilla, S; Moscardelli, S; Properzi, G; De Matteis, M A; Scorza Barcellona, P; Natali, P G; De Martino, C

    1987-08-01

    The postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light- and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.

  8. Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Day, I. S.

    2001-01-01

    BACKGROUND: Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants. RESULTS: Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication. CONCLUSIONS: Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

  9. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  10. Temporal embryonic transcription of chicken fast skeletal myosin heavy chain isoforms in the single comb white leghorn.

    PubMed

    Griffin, J; St-Pierre, N; Lilburn, M S; Wick, M

    2016-05-01

    There are numerous factors that can significantly influence embryonic development in poultry and thus make simple days of incubation (chronological age) a less than perfect metric for studying embryonic physiology. The developmental fast skeletal muscle myosin (MyHC), the predominant protein in the Pectoralis major (PM), is temporally expressed as a cadre of highly specific developmental isoforms. In the study described herein, a novel molecular technology (NanoString) was used to characterize the myosin isoform transcriptional patterns in the PM of Single Comb White Leghorn (SCWL) embryos. NanoString technology is based on quantitative analysis of the transcriptome through digital detection and quantification of target mRNA transcripts. Total RNA was isolated and gene transcription quantified using NanoString in embryonic muscle samples collected daily from 6 through 19 days of incubation. Data were analyzed using the LOESS smoothing function at a 95% confidence level. The temporal transcription of MyHC isoforms obtained in this study was consistent with the literature at higher specificity and resolution, thus validating NanoString for use in gene transcription analyses. The results support a hypothesis that the transcription patterns of the embryonic MyHC isoforms may be used as molecular clocks to further investigate the developmental relationships underlying embryonic fast skeletal muscle growth and development.

  11. Measurements of Myosin-II Motor Activity During Cytokinesis in Fission Yeast.

    PubMed

    Tang, Qing; Pollard, Luther W; Lord, Matthew

    2016-01-01

    Fission yeast myosin-II (Myo2p) represents the critical actin-based motor protein that drives actomyosin ring assembly and constriction during cytokinesis. We detail three different methods to measure Myo2p motor function. Actin-activated ATPases provide a readout of actomyosin ATPase motor activity in a bulk assay; actin filament motility assays reveal the speed and efficiency of myosin-driven actin filament gliding (when motors are anchored); myosin-bead motility assays reveal the speed and efficiency of myosin ensembles traveling along actin filaments (when actin is anchored). Collectively, these methods allow us to combine the standard in vivo approaches common to fission yeast with in vitro biochemical methods to learn more about the mechanistic action of myosin-II during cytokinesis.

  12. An Embryonic Myosin Isoform Enables Stretch Activation and Cyclical Power in Drosophila Jump Muscle

    PubMed Central

    Zhao, Cuiping; Swank, Douglas M.

    2013-01-01

    The mechanism behind stretch activation (SA), a mechanical property that increases muscle force and oscillatory power generation, is not known. We used Drosophila transgenic techniques and our new muscle preparation, the jump muscle, to determine if myosin heavy chain isoforms influence the magnitude and rate of SA force generation. We found that Drosophila jump muscles show very low SA force and cannot produce positive power under oscillatory conditions at pCa 5.0. However, we transformed the jump muscle to be moderately stretch-activatable by replacing its myosin isoform with an embryonic isoform (EMB). Expressing EMB, jump muscle SA force increased by 163% and it generated net positive power. The rate of SA force development decreased by 58% with EMB expression. Power generation is Pi dependent as >4 mM Pi was required for positive power from EMB. Pi increased EMB SA force, but not wild-type SA force. Our data suggest that when muscle expressing EMB is stretched, EMB is more easily driven backward to a weakly bound state than wild-type jump muscle. This increases the number of myosin heads available to rapidly bind to actin and contribute to SA force generation. We conclude that myosin heavy chain isoforms influence both SA kinetics and SA force, which can determine if a muscle is capable of generating oscillatory power at a fixed calcium concentration. PMID:23790374

  13. Myosin II Activity Softens Cells in Suspension

    PubMed Central

    Chan, Chii J.; Ekpenyong, Andrew E.; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J.; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-01-01

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  14. Cell Adhesion, Signaling and Myosin in Breast Cancer.

    DTIC Science & Technology

    1998-08-01

    calmodulin-binding myosins. Trends Cell Bio 5:310- 316. 4. de Lanerolle, P. and Paul, R.J. (1991) Myosin phosphorylation/ dephosphorylation and...muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus. J. Bio. Chem., 265:17876-17882 10

  15. The kinetics of mechanically coupled myosins exhibit group size-dependent regimes.

    PubMed

    Hilbert, Lennart; Cumarasamy, Shivaram; Zitouni, Nedjma B; Mackey, Michael C; Lauzon, Anne-Marie

    2013-09-17

    Naturally occurring groups of muscle myosin behave differently from individual myosins or small groups commonly assayed in vitro. Here, we investigate the emergence of myosin group behavior with increasing myosin group size. Assuming the number of myosin binding sites (N) is proportional to actin length (L) (N = L/35.5 nm), we resolve in vitro motility of actin propelled by skeletal muscle myosin for L = 0.2-3 μm. Three distinct regimes were found: L < 0.3 μm, sliding arrest; 0.3 μm ≤ L ≤ 1 μm, alternation between arrest and continuous sliding; L > 1 μm, continuous sliding. We theoretically investigated the myosin group kinetics with mechanical coupling via actin. We find rapid actin sliding steps driven by power-stroke cascades supported by postpower-stroke myosins, and phases without actin sliding caused by prepower-stroke myosin buildup. The three regimes are explained: N = 8, rare cascades; N = 15, cascade bursts; N = 35, continuous cascading. Two saddle-node bifurcations occur for increasing N (mono → bi → mono-stability), with steady states corresponding to arrest and continuous cascading. The experimentally measured dependence of actin sliding statistics on L and myosin concentration is correctly predicted.

  16. The Kinetics of Mechanically Coupled Myosins Exhibit Group Size-Dependent Regimes

    PubMed Central

    Hilbert, Lennart; Cumarasamy, Shivaram; Zitouni, Nedjma B.; Mackey, Michael C.; Lauzon, Anne-Marie

    2013-01-01

    Naturally occurring groups of muscle myosin behave differently from individual myosins or small groups commonly assayed in vitro. Here, we investigate the emergence of myosin group behavior with increasing myosin group size. Assuming the number of myosin binding sites (N) is proportional to actin length (L) (N = L/35.5 nm), we resolve in vitro motility of actin propelled by skeletal muscle myosin for L = 0.2–3 μm. Three distinct regimes were found: L < 0.3 μm, sliding arrest; 0.3 μm ≤ L ≤ 1 μm, alternation between arrest and continuous sliding; L > 1 μm, continuous sliding. We theoretically investigated the myosin group kinetics with mechanical coupling via actin. We find rapid actin sliding steps driven by power-stroke cascades supported by postpower-stroke myosins, and phases without actin sliding caused by prepower-stroke myosin buildup. The three regimes are explained: N = 8, rare cascades; N = 15, cascade bursts; N = 35, continuous cascading. Two saddle-node bifurcations occur for increasing N (mono → bi → mono-stability), with steady states corresponding to arrest and continuous cascading. The experimentally measured dependence of actin sliding statistics on L and myosin concentration is correctly predicted. PMID:24047998

  17. Myosin II Motor Activity in the Lateral Amygdala Is Required for Fear Memory Consolidation

    ERIC Educational Resources Information Center

    Gavin, Cristin F.; Rubio, Maria D.; Young, Erica; Miller, Courtney; Rumbaugh, Gavin

    2012-01-01

    Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have…

  18. Myosin I contributes to the generation of resting cortical tension.

    PubMed

    Dai, J; Ting-Beall, H P; Hochmuth, R M; Sheetz, M P; Titus, M A

    1999-08-01

    The amoeboid myosin I's are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin I's possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin I's, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin I's is not controlled by this stimulus. These results demonstrate that myosin I's work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

  19. Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber.

    PubMed

    Minoda, Hiroki; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-02-25

    Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

  20. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  1. Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

    PubMed Central

    Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

    2015-01-01

    MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

  2. Orbit/CLASP is required for myosin accumulation at the cleavage furrow in Drosophila male meiosis.

    PubMed

    Kitazawa, Daishi; Matsuo, Tatsuru; Kaizuka, Kana; Miyauchi, Chie; Hayashi, Daisuke; Inoue, Yoshihiro H

    2014-01-01

    Peripheral microtubules (MTs) near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR) in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.

  3. Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

    PubMed Central

    Hilbert, Lennart; Balassy, Zsombor; Zitouni, Nedjma B.; Mackey, Michael C.; Lauzon, Anne-Marie

    2015-01-01

    Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo—suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state. PMID:25650929

  4. Phosphate and ADP differently inhibit coordinated smooth muscle myosin groups.

    PubMed

    Hilbert, Lennart; Balassy, Zsombor; Zitouni, Nedjma B; Mackey, Michael C; Lauzon, Anne-Marie

    2015-02-03

    Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo--suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.

  5. Myosins as fundamental components during tumorigenesis: diverse and indispensable

    PubMed Central

    Li, Yan-Ruide; Yang, Wan-Xi

    2016-01-01

    Myosin is a kind of actin-based motor protein. As the crucial functions of myosin during tumorigenesis have become increasingly apparent, the profile of myosin in the field of cancer research has also been growing. Eighteen distinct classes of myosins have been discovered in the past twenty years and constitute a diverse superfamily. Various myosins share similar structures. They all convert energy from ATP hydrolysis to exert mechanical stress upon interactions with microfilaments. Ongoing research is increasingly suggesting that at least seven kinds of myosins participate in the formation and development of cancer. Myosins play essential roles in cytokinesis failure, chromosomal and centrosomal amplification, multipolar spindle formation and DNA microsatellite instability. These are all prerequisites of tumor formation. Subsequently, myosins activate various processes of tumor invasion and metastasis development including cell migration, adhesion, protrusion formation, loss of cell polarity and suppression of apoptosis. In this review, we summarize the current understanding of the roles of myosins during tumorigenesis and discuss the factors and mechanisms which may regulate myosins in tumor progression. Furthermore, we put forward a completely new concept of “chromomyosin” to demonstrate the pivotal functions of myosins during karyokinesis and how this acts to optimize the functions of the members of the myosin superfamily. PMID:27121062

  6. Muscle-Specific Myosin Heavy Chain Shifts in Response to a Long-Term High Fat/High Sugar Diet and Resveratrol Treatment in Nonhuman Primates

    PubMed Central

    Hyatt, Jon-Philippe K.; Nguyen, Lisa; Hall, Allison E.; Huber, Ashley M.; Kocan, Jessica C.; Mattison, Julie A.; de Cabo, Rafael; LaRocque, Jeannine R.; Talmadge, Robert J.

    2016-01-01

    Shifts in myosin heavy chain (MHC) expression within skeletal muscle can be induced by a host of stimuli including, but not limited to, physical activity, alterations in neural activity, aging, and diet or obesity. Here, we hypothesized that both age and a long-term (2 year) high fat/high sugar diet (HFS) would induce a slow to fast MHC shift within the plantaris, soleus, and extensor digitorum longus (EDL) muscles from rhesus monkeys. Furthermore, we tested whether supplementation with resveratrol, a naturally occurring compound that has been attributed with augmenting aerobic potential through mitochondrial proliferation, would counteract any diet-induced MHC changes by promoting a fast to slow isoform switch. In general, we found that MHC isoforms were not altered by aging during mid-life. The HFS diet had the largest impact within the soleus muscle where the greatest slow to fast isoform shifts were observed in both mRNA and protein indicators. As expected, long-term resveratrol treatment counteracted, or blunted, these diet-induced shifts within the soleus muscle. The plantaris muscle also demonstrated a fast-to-slow phenotypic response to resveratrol treatment. In conclusion, diet or resveratrol treatment impacts skeletal muscle phenotype in a muscle-specific manner and resveratrol supplementation may be one approach for promoting the fatigue-resistant MHC (type I) isoform especially if its expression is blunted as a result of a long-term high fat/sugar diet. PMID:26973542

  7. Muscle Fiber Type Specific Activation of the Slow Myosin Heavy Chain 2 Promoter by a Non-canonical E-box

    PubMed Central

    Weimer, Kristina; DiMario, Joseph X.

    2016-01-01

    Different mechanisms control skeletal muscle fiber type gene expression at specific times in vertebrate development. Embryonic myogenesis leading to formation of primary muscle fibers in avian species is largely directed by myoblast cell commitment to the formation of diverse fiber types. In contrast, development of different secondary fiber types during fetal myogenesis is partly determined by neural influences. In both primary and secondary chicken muscle fibers, differential expression of the slow myosin heavy chain 2 (MyHC2) gene distinguishes fast from fast/slow muscle fibers. This study focused on the transcriptional regulation of the slow MyHC2 gene in primary myotubes formed from distinct fast/slow and fast myogenic cell lineages. Promoter deletion analyses identified a discrete 86bp promoter segment that conferred fiber type, lineage-specific gene expression in fast/slow versus fast myoblast derived primary myotubes. Sequence analysis and promoter activity assays determined that this segment contains two functional cis-regulatory elements. One element is a non-canonical E-box, and electromobility shift assays demonstrated that both cis-elements interacted with the E-protein, E47. The results indicate that primary muscle fiber type specific expression of the slow MyHC2 gene is controlled by a novel mechanism involving a transcriptional complex that includes E47 at a non-canonical E-box. PMID:26707643

  8. In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats

    NASA Technical Reports Server (NTRS)

    Giger, J. M.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    2000-01-01

    In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

  9. Characterisation of isoform-specific tryptic peptides of rat cardiac myosin heavy chains using automated liquid chromatography-matrix assisted laser desorption ionisation (LC-MALDI) mass spectrometry.

    PubMed

    Burniston, Jatin G; Connolly, Joanne B

    2010-04-30

    Proteomics investigations using 2-dimensional electrophoresis (2-DE) cannot resolve the entire cardiac proteome because some proteins, including myosin heavy chains (MyHC), are insoluble in the buffers required for isoelectric focusing. Here, we report an automated mass spectrometry (MS) method complementary to 2-DE and capable of yielding important additional information. Rat myocardium was homogenised in standard lysis solution and centrifuged to produce a supernatant fraction, suitable for 2-DE. The pelleted fraction, which is normally discarded, was used for the current analysis. Proteins were digested with trypsin and the peptides fractionated by HPLC. Automated spotting of eluent fractions onto 384-well target plates and matrix-assisted laser desorption tandem time of flight (MALDI-ToF/ToF) MS were directed by dedicated software. Peptide ions were fragmented by collision-induced dissociation and the MS/MS spectra searched against the NCBI database using Mascot. This approach confidently identified 13 tryptic peptides specific to cardiac alpha-MyHC and 4 specific to beta-MyHC, which can be used to differentiate these highly homologous protein isoforms in future quantitative MS analyses.

  10. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    PubMed Central

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  11. Effects of chronic Akt/mTOR inhibition by rapamycin on mechanical overload-induced hypertrophy and myosin heavy chain transition in masseter muscle.

    PubMed

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2013-01-01

    To examine the effects of the Akt/mammalian target of rapamycin (mTOR) pathway on masseter muscle hypertrophy and myosin heavy chain (MHC) transition in response to mechanical overload, we analyzed the effects of bite-opening (BO) on the hypertrophy and MHC composition of masseter muscle of BO-rats treated or not treated with rapamycin (RAPA), a selective mTOR inhibitor. The masseter muscle weight in BO-rats was significantly greater than that in controls, and this increase was attenuated by RAPA treatment. Expression of slow-twitch MHC isoforms was significantly increased in BO-rats with/without RAPA treatment, compared with controls, but the magnitude of the increase was much smaller in RAPA-treated BO-rats. Phosphorylation of p44/42 MAPK (ERK1/2), which preserves fast-twitch MHC isoforms in skeletal muscle, was significantly decreased in BO-rats, but the decrease was abrogated by RAPA treatment. Calcineurin signaling is known to be important for masseter muscle hypertrophy and fast-to-slow MHC isoform transition, but expression of known calcineurin activity modulators was unaffected by RAPA treatment. Taken together, these results indicate that the Akt/mTOR pathway is involved in both development of masseter muscle hypertrophy and fast-to-slow MHC isoform transition in response to mechanical overload with inhibition of the ERK1/2 pathway and operates independently of the calcineurin pathway.

  12. Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs

    PubMed Central

    Cho, Eun‐Seok; Lee, Kyung‐Tai; Kim, Jun‐Mo; Lee, Si‐Woo; Jeon, Hyeon‐Jeong; Lee, Seung‐Hwan; Hong, Ki‐Chang

    2015-01-01

    Abstract We identified a potential molecular marker associated with meat quality traits in the myosin heavy chain 4, MYH4 gene of Landrace pigs. Sequencing revealed a single nucleotide polymorphism (SNP; g.‐1398G>T) in the 5' upstream region of MYH4. It was significantly associated with the number of type IIa muscle fibers and water‐holding capacity based on filter‐paper fluid uptake. The GG genotype groups had a greater number of type IIa fibers and a larger area composed of type IIa fibers than the other genotype group (P = 0.004 and P = 0.061, respectively). Expression level of MYH4 gene in the genotype TT or GT was higher than in genotype of GG (P < 0.0001). The T allele may enhance expression level of MYH4 gene and then the portion of IIb type fiber in the muscle be increased by the T allelle. Therefore, we suggest that the g.‐1398G>T in the 5' upstream region of the porcine MYH4 may be used as a molecular marker for meat quality traits, although its functional effect is not defined yet. PMID:26271027

  13. Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Chu, Wuying; Fu, Guihong; Bing, Shiyu; Meng, Tao; Zhou, Ruixue; Cheng, Jia; Zhao, Falan; Zhang, Hongfang; Zhang, Jianshe

    2010-03-01

    The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%-76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%-80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

  14. The genome of the diploid anuran Xenopus tropicalis contains a novel array of sarcoplasmic myosin heavy chain genes expressed in larval muscle and larynx

    PubMed Central

    Nasipak, Brian T.; Kelley, Darcy B.

    2010-01-01

    The sarcomeric myosin heavy chain (MyHC) proteins are a family of molecular motors responsible for the transduction of chemical energy into mechanical work in striated muscle. The vertebrate genome contains multiple copies of the MyHC gene, and expression of different isoforms correlates with differences in the physiological properties of muscle fibers. Most MyHC isoforms are found in two arrays, one containing the “fast-twitch” skeletal muscle isoforms and the other the “slow-twitch” or cardiac isoforms. To extend our understanding of MyHC evolution, we have examined the genome of the anuran Xenopus tropicalis. The X. tropicalis genome includes15 full-length MyHC genes organized in seven genomic locations. One unique array of MyHC genes is similar to the mammalian fast-skeletal array, but is not found in amniotes. The isoforms in this array are expressed during larval stages and in muscles of the adult larynx. Duplication of the fast-skeletal MyHC array appears to have led to expression divergence of muscle proteins in the larval and adult stages of the anuran life cycle. A striking similarity of gene order between regions flanking X. tropicalis MyHC arrays and human arrays was evident; genomic organization of MyHC isoforms may thus be highly conserved across tetrapods. PMID:18551304

  15. Altered expression of pectoral myosin heavy chain isoforms corresponds to migration status in the white-crowned sparrow (Zonotrichia leucophrys gambelii)

    PubMed Central

    Welch, Kenneth C.; Ramenofsky, Marilyn

    2016-01-01

    Birds undergo numerous changes as they progress through life-history stages, yet relatively few studies have examined how birds adapt to both the dynamic energetic and mechanical demands associated with such transitions. Myosin heavy chain (MyHC) expression, often linked with muscle fibre type, is strongly correlated with a muscle's mechanical power-generating capability, thus we examined several morphological properties, including MyHC expression of the pectoralis, in a long-distance migrant, the white-crowned sparrow (Zonotrichia leucophrys gambelii) throughout the progression from winter, spring departure and arrival on breeding grounds. White-crowned sparrows demonstrated significant phenotypic flexibility throughout the seasonal transition, including changes in prealternate moult status, lipid fuelling, body condition and flight muscle morphology. Pectoral MyHC expression also varied significantly over the course of the study. Wintering birds expressed a single, newly classified adult fast 2 isoform. At spring departure, pectoral isoform expression included two MyHC isoforms: the adult fast 2 isoform along with a smaller proportion of a newly present adult fast 1 isoform. By spring arrival, both adult fast isoforms present at departure remained, yet expression had shifted to a greater relative proportion of the adult fast 1 isoform. Altering pectoral MyHC isoform expression in preparation for and during spring migration may represent an adaptation to modulate muscle mechanical output to support long-distance flight. PMID:28018664

  16. Visualization of an unstable coiled coil from the scallop myosin rod.

    PubMed

    Li, Yu; Brown, Jerry H; Reshetnikova, Ludmilla; Blazsek, Antal; Farkas, László; Nyitray, László; Cohen, Carolyn

    2003-07-17

    Alpha-helical coiled coils in muscle exemplify simplicity and economy of protein design: small variations in sequence lead to remarkable diversity in cellular functions. Myosin II is the key protein in muscle contraction, and the molecule's two-chain alpha-helical coiled-coil rod region--towards the carboxy terminus of the heavy chain--has unusual structural and dynamic features. The amino-terminal subfragment-2 (S2) domains of the rods can swing out from the thick filament backbone at a hinge in the coiled coil, allowing the two myosin 'heads' and their motor domains to interact with actin and generate tension. Most of the S2 rod appears to be a flexible coiled coil, but studies suggest that the structure at the N-terminal region is unstable, and unwinding or bending of the alpha-helices near the head-rod junction seems necessary for many of myosin's functional properties. Here we show the physical basis of a particularly weak coiled-coil segment by determining the 2.5-A-resolution crystal structure of a leucine-zipper-stabilized fragment of the scallop striated-muscle myosin rod adjacent to the head-rod junction. The N-terminal 14 residues are poorly ordered; the rest of the S2 segment forms a flexible coiled coil with poorly packed core residues. The unusual absence of interhelical salt bridges here exposes apolar core atoms to solvent.

  17. Calcineurin-NFAT Signaling and Neurotrophins Control Transformation of Myosin Heavy Chain Isoforms in Rat Soleus Muscle in Response to Aerobic Treadmill Training

    PubMed Central

    Liu, Wenfeng; Chen, Gan; Li, Fanling; Tang, Changfa; Yin, Dazhong

    2014-01-01

    This study elucidated the role of CaN-NFAT signaling and neurotrophins on the transformation of myosin heavy chain isoforms in the rat soleus muscle fiber following aerobic exercise training. To do so, we examined the content and distribution of myosin heavy chain (MyHC) isoforms in the rat soleus muscle fiber, the activity of CaN and expression of NFATc1 in these fibers, and changes in the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neutrophin-3 (NT-3) in the soleus and striatum following high-and medium-intensity aerobic treadmill training. Specific pathogen-free 2 month old male Sprague-Dawley (SD) rats were randomly divided into three groups: Control group (Con, n = 8), moderate-intensity aerobic exercise group (M-Ex, n = 8) and high-intensity aerobic exercise group (H-Ex, n = 8). We used ATPase staining to identify the muscle fiber type I and II, SDS-PAGE to separate and analyze the isoforms MyHCI, MyHCIIA, MyHCIIB and MyHCIIx, and performed western blots to determine the expression of NFATc1, NGF, BDNF and NT-3. CaN activity was measured using a colorimetric assay. In the soleus muscle, 8 weeks of moderate-intensity exercise can induce transformation of MyHC IIA and MyHC IIB to MyHC IIX and MyHC I (p < 0.01), while high-intensity treadmill exercise can induce transform MyHC IIx to MyHC IIB, MyHC IIA and MyHC I (p < 0.01). In comparison to the control group, CaN activity and NFATcl protein level were significantly increased in both the M-Ex and H-Ex groups (p < 0.05, p < 0.01), with a more pronounced upregulation in the M-Ex group (p < 0.05). Eight weeks of moderate- and high-intensity aerobic exercise induced the expression of NGF, BDNF and NT-3 in the soleus muscle and the striatum (p < 0.01), with the most significant increase in the H-Ex group (p < 0.01). In the rat soleus muscle, (1) CaN–NFATcl signaling contributes to the conversion of MyHC I isoform in response to moderate-intensity exercise; (2) Neurotrophins

  18. Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

    PubMed Central

    Brown, David M.; Brameld, John M.; Parr, Tim

    2014-01-01

    Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter. PMID:25469802

  19. Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading.

    PubMed

    Pandorf, Clay E; Haddad, Fadia; Wright, Carola; Bodell, Paul W; Baldwin, Kenneth M

    2009-07-01

    Recent advances in chromatin biology have enhanced our understanding of gene regulation. It is now widely appreciated that gene regulation is dependent upon post-translational modifications to the histones which package genes in the nucleus of cells. Active genes are known to be associated with acetylation of histones (H3ac) and trimethylation of lysine 4 in histone H3 (H3K4me3). Using chromatin immunoprecipitation (ChIP), we examined histone modifications at the myosin heavy chain (MHC) genes expressed in fast vs. slow fiber-type skeletal muscle, and in a model of muscle unloading, which results in a shift to fast MHC gene expression in slow muscles. Both H3ac and H3K4me3 varied directly with the transcriptional activity of the MHC genes in fast fiber-type plantaris and slow fiber-type soleus. During MHC transitions with muscle unloading, histone H3 at the type I MHC becomes de-acetylated in correspondence with down-regulation of that gene, while upregulation of the fast type IIx and IIb MHCs occurs in conjunction with enhanced H3ac in those MHCs. Enrichment of H3K4me3 is also increased at the type IIx and IIb MHCs when these genes are induced with muscle unloading. Downregulation of IIa MHC, however, was not associated with corresponding loss of H3ac or H3K4me3. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of types I, IIx and IIb MHC genes are sensitive to histone modifications both in different muscle fiber-types and in response to altered loading states.

  20. Contractile properties of motor units and expression of myosin heavy chain isoforms in rat fast-type muscle after volitional weight-lifting training.

    PubMed

    Łochyński, Dawid; Kaczmarek, Dominik; Mrówczyński, Włodzimierz; Warchoł, Wojciech; Majerczak, Joanna; Karasiński, Janusz; Korostyński, Michał; Zoladz, Jerzy A; Celichowski, Jan

    2016-10-01

    Dynamic resistance training increases the force and speed of muscle contraction, but little is known about modifications to the contractile properties of the main physiological types of motor units (MUs) that contribute to these muscle adaptations. Although the contractile profile of MU muscle fibers is tightly coupled to myosin heavy chain (MyHC) protein expression, it is not well understood if MyHC transition is a prerequisite for modifications to the contractile characteristics of MUs. In this study, we examined MU contractile properties, the mRNA expression of MyHC, parvalbumin, and sarcoendoplasmic reticulum Ca(2+) pump isoforms, as well as the MyHC protein content after 5 wk of volitional progressive weight-lifting training in the medial gastrocnemius muscle in rats. The training had no effect on MyHC profiling or Ca(2+)-handling protein gene expression. Maximum force increased in slow (by 49%) and fast (by 21%) MUs. Within fast MUs, the maximum force increased in most fatigue-resistant and intermediate but not most fatigable MUs. Twitch contraction time was shortened in slow and fast fatigue-resistant MUs. Twitch half-relaxation was shortened in fast most fatigue-resistant and intermediate MUs. The force-frequency curve shifted rightward in fast fatigue-resistant MUs. Fast fatigable MUs fatigued less within the initial 15 s while fast fatigue-resistant units increased the ability to potentiate the force within the first minute of the standard fatigue test. In conclusion, at the early stage of resistance training, modifications to the contractile characteristics of MUs appear in the absence of MyHC transition and the upregulation of Ca(2+)-handling genes.

  1. Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome virus.

    PubMed

    Sun, Yinyan; Qi, Yonghe; Liu, Chenxuan; Gao, Wenqing; Chen, Pan; Fu, Liran; Peng, Bo; Wang, Haimin; Jing, Zhiyi; Zhong, Guocai; Li, Wenhui

    2014-01-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection.

  2. Exogenous apelin changes alpha and beta myosin heavy chain mRNA expression and improves cardiac function in PTU-induced hypothyroid rats.

    PubMed

    Faraji Shahrivar, Farzaneh; Badavi, Mohammad; Dianat, Mahin; Mard, Ali; Ahangarpour, Akram; Samarbaf-Zadeh, Alireza

    2016-12-20

    The most important conditions associated with hypothyroidism is the cardiac dysfunction. Apelin is an endogenous ligand, involved in energy storage and metabolism which improves cardiac contractility. This study was done to evaluate the effects of apelin, l-Thyroxin (T4) or a combination of both, on cardiac function and mRNA expression of two contractile proteins, α and β myosin heavy chain (α-MHC and β-MHC), in 6-propyl-2-thiouracil (PTU)-induced hypothyroid rats. Forty male Wistar rats were randomly assigned into five groups: Ctrl (Control), and 4 hypothyroid groups (H, HA, HT, and HAT). The Hypothyroid (H) group received 0.05% PTU in the drinking water for six weeks; the next 3 groups, along with PTU, received apelin (HA, 200μg/kg/day, ip), T4 (HT, 20μg/kg/day, gavage), or a combination of both drugs (HAT) for the last 2weeks (weeks 5 and 6). TSH and T4 were measured using ELISA kit. Isolated hearts of animals were perfused in Langendorff apparatus and left ventricular developed pressure, cardiac contractility, heart rate, rate pressure product and perfusion pressure were assessed using PowerLab ADInstruments. In addition α-MHC and β-MHC mRNA expression were evaluated by RT-PCR method in heart tissue. Apelin alone or accompanied by T4 significantly increased cardiac contractility and performance as compared to hypothyroid group. Apelin also significantly increased the alpha-MHC mRNA expression and in the presence of T4 significantly decreased beta-MHC mRNA expression. It seems that apelin alone may improve cardiac function in hypothyroid rats via genomic pathways.

  3. Multiple positive and negative 5' regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene.

    PubMed Central

    Bouvagnet, P F; Strehler, E E; White, G E; Strehler-Page, M A; Nadal-Ginard, B; Mahdavi, V

    1987-01-01

    To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins. Images PMID:2830491

  4. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    SciTech Connect

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  5. Differential muscular myosin heavy chain expression of the pectoral and pelvic girdles during early growth in the king penguin (Aptenodytes patagonicus) chick.

    PubMed

    Erbrech, Aude; Robin, Jean-Patrice; Guérin, Nathalie; Groscolas, René; Gilbert, Caroline; Martrette, Jean-Marc

    2011-06-01

    Continuous growth, associated with a steady parental food supply, is a general pattern in offspring development. So that young chicks can acquire their locomotor independence, this period is usually marked by a fast maturation of muscles, during which different myosin heavy chain (MyHC) isoforms are expressed. However, parental food provisioning may fluctuate seasonally, and offspring therefore face a challenge to ensure the necessary maturation of their tissues when energy is limited. To address this trade-off we investigated muscle maturation in both the pectoral and pelvic girdles of king penguin chicks. This species has an exceptionally long rearing period (1 year), which is prolonged when parental food provisioning is drastically reduced during the sub-Antarctic winter. Approximately 1 month post hatching, chicks acquire a functional pedestrian locomotion, which uses pelvic muscles, whereas swimming, which uses the pectoral muscles, only occurs 1 year later. We therefore tested the hypothesis that the MyHC content of the leg muscles reaches a mature state before those of the pectoral muscles. We found that leg muscle MyHC composition changed with the progressive acquisition of pedestrian locomotion, whereas pectoral muscle fibres reached their mature MyHC profile as early as hatching. Contrary to our predictions, the acquisition of the adult profile in pectoral muscles could be related to an early maturation of the contractile muscular proteins, presumably associated with early thermoregulatory capacities of chicks, necessary for survival in their cold environment. This differential maturation appears to reconcile both the locomotor and environmental constraints of king penguin chicks during growth.

  6. Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A*

    PubMed Central

    Yang, Yidai; Ye, Qilu; Jia, Zongchao; Côté, Graham P.

    2015-01-01

    The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min−1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PMID:26260792

  7. Comparisons of different myosin heavy chain types, AMPK, and PGC-1α gene expression in the longissimus dorsi muscles in Bama Xiang and Landrace pigs.

    PubMed

    Huang, Y N; Ao, Q W; Jiang, Q Y; Guo, Y F; Lan, G Q; Jiang, H S

    2016-07-14

    Bama Xiang and Landrace pigs are the local fatty and lean breeds, respectively, in China. We compared differences in carcass traits, meat quality traits, and myosin heavy chain (MyHC) types in the longissimus dorsi muscles between Bama Xiang and Landrace pigs. This was done in pigs of the same age, using real-time PCR, to investigate the relationship between MyHC fiber types and carcass characteristics, meat quality traits, and the key factors regulating muscle fiber type. Bama Xiang pigs exhibited smaller size and slower growth than Landrace pigs (P < 0.01). We found that the superior meat quality, especially the high intramuscular fat (IMF) content in Bama Xiang pig, was related to elevated type I oxidative muscle fiber content (P < 0.01). In contrast, Landrace pig muscle had a higher glycolytic type IIb muscle fiber content (P < 0.01). MyHC I gene expression was significantly positively correlated with backfat thickness and IMF content (P < 0.01). MyHC IIb was significantly negatively correlated with IMF content (P < 0.05), and positively correlated with carcass yield (P < 0.05). AMP-activated protein kinase and peroxisome proliferator-activated receptor-g coactivator-1a are suggested to be the two key factors regulating muscle fiber type in pigs. Our results indicate that muscle fiber composition is one of the key differences leading to the differences of meat quality between Bama Xiang and Landrace pigs. These results may provide a theoretical basis for further studies of the molecular mechanism underlying the excellent meat quality of the Bama Xiang pig.

  8. Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression.

    PubMed Central

    Gupta, M P; Amin, C S; Gupta, M; Hay, N; Zak, R

    1997-01-01

    The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation. PMID:9199327

  9. Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation.

    PubMed

    Ohnuki, Yoshiki; Umeki, Daisuke; Mototani, Yasumasa; Jin, Huiling; Cai, Wenqian; Shiozawa, Kouichi; Suita, Kenji; Saeki, Yasutake; Fujita, Takayuki; Ishikawa, Yoshihiro; Okumura, Satoshi

    2014-12-15

    The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.

  10. MicroRNA-27a Regulates Beta Cardiac Myosin Heavy Chain Gene Expression by Targeting Thyroid Hormone Receptor β1 in Neonatal Rat Ventricular Myocytes▿

    PubMed Central

    Nishi, Hitoo; Ono, Koh; Horie, Takahiro; Nagao, Kazuya; Kinoshita, Minako; Kuwabara, Yasuhide; Watanabe, Shin; Takaya, Tomohide; Tamaki, Yodo; Takanabe-Mori, Rieko; Wada, Hiromichi; Hasegawa, Koji; Iwanaga, Yoshitaka; Kawamura, Teruhisa; Kita, Toru; Kimura, Takeshi

    2011-01-01

    MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene expression and play important roles in gene regulation in the heart. To examine the role of miRNAs in the expression of the two isoforms of the cardiac myosin heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility, endogenous miRNAs were downregulated in neonatal rat ventricular myocytes (NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer, an essential enzyme for miRNA biosynthesis, and MHC expression levels were examined. As a result, Dicer siRNA could downregulate endogenous miRNAs simultaneously and the β-MHC gene but not α-MHC, which implied that specific miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC protein was downregulated by silencing of endogenous miR-27a. Through a bioinformatics screening using TargetScan, we identified thyroid hormone receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation via thyroid hormone signaling and to be upregulated during the differentiation of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC gene expression by targeting TRβ1 in cardiomyocytes. PMID:21149577

  11. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

    NASA Astrophysics Data System (ADS)

    Hariadi, R. F.; Sommese, R. F.; Adhikari, A. S.; Taylor, R. E.; Sutton, S.; Spudich, J. A.; Sivaramakrishnan, S.

    2015-08-01

    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

  12. Asymmetric Myosin Binding to the Thin Filament as Revealed by a Fluorescent Nanocircuit

    PubMed Central

    Coffee Castro-Zena, Pilar G.; Root, Douglas D.

    2013-01-01

    The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the prepowerstroke orientation when bound near troponin, but is predominantly in the postpowerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament. PMID:23274408

  13. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level.

  14. In Vitro and In Vivo Single Myosin Step-Sizes in Striated Muscle a

    PubMed Central

    Burghardt, Thomas P.; Sun, Xiaojing; Wang, Yihua; Ajtai, Katalin

    2016-01-01

    Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a “second characterization” is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover “bottom-up” and “top-down” assaying of myosin characteristics. PMID:26728749

  15. Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes.

    PubMed

    Salas-Cortes, Laura; Ye, Fei; Tenza, Danièle; Wilhelm, Claire; Theos, Alexander; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

    2005-10-15

    Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.

  16. Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase.

    PubMed

    Schuster, Martin; Treitschke, Steffi; Kilaru, Sreedhar; Molloy, Justin; Harmer, Nicholas J; Steinberg, Gero

    2012-01-04

    Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.

  17. Myosin-V: a class of unconventional molecular motors.

    PubMed

    Larson, R E

    1996-03-01

    In this review we focus on the biochemical and structural properties of the myosin-V class of unconventional myosins as an example of the diversity of molecular motors within the myosin superfamily. A member of this class was first identified as a novel calmodulin-binding protein in mammalian brain (Larson RE, Pitta DE and Ferro JA (1988). Brazilian Journal of Medical and Biological Research, 21: 213-217). To date, the myosin-V class is represented by two molecules from yeast, one from nematodes, several from vertebrates (chickens, rats, mice and humans) and possibly one from plants. The domain structure of these myosins features a highly conserved head containing the ATP-hydrolysis and actin-binding sites, an extended neck composed of six tandem IQ-motifs which are sites for calmodulin binding and a large tail which has coiled-coil segments intercalated with globular regions of as yet unknown function. Biochemical studies on purified myosin-V from vertebrate brains and the description of myosin-V mutants in yeast and mice have made myosin-V one of the best characterized, unconventional myosin classes at the present time, surpassed only by the well-studied myosin-I class.

  18. Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

    SciTech Connect

    Minoda, Hiroki; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-02-25

    Research highlights: {yields} We succeeded in recording structural changes of hydrated myosin cross-bridges. {yields} We succeeded in position-marking the cross-bridges with site-directed antibodies. {yields} We recorded cross-bridge movement at different regions in individual cross-bridge. {yields} The movement was smallest at the cross-bridge-subfragment two boundary. {yields} The results provide evidence for the cross-bridge lever arm mechanism. -- Abstract: Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

  19. Rare, Non-Synonymous Variant in the Smooth Muscle-Specific Isoform of Myosin Heavy Chain, MYH11, R247C, Alters Force Generation in the Aorta and Phenotype of Smooth Muscle Cells

    PubMed Central

    Kuang, Shao-Qing; Kwartler, Callie S.; Byanova, Katerina L.; Pham, John; Gong, Limin; Prakash, Siddharth K.; Huang, Jian; Kamm, Kristine E.; Stull, James T.; Sweeney, H. Lee; Milewicz, Dianna M.

    2013-01-01

    Rationale Mutations in MYH11 cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, non-synonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. Objective To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). Methods and Results The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11R247C/R247C mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11R247C/R247C mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11R247C/R247C mice were de-differentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11R247C/R247C SMCs to a Rho activator rescued the de-differentiated phenotype of the SMCs. Conclusions These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury. PMID:22511748

  20. Characterization of sea urchin unconventional myosins and analysis of their patterns of expression during early embryogenesis.

    PubMed

    Sirotkin, V; Seipel, S; Krendel, M; Bonder, E M

    2000-10-01

    Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes. These events may involve the activities of multiple members of the superfamily of myosin motor proteins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes Igamma, II, V, VI, VII, IX, and amoeboid-type I, and the remaining four may be from novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were either completely or partially cloned and their molecular structures characterized. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I shared a high degree of sequence identity with their respective family members from vertebrates and they retained their class-specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during development revealed that each myosin mRNA displayed a distinct temporal pattern of expression, suggesting that myosins might be involved in specific events of early embryogenesis. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression. The presence of multiple myosin mRNAs in eggs and embryos provides insight into the potential involvement of multiple specific motor proteins in the actin-dependent events of embryo development.

  1. Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

    PubMed Central

    Hilbert, Lennart; Bates, Genevieve; Roman, Horia N.; Blumenthal, Jenna L.; Zitouni, Nedjma B.; Sobieszek, Apolinary; Mackey, Michael C.; Lauzon, Anne-Marie

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics. PMID:24204225

  2. Biochemical and bioinformatic analysis of the myosin-XIX motor domain.

    PubMed

    Adikes, Rebecca C; Unrath, William C; Yengo, Christopher M; Quintero, Omar A

    2013-05-01

    Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial myosin transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-co-sedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment.

  3. Mechanical coupling between myosin molecules causes differences between ensemble and single-molecule measurements.

    PubMed

    Walcott, Sam; Warshaw, David M; Debold, Edward P

    2012-08-08

    In contracting muscle, individual myosin molecules function as part of a large ensemble, hydrolyzing ATP to power the relative sliding of actin filaments. The technological advances that have enabled direct observation and manipulation of single molecules, including recent experiments that have explored myosin's force-dependent properties, provide detailed insight into the kinetics of myosin's mechanochemical interaction with actin. However, it has been difficult to reconcile these single-molecule observations with the behavior of myosin in an ensemble. Here, using a combination of simulations and theory, we show that the kinetic mechanism derived from single-molecule experiments describes ensemble behavior; but the connection between single molecule and ensemble is complex. In particular, even in the absence of external force, internal forces generated between myosin molecules in a large ensemble accelerate ADP release and increase how far actin moves during a single myosin attachment. These myosin-induced changes in strong binding lifetime and attachment distance cause measurable properties, such as actin speed in the motility assay, to vary depending on the number of myosin molecules interacting with an actin filament. This ensemble-size effect challenges the simple detachment limited model of motility, because even when motility speed is limited by ADP release, increasing attachment rate can increase motility speed.

  4. Modulation of myosin filament organization by C-protein family members.

    PubMed

    Seiler, S H; Fischman, D A; Leinwand, L A

    1996-01-01

    We have analyzed the interactions between two types of sarcomeric proteins: myosin heavy chain (MyHC) and members of an abundant thick filament-associated protein family (myosin-binding protein; MyBP). Previous work has demonstrated that when MyHC is transiently transfected into mammalian nonmuscle COS cells, the expressed protein forms spindle-shaped structures consisting of bundles of myosin thick filaments. Co-expression of MyHC and MyBP-C or -H modulates the MyHC structures, resulting in dramatically longer cables consisting of myosin and MyBP encircling the nucleus. Immunoelectron microscopy indicates that these cable structures are more uniform in diameter than the spindle structures consisting solely of MyHC, and that the myosin filaments are compacted in the presence of MyBP. Deletion analysis of MyBP-H indicates that cable formation is dependent on the carboxy terminal 24 amino acids. Neither the MyHC spindles nor the MyHC/MyBP cables associate with the endogenous actin cytoskeleton of the COS cell. While there is no apparent co-localization between these structures and the microtubule network, colchicine treatment of the cells promotes the formation of longer assemblages, suggesting that cytoskeletal architecture may physically impede or regulate polymer formation/extension. The data presented here contribute to a greater understanding of the interactions between the MyBP family and MyHC, and provide additional evidence for functional homology between MyBP-C and MyBP-H.

  5. Cardiomyopathy-related mutation (A30V) in mouse cardiac troponin T divergently alters the magnitude of stretch activation in α- and β-myosin heavy chain fibers.

    PubMed

    Mickelson, Alexis V; Gollapudi, Sampath K; Chandra, Murali

    2017-01-01

    The present study investigated the functional consequences of the human hypertrophic cardiomyopathy (HCM) mutation A28V in cardiac troponin T (TnT). The A28V mutation is located within the NH2 terminus of TnT, a region known to be important for full activation of cardiac thin filaments. The functional consequences of the A28V mutation in TnT remain unknown. Given how α- and β-myosin heavy chain (MHC) isoforms differently alter the functional effect of the NH2 terminus of TnT, we hypothesized that the A28V-induced effects would be differently modulated by α- and β-MHC isoforms. Recombinant wild-type mouse TnT (TnTWT) and the mouse equivalent of the human A28V mutation (TnTA30V) were reconstituted into detergent-skinned cardiac muscle fibers extracted from normal (α-MHC) and transgenic (β-MHC) mice. Dynamic and steady-state contractile parameters were measured in reconstituted muscle fibers. Step-like length perturbation experiments demonstrated that TnTA30V decreased the magnitude of the muscle length-mediated recruitment of new force-bearing cross bridges (ER) by 30% in α-MHC fibers. In sharp contrast, TnTA30V increased ER by 55% in β-MHC fibers. Inferences drawn from other dynamic contractile parameters suggest that directional changes in ER in TnTA30V + α-MHC and TnTA30V + β-MHC fibers result from a divergent impact on cross bridge-regulatory unit (troponin-tropomyosin complex) cooperativity. TnTA30V-mediated effects on Ca(2+)-activated maximal tension and instantaneous muscle fiber stiffness (ED) were also divergently affected by α- and β-MHC. Our study demonstrates that TnTA30V + α-MHC and TnTA30V + β-MHC fibers show contrasting contractile phenotypes; however, only the observations from β-MHC fibers are consistent with the clinical data for A28V in humans.

  6. Dynamics of myosin heavy chain isoform transition in the longissimus muscle of domestic and wild pigs during growth: a comparative study.

    PubMed

    Fazarinc, G; Vrecl, M; Škorjanc, D; Čehovin, T; Čandek-Potokar, M

    2017-01-01

    Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characterized by an accelerated transformation of the fast to slow MyHC during the first hours postpartum, followed by differentiation towards oxidative myofibers in which type I, IIa and IIx MyHCs predominated. In the DP, the transformation shifted towards glycolytic myofibers that expressed MyHC-IIb. The expression of foetal MyHC was higher in the DP than in the WP at 1 day of age, and the decline in the foetal MyHC during the first 3 weeks was more rapid in the WP than in the DP denoting an accelerated early postnatal muscle maturation in WP than DP piglets. All foetal MyHC-positive myofibers co-expressed IIa isoform, but not vice versa. The intense myofiber hypertrophy was evident from 3 weeks until 7 months of age. In this period, the myofiber cross-sectional area increased up to 10- and 20-fold in the WP and the DP, respectively. In the DP, the hypertrophy of all myofiber types was more pronounced than in the WP, particularly the hypertrophy of IIx and IIb myofibers. To summarize, the

  7. Activation of the beta myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway.

    PubMed

    Meissner, Joachim D; Umeda, Patrick K; Chang, Kin-Chow; Gros, Gerolf; Scheibe, Renate J

    2007-04-01

    Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast-to-slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T-cells (NFAT) isoform c1, whose dephosphorylation by the calcium/calmodulin-dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast-type MyHCII protein undergo fast-to-slow transformation following calcium-ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) beta promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCbeta promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc1, myocyte enhancer factor-2D (MEF-2D) and the myogenic transcription factor MyoD transactivated the MyHCbeta promoter in calcium-ionophore-treated myotubes in a calcineurin-dependent manner. To elucidate the mechanism involved in regulating MyHCbeta gene expression, we analyzed the -2.4-kb MyHCbeta promoter construct for cis-regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcoIP) assays, we demonstrated calcium-ionophore-induced binding of NFATc1 to a NFAT consensus site adjacent to a MyoD-binding E-box. At their respective binding sites, both NFATc1 and MyoD recruited the transcriptional coactivator p300, and in turn, MEF-2D bound to the MyoD complex. The calcium-ionophore-induced effects on the MyHCbeta promoter were shown to be calcineurin-dependent. Together, our findings demonstrate calcium-ionophore-induced activation of the beta MyHC promoter by

  8. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model

    PubMed Central

    Gupta, Jyoti; Pathak, Manisha; Misra, Sweta; Misra-Bhattacharya, Shailja

    2015-01-01

    We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing

  9. Calcium and cargoes as regulators of myosin 5a activity

    SciTech Connect

    Sellers, James R. Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-04-25

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

  10. Emergent systems energy laws for predicting myosin ensemble processivity.

    PubMed

    Egan, Paul; Moore, Jeffrey; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

    2015-04-01

    In complex systems with stochastic components, systems laws often emerge that describe higher level behavior regardless of lower level component configurations. In this paper, emergent laws for describing mechanochemical systems are investigated for processive myosin-actin motility systems. On the basis of prior experimental evidence that longer processive lifetimes are enabled by larger myosin ensembles, it is hypothesized that emergent scaling laws could coincide with myosin-actin contact probability or system energy consumption. Because processivity is difficult to predict analytically and measure experimentally, agent-based computational techniques are developed to simulate processive myosin ensembles and produce novel processive lifetime measurements. It is demonstrated that only systems energy relationships hold regardless of isoform configurations or ensemble size, and a unified expression for predicting processive lifetime is revealed. The finding of such laws provides insight for how patterns emerge in stochastic mechanochemical systems, while also informing understanding and engineering of complex biological systems.

  11. Reconstitution of a Minimal Actin Cortex by Coupling Actin Filaments to Reconstituted Membranes.

    PubMed

    Vogel, Sven K

    2016-01-01

    A thin layer of actin filaments in many eukaryotic cell types drives pivotal aspects of cell morphogenesis and is generally cited as the actin cortex. Myosin driven contractility and actin cytoskeleton membrane interactions form the basis of fundamental cellular processes such as cytokinesis, cell migration, and cortical flows. How the interplay between the actin cytoskeleton, the membrane, and actin binding proteins drives these processes is far from being understood. The complexity of the actin cortex in living cells and the hardly feasible manipulation of the omnipotent cellular key players, namely actin, myosin, and the membrane, are challenging in order to gain detailed insights about the underlying mechanisms. Recent progress in developing bottom-up in vitro systems where the actin cytoskeleton is combined with reconstituted membranes may provide a complementary route to reveal general principles underlying actin cortex properties. In this chapter the reconstitution of a minimal actin cortex by coupling actin filaments to a supported membrane is described. This minimal system may be very well suited to study for example protein interactions on membrane bound actin filaments in a very controlled and quantitative manner as it may be difficult to perform in living systems.

  12. The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm

    PubMed Central

    1995-01-01

    The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three- dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. We propose that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form. PMID:7790355

  13. Myosin is reversibly inhibited by S-nitrosylation.

    PubMed

    Nogueira, Leonardo; Figueiredo-Freitas, Cicero; Casimiro-Lopes, Gustavo; Magdesian, Margaret H; Assreuy, Jamil; Sorenson, Martha M

    2009-11-11

    Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO* donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO* or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions.

  14. An unconventional myosin required for cell polarization and chemotaxis.

    PubMed

    Breshears, Laura M; Wessels, Deborah; Soll, David R; Titus, Margaret A

    2010-04-13

    MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.

  15. Structure of androcam supports specialized interactions with myosin VI

    PubMed Central

    Joshi, Mehul K.; Moran, Sean; Beckingham, Kathleen M.; MacKenzie, Kevin R.

    2012-01-01

    Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca2+ and is locked in a “closed” conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca2+-regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin-mediated Ca2+ signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins. PMID:22851764

  16. A role for myosin II in mammalian mitochondrial fission.

    PubMed

    Korobova, Farida; Gauvin, Timothy J; Higgs, Henry N

    2014-02-17

    Mitochondria are dynamic organelles, undergoing both fission and fusion regularly in interphase cells. Mitochondrial fission is thought to be part of a quality-control mechanism whereby damaged mitochondrial components are segregated from healthy components in an individual mitochondrion, followed by mitochondrial fission and degradation of the damaged daughter mitochondrion. Fission also plays a role in apoptosis. Defects in mitochondrial dynamics can lead to neurodegenerative diseases such as Alzheimer's disease. Mitochondrial fission requires the dynamin GTPase Drp1, which assembles in a ring around the mitochondrion and appears to constrict both outer and inner mitochondrial membranes. However, mechanisms controlling Drp1 assembly on mammalian mitochondria are unclear. Recent results show that actin polymerization, driven by the endoplasmic reticulum-bound formin protein INF2, stimulates Drp1 assembly at fission sites. Here, we show that myosin II also plays a role in fission. Chemical inhibition by blebbistatin or small interfering RNA (siRNA)-mediated suppression of myosin IIA or myosin IIB causes an increase in mitochondrial length in both control cells and cells expressing constitutively active INF2. Active myosin II accumulates in puncta on mitochondria in an actin- and INF2-dependent manner. In addition, myosin II inhibition decreases Drp1 association with mitochondria. Based on these results, we propose a mechanistic model in which INF2-mediated actin polymerization leads to myosin II recruitment and constriction at the fission site, enhancing subsequent Drp1 accumulation and fission.

  17. Non-muscle myosins in tumor progression, cancer cell invasion and metastasis

    PubMed Central

    Ouderkirk, J. L.; Krendel, M.

    2014-01-01

    The actin cytoskeleton, which regulates cell polarity, adhesion, and migration, can influence cancer progression, including initial acquisition of malignant properties by normal cells, invasion of adjacent tissues, and metastasis to distant sites. Actin-dependent molecular motors, myosins, play key roles in regulating tumor progression and metastasis. In this review, we examine how non-muscle myosins regulate neoplastic transformation and cancer cell migration and invasion. Members of the myosin superfamily can act as either enhancers or suppressors of tumor progression. This review summarizes the current state of knowledge on how mutations or epigenetic changes in myosin genes and changes in myosin expression may affect tumor progression and patient outcomes and discusses the proposed mechanisms linking myosin inactivation or upregulation to malignant phenotype, cancer cell migration, and metastasis. PMID:25087729

  18. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

    PubMed Central

    1987-01-01

    Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg- ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin. PMID:2958483

  19. Recombinant alpha-actin for specific fluorescent labeling.

    PubMed

    Iwane, Atsuko H; Morimatsu, Masatoshi; Yanagida, Toshio

    2009-01-01

    Until recently, actin was thought to act merely as a passive track for its motility partner, myosin, during actomyosin interactions. Yet a recent report having observed dynamical conformational changes in labeled skeletal muscle alpha-actin suggests that actin has a more active role. Because the labeling technique was still immature, however, conclusions regarding the significance of the different conformations are difficult to make. Here, we describe the preparation of fully active alpha-actin obtained from a baculovirus expression system. We developed alpha-actin recombinants, of which subdomains 1 and 2 have specific sites for fluorescent probes. This specific labeling technique offers to significantly expand the information acquired from actin studies.

  20. Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.

    PubMed

    Billington, Neil; Beach, Jordan R; Heissler, Sarah M; Remmert, Kirsten; Guzik-Lendrum, Stephanie; Nagy, Attila; Takagi, Yasuharu; Shao, Lin; Li, Dong; Yang, Yi; Zhang, Yingfan; Barzik, Melanie; Betzig, Eric; Hammer, John A; Sellers, James R

    2015-03-30

    Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

  1. Ca(2+)-dependent phosphorylation of the tail domain of myosin-V, a calmodulin-binding myosin in vertebrate brain.

    PubMed

    Coelho, M V; Larson, R E

    1993-05-01

    1. Myosin-V from vertebrate brain is a novel molecular motor with a myosin-like head domain, a calmodulin-binding neck region and a unique tail domain of unknown function. Previous studies showed brain myosin-V to be a phosphoprotein substrate for Ca2+/calmodulin-dependent protein kinase associated with actomyosin. In the present study we describe the preparation of a specific actin-cytoskeletal fraction which is enriched in brain myosin-V. 2. We show that Ca2+/calmodulin-dependent protein kinase activity is also associated with this preparation and phosphorylates brain myosin-V. 3. Calpain, a Ca(2+)-dependent protease, generates a M(r) 80,000 fragment from the COOH terminal region of brain myosin-V containing most or all of the phosphorylation sites. 4. These results suggest that the unique tail domain of this novel myosin is subject to Ca2+ control via phosphorylation by kinase activity associated with the actin cytoskeleton.

  2. Tropomyosin movement on F-actin during muscle activation explained by energy landscapes.

    PubMed

    Orzechowski, Marek; Moore, Jeffrey R; Fischer, Stefan; Lehman, William

    2014-03-01

    Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced "open-state" position. This indicates that spontaneous movement of tropomyosin away from its energetic "ground-state" to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations.

  3. Actomyosin content of Physarum plasmodia and detection of immunological cross-reactions with myosins from related species

    PubMed Central

    1976-01-01

    The content of myosin in plasmodia of the myxomycete Physarum polycephalum was measured by an immunological technique, quantitative microcomplement (C') fixation. Migrating plasmodia (starved after growth on rolled oats) contained 0.60 +/- 0.08 (SD) mg myosin per g fresh plasmodia. Myosin comprised 0.77% +/- 0.05 (SD) of the total plasmodial protein. When total plasmodial proteins were separated by electrophoresis on SDS-polyacrylamide gels, a large amount of protein appeared in a band comigrating with muscle actin. Densitometry performed after Coomassie blue staining indicated that as much as 15- 25% of the total protein in the plasmodium could be actin. This gives an actin/myosin ratio by weight in the myxomycete plasmodium as high as 19-33, a very "actin-rich" actomyosin compared with rabbit skeletal muscle actomyosin with an actin/myosin ratio of 0.6. Starvation stimulates rapid migration and is correlated with a higher percent of both myosin and actin in the total protein of the plasmodium compared with normally growing cultures. Immunological cross-reaction of myosins from a variety of species was measured by C' fixation using an antiserum produced against purified native myosin from P. polycephalum. Although myxomycete and vertebrate striated muscle myosins have very similar morphological and biochemical properties, and apparently possess similar binding properties to F-actin, only myosins from myxomycetes in the order Physarales, rather closely related to P. polycephalum, gave detectable cross-reactions. This finding suggests that many amino acid sequences in myosin have been variable during evolution. PMID:944188

  4. Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI.

    PubMed

    Ménétrey, Julie; Isabet, Tatiana; Ropars, Virginie; Mukherjea, Monalisa; Pylypenko, Olena; Liu, Xiaoyan; Perez, Javier; Vachette, Patrice; Sweeney, H Lee; Houdusse, Anne M

    2012-10-12

    Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

  5. Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells*

    PubMed Central

    Kwartler, Callie S.; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V.; Walker, Lori; Hill, Joseph A.; Epstein, Henry F.; Taegtmeyer, Heinrich; Milewicz, Dianna M.

    2014-01-01

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  6. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications.

  7. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  8. In vivo myosin step-size from zebrafish skeletal muscle

    PubMed Central

    Ajtai, Katalin; Sun, Xiaojing; Takubo, Naoko; Wang, Yihua

    2016-01-01

    Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the ‘bottom-up’ myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive ‘top-down’ phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle. PMID:27249818

  9. Tension regulates myosin dynamics during Drosophila embryonic wound repair.

    PubMed

    Kobb, Anna B; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo

    2017-02-15

    Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle myosin II are redistributed in the cells adjacent to the wound, forming a supracellular purse string around the lesion. Purse string contraction coordinates cell movements and drives rapid wound closure. By using fluorescence recovery after photobleaching in Drosophila embryos, we found that myosin turns over as the purse string contracts. Myosin turnover at the purse string was slower than in other actomyosin networks that had a lower level of contractility. Mathematical modelling suggested that myosin assembly and disassembly rates were both reduced by tension at the wound edge. We used laser ablation to show that tension at the purse string increased as wound closure progressed, and that the increase in tension was associated with reduced myosin turnover. Reducing purse string tension by laser-mediated severing resulted in increased turnover and loss of myosin. Finally, myosin motor activity was necessary for its stabilization around the wound and for rapid wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair.

  10. Drosophila non-muscle myosin II motor activity determines the rate of tissue folding.

    PubMed

    Vasquez, Claudia G; Heissler, Sarah M; Billington, Neil; Sellers, James R; Martin, Adam C

    2016-12-30

    Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos.

  11. Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

    PubMed

    Raposo, G; Cordonnier, M N; Tenza, D; Menichi, B; Dürrbach, A; Louvard, D; Coudrier, E

    1999-05-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

  12. Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

    PubMed Central

    Raposo, Graça; Cordonnier, Marie-Neige; Tenza, Danièle; Menichi, Bernadette; Dürrbach, Antoine; Louvard, Daniel; Coudrier, Evelyne

    1999-01-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes. PMID:10233157

  13. Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking.

    PubMed

    Li, Jianchao; Lu, Qing; Zhang, Mingjie

    2016-08-01

    Unconventional myosins are a superfamily of actin-based molecular motors playing diverse roles including cellular trafficking, mechanical supports, force sensing and transmission, etc. The variable neck and tail domains of unconventional myosins function to bind to specific cargoes including proteins and lipid vesicles and thus are largely responsible for the diverse cellular functions of myosins in vivo. In addition, the tail regions, together with their cognate cargoes, can regulate activities of the motor heads. This review outlines the advances made in recent years on cargo recognition and cargo binding-induced regulation of the activity of several unconventional myosins including myosin-I, V, VI and X in cellular trafficking. We approach this topic by describing a series of high-resolution structures of the neck and tail domains of these unconventional myosins either alone or in complex with their specific cargoes, and by discussing potential implications of these structural studies on cellular trafficking of these myosin motors.

  14. Myosin superfamily: The multi-functional and irreplaceable factors in spermatogenesis and testicular tumors.

    PubMed

    Li, Yan-Ruide; Yang, Wan-Xi

    2016-01-15

    Spermatogenesis is a fundamental process in sexual development and reproduction, in which the diploid spermatogonia transform into haploid mature spermatozoa. This process is under the regulation of multiple factors and pathway. Myosin has been implicated in various aspects during spermatogenesis. Myosins constitute a diverse superfamily of actin-based molecular motors that translocate along microfilament in an ATP-dependent manner, and six kinds of myosins have been proved that function during spermatogenesis. In mitosis and meiosis, myosins play an important role in spindle assembly and positioning, karyokinesis and cytokinesis. During spermiogenesis, myosins participate in acrosomal formation, nuclear morphogenesis, mitochondrial translocation and spermatid individualization. In this review, we summarize current understanding of the functions of myosin in spermatogenesis and some reproductive system diseases such as testicular tumors and prostate cancer, and discuss the roles of possible upstream molecules which regulate myosin in these processes.

  15. Myosin I Is Required for Hypha Formation in Candida albicans†

    PubMed Central

    Oberholzer, U.; Marcil, A.; Leberer, E.; Thomas, D. Y.; Whiteway, M.

    2002-01-01

    The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the “TEDS-rule” site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5S366D were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation. PMID:12455956

  16. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  17. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation.

    PubMed

    Walcott, Sam; Kad, Neil M

    2015-11-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies.

  18. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation

    PubMed Central

    Walcott, Sam; Kad, Neil M.

    2015-01-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies. PMID:26536123

  19. Competition and compensation: dissecting the biophysical and functional differences between the class 3 myosin paralogs, myosins 3a and 3b.

    PubMed

    Manor, Uri; Grati, M'hamed; Yengo, Christopher M; Kachar, Bechara; Gov, Nir S

    2012-01-01

    Stereocilia are actin protrusions with remarkably well-defined lengths and organization. A flurry of recent papers has reported multiple myosin motor proteins involved in regulating stereocilia structures by transporting actin-regulatory cargo to the tips of stereocilia. In our recent paper, we show that two paralogous class 3 myosins--Myo3a and Myo3b--both transport the actin-regulatory protein Espin 1 (Esp1) to stereocilia and filopodia tips in a remarkably similar, albeit non-identical fashion. (1) Here we present experimental and computational data that suggests that subtle differences between these two proteins' biophysical and biochemical properties can help us understand how these myosin species target and regulate the lengths of actin protrusions.

  20. Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells.

    PubMed

    Yi, Jason; Wu, Xufeng S; Crites, Travis; Hammer, John A

    2012-03-01

    Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.

  1. Disease causing mutations of troponin alter regulated actin state distributions.

    PubMed

    Chalovich, Joseph M

    2012-12-01

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

  2. Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

    PubMed Central

    Bird, Jonathan E.; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R.; Friedman, Thomas B.

    2014-01-01

    Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s−1 at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (kcat ∼6 s−1) was similar to the actin-detachment rate (kdet = 6.2 s−1) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

  3. Novel Familial Dilated Cardiomyopathy Mutation in MYL2 Affects the Structure and Function of Myosin Regulatory Light Chain

    PubMed Central

    Huang, Wenrui; Liang, Jingsheng; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Zhou, Zhiqun; Morales, Ana; McBride, Kim L.; Fitzgerald-Butt, Sara M.; Hershberger, Ray E.; Szczesna-Cordary, Danuta

    2015-01-01

    Dilated Cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. In this report we describe a novel DCM mutation identified for the first time in the myosin regulatory light chain (RLC), replacing Aspartic Acid at position 94 with Alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To gain insight into the functional significance of this pathogenic MYL2 variant, we have cloned and purified the human ventricular RLC wild-type (WT) and D94A-mutant proteins and performed in vitro experiments using RLC-exchanged porcine cardiac preparations. The mutation was observed to induce a reduction in the α-helical content of the RLC and imposed intra-molecular rearrangements. The Ca2+-calmodulin-activated myosin light chain kinase phosphorylation of RLC was not affected by D94A. The mutation was seen to impair the binding of RLC to the MHC (myosin heavy chain), and its incorporation into the RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared to ATPase of WT. No changes in myofibrillar ATPase-pCa relationship were observed in WT- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle with no change in calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with WT. Our data indicate that subtle structural rearrangements in the RLC molecule followed by its impaired interaction with the MHC may trigger functional abnormalities contributing to the DCM phenotype. PMID:25825243

  4. The Globular Tail Domain of Myosin-5a Functions as a Dimer in Regulating the Motor Activity.

    PubMed

    Zhang, Wen-Bo; Yao, Lin-Lin; Li, Xiang-Dong

    2016-06-24

    Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function.

  5. Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP

    PubMed Central

    Sugi, Haruo; Yamaguchi, Maki; Ohno, Tetsuo; Kobayashi, Takakazu; Chaen, Shigeru; Okuyama, Hiroshi

    2016-01-01

    During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles. PMID:27583360

  6. Purification and partial characterization of myosin II from rat testis.

    PubMed

    Dias, Decivaldo dos Santos; Coelho, Milton Vieira

    2007-10-01

    The intent, in this work, was to isolate rat testis myosin II. Testis 40,000 x g x 40' supernatant was frozen at -20 degrees C for 48 h and, after it was thawed and centrifuged. The precipitate, after washed twice, was enriched in three polypeptides bands: p205, p43 and one that migrated together with the front of the gel. These polypeptides were solubilized in pH 10.8 at 27 degrees C and separated in Sephacryl S-400 column. Three low weight polypeptides co-eluted together with p205. The p205 was marked with anti-myosin II, possess actin-stimulated Mg-ATPase activity and co-sedimented with F-actin in the absence, but not in the presence, of ATP. In the present study, we have been developing a method for purification of myosin II from rat testis.

  7. Leveraging the membrane-cytoskeleton interface with myosin-1

    PubMed Central

    McConnell, Russell E.; Tyska, Matthew J.

    2010-01-01

    Class 1 myosins are small motor proteins with the ability to simultaneously bind to actin filaments and cellular membranes. Given their ability to generate mechanical force, and their high prevalence in many cell types, these molecules are well positioned to carry out a number of important biological functions at the interface of membrane and the actin cytoskeleton. Indeed, recent studies implicate these motors in endocytosis, exocytosis, release of extracellular vesicles, and the regulation of tension between membrane and the cytoskeleton. Many class 1 myosins also exhibit a load-dependent mechano-chemical cycle that enables them to maintain tension for long periods of time without hydrolyzing ATP. These properties put myosins-1 in a unique position to regulate dynamic membrane-cytoskeleton interactions and respond to physical forces during these events. PMID:20471271

  8. Approaches to myosin modelling in a two-phase flow model for cell motility

    NASA Astrophysics Data System (ADS)

    Kimpton, L. S.; Whiteley, J. P.; Waters, S. L.; Oliver, J. M.

    2016-04-01

    A wide range of biological processes rely on the ability of cells to move through their environment. Mathematical models have been developed to improve our understanding of how cells achieve motion. Here we develop models that explicitly track the cell's distribution of myosin within a two-phase flow framework. Myosin is a small motor protein which is important for contracting the cell's actin cytoskeleton and enabling cell motion. The two phases represent the actin network and the cytosol in the cell. We start from a fairly general description of myosin kinetics, advection and diffusion in the two-phase flow framework, then identify a number of sub-limits of the model that may be relevant in practice, two of which we investigate further via linear stability analyses and numerical simulations. We demonstrate that myosin-driven contraction of the actin network destabilizes a stationary steady state leading to cell motion, but that rapid diffusion of myosin and rapid unbinding of myosin from the actin network are stabilizing. We use numerical simulation to investigate travelling-wave solutions relevant to a steadily gliding cell and we consider a reduction of the model in which the cell adheres strongly to the substrate on which it is crawling. This work demonstrates that a number of existing models for the effect of myosin on cell motility can be understood as different sub-limits of our two-phase flow model.

  9. Nonmuscle myosin IIB as a therapeutic target for the prevention of relapse to methamphetamine use.

    PubMed

    Young, E J; Blouin, A M; Briggs, S B; Sillivan, S E; Lin, L; Cameron, M D; Rumbaugh, G; Miller, C A

    2016-05-01

    Memories associated with drug use increase vulnerability to relapse in substance use disorder (SUD), and there are no pharmacotherapies for the prevention of relapse. Previously, we reported a promising finding that storage of memories associated with methamphetamine (METH), but not memories for fear or food reward, is vulnerable to disruption by actin depolymerization in the basolateral amygdala complex (BLC). However, actin is not a viable therapeutic target because of its numerous functions throughout the body. Here we report the discovery of a viable therapeutic target, nonmuscle myosin IIB (NMIIB), a molecular motor that supports memory by directly driving synaptic actin polymerization. A single intra-BLC treatment with Blebbistatin (Blebb), a small-molecule inhibitor of class II myosin isoforms, including NMIIB, produced a long-lasting disruption of context-induced drug seeking (at least 30 days). Further, postconsolidation genetic knockdown of Myh10, the heavy chain of the most highly expressed NMII in the BLC, was sufficient to produce METH-associated memory loss. Blebb was found to be highly brain penetrant. A single systemic injection of the compound selectively disrupted the storage of METH-associated memory and reversed the accompanying increase in BLC spine density. This effect was specific to METH-associated memory, as it had no effect on an auditory fear memory. The effect was also independent of retrieval, as METH-associated memory was disrupted 24 h after a single systemic injection of Blebb delivered in the home cage. Together, these results argue for the further development of small-molecule inhibitors of NMII as potential therapeutics for the prevention of SUD relapse triggered by drug associations.

  10. The expression pattern and cellular localisation of Myosin VI during the Drosophila melanogaster life cycle.

    PubMed

    Millo, Hadas; Bownes, Mary

    2007-02-01

    Myosin VI is a motor protein which is necessary for the morphogenesis of epithelial tissues during Drosophila development. The spatial and temporal expression of Myosin VI was examined by expressing a GFP (Green Fluorescent Protein) tagged Myosin VI molecule (PGM), under the control of a Myosin VI-Gal4 line. PGM was present in tissues that were shown previously to express Myosin VI, such as the ovarian follicle epithelium, and the individualization complex; and in other tissues, including the trachea, the midgut, the salivary glands and the imaginal discs. The GFP-tagged Myosin V1 rescued the male sterile phenotype of Jaguar showing it is functional in vivo. Within individual cells, the role of the head and neck domain and the tail domain in targeting of the Myosin V1 molecule was examined by investigating the localisation of the separate domains tagged to GFP. In salivary glands and follicle cells the head and neck domains were concentrated in the cell nucleus, where the minus end of each actin filament is located. We found that the tail domain anchors the whole molecule outside of the nucleus. Similarly, in the individualization complex in the testes, the tail anchors the whole molecule to the base of the complex while the separated head with neck domain becomes scattered along the entire actin molecule suggesting the cellular location may be determined by cargo proteins that bind to the tail domain rather than by the movement of Myosin VI along the actin filaments.

  11. Myosin V is a biological Brownian machine

    PubMed Central

    Fujita, Keisuke; Iwaki, Mitsuhiro

    2014-01-01

    Myosin V is a vesicle transporter that unidirectionally walks along cytoskeletal actin filaments by converting the chemical energy of ATP into mechanical work. Recently, it was found that myosin V force generation is a composition of two processes: a lever-arm swing, which involves a conformational change in the myosin molecule, and a Brownian search-and-catch, which involves a diffusive “search” by the motor domain that is followed by an asymmetric “catch” in the forward actin target such that Brownian motion is rectified. Here we developed a system that combines optical tweezers with DNA nano-material to show that the Brownian search-and-catch mechanism is the energetically dominant process at near stall force, providing 13 kBT of work compared to just 3 kBT by the lever-arm swing. Our result significantly reconsiders the lever-arm swinging model, which assumes the swing dominantly produces work (>10 kBT), and sheds light on the Brownian search-and-catch as a driving process. PMID:27493501

  12. Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages

    SciTech Connect

    Mykles, D.L.

    1985-01-01

    Fast and slow muscles from the claws and abdomen of the American lobster Homarus americanus were examined for adenosine triphosphatase (ATPase) activity and for differences in myofibrillar proteins. Both myosin and actomyosin ATPase were correlated with fiber composition and contractile speed. Four distinct patterns of myofibrilla proteins observed in sodium dodecyl sulfate-polyacrylamide gels were distinguished by different assemblages of regulatory and contractile protein variants. A total of three species of troponin-T, five species of troponin-I, and three species of troponin-C were observed. Lobster myosins contained two groups of light chains (LC), termed alpha and beta. There were three ..cap alpha..-LC variants and two ..beta..-LC variants. There were no apparent differences in myosin heavy chain, actin, and tropomyosin. Only paramyosin showed a pattern completely consistent with muscle fiber type: slow fibers contained a species (105 kD) slightly smaller than the principle variant (110 kD) in fast fibers. It is proposed that the type of paramyosin present could provide a biochemical marker to identify the fiber composition of muscles that have not been fully characterized. The diversity of troponin and myosin LC variants suggests that subtle differences in physiological performance exist within the broader categories of fast- and slow-twitch muscles. 31 references, 6 figures, 2 tables.

  13. Modeling Hand-Over-Hand and Inchworm Steps in Myosin VI

    NASA Astrophysics Data System (ADS)

    Jack, Amanda; Lowe, Ian; Tehver, Riina

    Myosin VI is a molecular motor protein that moves along actin filaments to transport cargo within a cell. There is much experimental evidence that the myosin VI dimer moves ``hand-over-hand'' along actin; however, recent experiments suggest that the protein can also move via an ``inchworm'' mechanism. We created a mechanochemical kinetic model to predict myosin VI's behavior under different ATP, ADP, and force conditions, taking these alternative mechanisms into account. Our model's calculations agree well with experimental results and can also be used to predict myosin VI's behavior outside experimentally tested regimes, such as under forward force. We also predict an optimized motor function for the protein around physiological (-2 pN) load and anchoring under -3 pN load. By using our model to predict myosin VI's response to environmental change, we can gain insight into the behavior of a protein that can be difficult to observe experimentally.

  14. Catch-bond behaviour facilitates membrane tubulation by non-processive myosin 1b

    NASA Astrophysics Data System (ADS)

    Yamada, Ayako; Mamane, Alexandre; Lee-Tin-Wah, Jonathan; di Cicco, Aurélie; Prévost, Coline; Lévy, Daniel; Joanny, Jean-François; Coudrier, Evelyne; Bassereau, Patricia

    2014-04-01

    Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.

  15. Still and rotating myosin clusters determine cytokinetic ring constriction

    PubMed Central

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  16. Axial disposition of myosin heads in isometrically contracting muscles.

    PubMed

    Juanhuix, J; Bordas, J; Campmany, J; Svensson, A; Bassford, M L; Narayanan, T

    2001-03-01

    Meridional x-ray diffraction diagrams, recorded with high angular resolution, from muscles contracting at the plateau of isometric tension show that the myosin diffraction orders are clusters of peaks. These clusters are due to pronounced interference effects between the myosin diffracting units on either side of the M-line. A theoretical analysis based on the polarity of the myosin (and actin) filaments shows that it is possible to extract phase information from which the axial disposition of the myosin heads can be determined. The results show that each head in a crown pair has a distinct structural disposition. It appears that only one of the heads in the pair stereospecifically interacts with the thin filament at any one time.

  17. Axial disposition of myosin heads in isometrically contracting muscles.

    PubMed Central

    Juanhuix, J; Bordas, J; Campmany, J; Svensson, A; Bassford, M L; Narayanan, T

    2001-01-01

    Meridional x-ray diffraction diagrams, recorded with high angular resolution, from muscles contracting at the plateau of isometric tension show that the myosin diffraction orders are clusters of peaks. These clusters are due to pronounced interference effects between the myosin diffracting units on either side of the M-line. A theoretical analysis based on the polarity of the myosin (and actin) filaments shows that it is possible to extract phase information from which the axial disposition of the myosin heads can be determined. The results show that each head in a crown pair has a distinct structural disposition. It appears that only one of the heads in the pair stereospecifically interacts with the thin filament at any one time. PMID:11222303

  18. Expression of myosin isoforms in the smooth muscle of human corpus cavernosum.

    PubMed

    Koi, P T; Milhoua, P M; Monrose, V; Melman, A; DiSanto, M E

    2007-01-01

    The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.

  19. Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin.

    PubMed

    Zhang, Ying; Kawamichi, Hozumi; Tanaka, Hideyuki; Yoshiyama, Shinji; Kohama, Kazuhiro; Nakamura, Akio

    2012-08-01

    We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

  20. Mutations in Myosin Light Chain Kinase Cause Familial Aortic Dissections

    PubMed Central

    Wang, Li; Guo, Dong-chuan; Cao, Jiumei; Gong, Limin; Kamm, Kristine E.; Regalado, Ellen; Li, Li; Shete, Sanjay; He, Wei-Qi; Zhu, Min-Sheng; Offermanns, Stephan; Gilchrist, Dawna; Elefteriades, John; Stull, James T.; Milewicz, Dianna M.

    2010-01-01

    Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections. PMID:21055718

  1. Myosin-10 independently influences mitotic spindle structure and mitotic progression.

    PubMed

    Sandquist, Joshua C; Larson, Matthew E; Hine, Ken J

    2016-06-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc.

  2. Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease

    PubMed Central

    Hering, Tanja; Braubach, Peter; Landwehrmeyer, G. Bernhard; Lindenberg, Katrin S.

    2016-01-01

    Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers. PMID:27820862

  3. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  4. Activated full-length myosin-X moves processively on filopodia with large steps toward diverse two-dimensional directions

    PubMed Central

    Sato, Osamu; Jung, Hyun Suk; Komatsu, Satoshi; Tsukasaki, Yoshikazu; Watanabe, Tomonobu M.; Homma, Kazuaki; Ikebe, Mitsuo

    2017-01-01

    Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10FullLZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M101–979HMM). M10FullLZ localizes at the tip of filopodia like myosin-X full-length (M10Full). M10FullLZ moves on actin filaments in the presence of PI(3,4,5)P3, an activator of myosin-X. Single molecule motility analysis revealed that the step sizes of both M10FullLZ and BAP-M101–979HMM are widely distributed on single actin filaments that is consistent with electron microscopy observation. M10FullLZ moves on filopodial actin bundles of cells with a mean step size (~36 nm), similar to the step size on single actin filaments (~38 nm). Cartesian plot analysis revealed that M10FullLZ meandered on filopodial actin bundles to both x- and y- directions. These results suggest that the lever-arm of full-length myosin-X is flexible enough to processively steps on different actin filaments within the actin bundles of filopodia. This characteristic of myosin-X may facilitate actin filament convergence for filopodia production. PMID:28287133

  5. UCS protein Rng3p is essential for myosin-II motor activity during cytokinesis in fission yeast.

    PubMed

    Stark, Benjamin C; James, Michael L; Pollard, Luther W; Sirotkin, Vladimir; Lord, Matthew

    2013-01-01

    UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.

  6. Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts.

    PubMed

    Chang, Wakam; Folker, Eric S; Worman, Howard J; Gundersen, Gregg G

    2013-12-01

    In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.

  7. Force Generation by Membrane-Associated Myosin-I

    PubMed Central

    Pyrpassopoulos, Serapion; Arpağ, Göker; Feeser, Elizabeth A.; Shuman, Henry; Tüzel, Erkan; Ostap, E. Michael

    2016-01-01

    Vertebrate myosin-IC (Myo1c) is a type-1 myosin that links cell membranes to the cytoskeleton via its actin-binding motor domain and its phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding tail domain. While it is known that Myo1c bound to PtdIns(4,5)P2 in fluid-lipid bilayers can propel actin filaments in an unloaded motility assay, its ability to develop forces against external load on actin while bound to fluid bilayers has not been explored. Using optical tweezers, we measured the diffusion coefficient of single membrane-bound Myo1c molecules by force-relaxation experiments, and the ability of ensembles of membrane-bound Myo1c molecules to develop and sustain forces. To interpret our results, we developed a computational model that recapitulates the basic features of our experimental ensemble data and suggests that Myo1c ensembles can generate forces parallel to lipid bilayers, with larger forces achieved when the myosin works away from the plane of the membrane or when anchored to slowly diffusing regions. PMID:27156719

  8. Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport

    PubMed Central

    Waterman-Storer, Clare; Duey, Devin Y.; Weber, Kari L.; Keech, John; Cheney, Richard E.; Salmon, E.D.; Bement, William M.

    2000-01-01

    Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid (∼250–300 nm/s) jerking and slow (∼50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings. PMID:10908578

  9. Bacterial nucleators: actin' on actin

    PubMed Central

    Bugalhão,